Compositions for blood-brain barrier transport

FIELD OF THE INVENTION

0001Described herein are compositions and methods for modulating blood-brain barrier transport of compounds. In addition, described herein are screening assays for identifying compounds that are useful for modulating transport across the blood-brain barrier.

BACKGROUND OF THE INVENTION

0002In the early 1980's, melanotransferrin (MTf) was identified as an oncofetal antigen that was either not expressed, or only slightly expressed in normal tissues, but was found in much larger amounts in neoplastic cells (especially malignant melanoma cells) and fetal tissues (<nplcit id="ncit0001" npl-type="s"><text>Woodbury, et al, P.N.A.S. USA, 77:2183-2187 (1980</text></nplcit>)). More recently, there have been additional reports of human MTf being identified in normal tissues, including sweat gland ducts, liver endothelial cells and the endothelium and reactive microglia of the brain (<nplcit id="ncit0002" npl-type="s"><text>Jefferies, et aL, Brain Res., 712:122-126 (1996</text></nplcit>); and <nplcit id="ncit0003" npl-type="s"><text>Rothenberger, et al., Brain Res., 712-117-121 (1996</text></nplcit>)). Interestingly, normal serum contains very low levels of soluble circulating MTf, but increased soluble serum MTf has been found in patients with advanced Alzheimer's Disease (<nplcit id="ncit0004" npl-type="s"><text>Kennard, et al., Nat. Med., 2:1230-1235 (1996</text></nplcit>); <patcit id="pcit0001" dnum="US5981194A"><text>U.S. Patent No. 5,981,194</text></patcit>)

0003The biochemical role and metabolism of MTf has proven difficult to elucidate. Based on appearances, MTf is deceptively similar to transferrin (Tf) and lactotransferrin (lactoferrin or Lf). In humans, these proteins share a 37-39% amino acid sequence homology. In particular, each of these proteins reversibly binds iron, and their N-terminal iron binding domains are quite similar (<nplcit id="ncit0005" npl-type="s"><text>Baker, et al., TIBS, 12:350-353 (1987</text></nplcit>)).

0004However, functional parallels between these proteins have not been confirmed. For one thing, unlike Tf and Lf, MTf exists in both a membrane bound form and a serum soluble form. Further, in contrast to Tf and Lf, no cellular receptor for MTf has been identified. Serum soluble Tf is known to be taken into cells in an energy-dependent process mediated by the transferrin receptor (Tf-R) (<nplcit id="ncit0006" npl-type="s"><text>Cook, et al., Annu. Rev. Med, 44:63-74 (1993</text></nplcit>)). Lf internalization is also likely to be mediated by a receptor mediated process (<nplcit id="ncit0007" npl-type="s"><text>Fillebeen, et al., J. Biol. Chem., 274(11):7011-7017 (1999</text></nplcit>)). Two known receptors for Lf are LRP1 and RAGE, although others may exist (<nplcit id="ncit0008" npl-type="s"><text>Meilinger, et al., FEBS Letters, 360:70-74 (1995</text></nplcit>); <nplcit id="ncit0009" npl-type="s"><text>Schmidt, J. Biol. Chem., 269(13):9882-9888 (1994</text></nplcit>)).

0005Although it has been postulated that MTf is an alternate ligand for Tf-R (<i>see,</i><patcit id="pcit0002" dnum="US5981194A"><text>U.S. Patent No. 5,981,194</text></patcit>), no published data confirms this finding. Further, although various studies have confirmed iron transport into cells by membrane bound MTf, it occurs only in cells where membrane bound MTf is overexpressed well beyond physiological levels (<nplcit id="ncit0010" npl-type="s"><text>Richardson, Eur. J. Biochem., 267:1290-1298 (2000</text></nplcit>)).

0006Therapeutic and diagnostic agents conjugated to soluble MTf are the basis of recently filed <patcit id="pcit0003" dnum="US226242P" dnum-type="L"><text>U.S. Provisional Patent Application Nos. 60/226,242</text></patcit> and <patcit id="pcit0004" dnum="US60226254P" dnum-type="L"><text>60/226,254</text></patcit>, the teachings of which are incorporated herein by reference for all purposes.

0007The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of pharmacological agents to cerebral tissues, necessitating the use of vectors for their transit. Blood-brain barrier (BBB) permeability is frequently a rate-limiting factor for the penetration of drugs or peptides into the central nervous system (CNS) (see <nplcit id="ncit0011" npl-type="s"><text>Pardridge, W.M. J. Neurovirol. 5: 556-569 (1999</text></nplcit>); <nplcit id="ncit0012" npl-type="s"><text>Bickel, U., Yoshikawa, T. &amp; Pardridge, W.M. Adv. Drug Deliv. Rev. 46: 247-279 (2001</text></nplcit>)). The brain is shielded against potentially toxic substances by the BBB, which is formed by brain capillary endothelial cells that are closely sealed by tight junctions. In addition, brain capillaries possess few fenestrae and few endocytic vesicles, compared to the capillaries of other organs (see <nplcit id="ncit0013" npl-type="s"><text>Pardridge, W.M. J. Neurovirol. 5: 556-569 (1999</text></nplcit>)). There is little transit across the BBB of large, hydrophilic molecules aside from some specific proteins such as transferrin, lactoferrin and low-density lipoproteins, which are taken up by receptor-mediated endocytosis (see <nplcit id="ncit0014" npl-type="s"><text>Pardridge, W.M. J. Neurovirol. 5: 556-569 (1999</text></nplcit>); <nplcit id="ncit0015" npl-type="s"><text>Tsuji, A. &amp; Tamai, I. Adv.Drug Deliv.Rev. 36: 277-290 (1999</text></nplcit>); <nplcit id="ncit0016" npl-type="s"><text>Kusuhara, H. &amp; Sugiyama, Y. Drug Discov. Today 6:150-156 (2001</text></nplcit>); <nplcit id="ncit0017" npl-type="s"><text>Dehouck, B. et al. J. Cell. Biol. 138: 877-889 (1997</text></nplcit>); and <nplcit id="ncit0018" npl-type="s"><text>Fillebeen, C. et al. J. Biol. Chem. 274: 7011-7017 (1999</text></nplcit>).

0008<patcit id="pcit0005" dnum="WO0004926A"><text>WO 00/04926</text></patcit> describes methods and compositions for treating secondary tissue damage and other inflammatory conditions.

0009In order to understand and improve the delivery of therapeutic agents into cells, it is highly desirable to understand the receptors and metabolic basis of MTf activity. It is an object described herein to identify the receptor for MTf, and to provide methods and compounds for improving the delivery of therapeutic and diagnostic agents into cells, in particular, therapeutic and diagnostic agents conjugated to MTf, and particularly their delivery across the blood-brain barrier.

(2) BRIEF SUMMARY OF THE INVENTION

0010The present application is in its broadest sense defined by the appended claims.

0011Described herein are compositions and methods for modulating trancystosis, endocytosis, and blood-brain barrier transport of compounds. In addition, described herein are screening assays for identifying compounds that are useful for modulating transport across the blood brain barrier and for delivering active agents conjugated to such agents which undergo endocytosis or transcytosis upon binding to the LRP (e.g., LRP1 and LRP1B) receptor.

0012Described herein are methods for screening compounds for the ability to modulate the endocytosis or transcytosis of p97 and p97 conjugated to active agents by asssessing their ability to bind a low density lipoprotein receptor related protein (LRP). In another aspect, described herein are modulators of the endocytosis or transcytosis of p97 and p97 conjugated to active agents. In another aspect, described herein are conjugates of compounds (e.g., Ligands) which bind to the LRP receptor and which undergo endocytosis or transcytosis upon binding to the LRP receptor. In another aspect, described herein are methods of determining the capability of a given cell, cell type, or tissue to endocytose or transcytose p97 or a p97 conjugate to an active agent by measuring the expression of an LRP receptor nucleic acid or protein of the cell, cell type, or tissue.

0013In another aspect, described herein are ligands and modulators of the LRP family of receptors for use as modulators of p97 transcytosis. In another aspect, described herein are conjugates of LRP receptor ligands for use in delivering therapeutic agents across the blood brain barrier and/or to an intracellular compartment, particularly the lysosome. In another aspect, described herein are methods for modulating the expression of LRP receptors by administering p97.

0014As described, the receptor is the LRP1 receptor. As described the receptor is the LRP1B receptor.

0015Disclosed herein is a method for identifying a compound that modulates or binds to a melanotransferrin receptor ("MTf-R" or "p97 receptor"), the method comprises contacting the compound with the p97 receptor and determining the effect of LRP receptor ligands (e.g., lactoferrin, p97, BSA, aprotinin, and RAP) on the interaction of the compound with the receptor. One or more LRP receptor ligands (e.g., lactoferrin, RAP, BSA, aprotinin, and p97) are individually contacted with the p97 receptor and the effect of the compound on the interaction of each compound with the p97 receptor is respectively determined. The functional effect is an effect on transcytosis or on endocytosis. The effect is on receptor binding as measured by the changes in the binding, for instance, of RAP, p97, or lactoferrin to the p97 receptor. The method is a high throughput screening assay. The receptor is the LRP1 receptor. The receptor is the LRP1B receptor.

0016Disclosed herein is a method for identifying a selective modulator of p97 receptor activity, by contacting the modulator and a ligand of the LDL-R receptor family ligand with the melanotransferrin receptor and determining the ability of one to affect the binding or interaction of the other with the melanotransferrin receptor. The LDL-R receptor ligand is selected from PAI-1 (plasminogen aativator inhibitor type-1), plasminogen, pro-uPA (pro-urokinase plasminogen activator), tissue factor inhibitor, tPA (tissue type plasminogen activator), activated α₂-macroglobulin α₁-chymotrypsin, cathepsin G, lactoferrin, RAP (receptor associated protein), thyroglobulin, circumsporozite protein, saposin, gentamycin, polymixin B, pseudomonas exotoxin A, seminal vesicle secretory protein A, thrombospondin -1,β-VLDL, chylomicron remnants, IDL, Lp(a), VLDL (very low density lipoprotein), ApoB100 (apolipoprotein B 100), and Apolipoprotein E (Apo E). Alternatively, a plurality of such ligands are selected and the selectivity of the compound for the receptor is assessed according to how similarly to p97 the compound's interaction with the receptor is affected by such ligands. The LDL-R receptor ligand is a LRP receptor ligand, more preferred, the LRP receptor ligand is an LRP1 receptor ligand or an LRP1B receptor ligand. The effect on binding is assessed indirectly by a functional effect (e.g., transcytosis, endocytosis). In another example, the effect is on receptor binding as measured by the changes in the binding, for instance, of RAP, p97, or lactoferrin from the p97 receptor. In certain examples, the method is a high throughput screening assay. In a further example, the method is a BioCore method. In a preferred example, the receptor is the LRP 1 receptor. In a more preferred example, the receptor is the LRP1B receptor. In another example, the screening assay is measures the competitive displacement of a ligand, preferably p97, of the p97 receptor,

0017In one example, the melanotransferrin receptor modulator or ligand has neurological activity such that it is useful in the treatment, prophylaxis or diagnosis of a neurological disorder. In other examples, the melanotransferrin receptor modulator or ligand is useful for the modulation of the uptake of melanotransferrin conjugated therapeutic agents into the brain. In still other examples, the compound is useful for reducing a neurological side-effect of such a therapeutic agent. In a preferred example, the disease or disorder is Alzheimer's disease.

0018In one example, the p97 modulatory compound has neurological activity such that it is useful in the treatment, prophylaxis or diagnosis of a neurological disorder. In other examples, the compound is useful for the modulation of the uptake of melanotransferrin conjugated therapeutic agents into the brain. In still other examples, the compound is useful for reducing a neurological side-effect of a therapeutic agent. In another example, disclosed herein is a method of treating a neurological disorder in a patient, the method comprising administering to the patient a therapeutically effective amount of the modulatory campound.

0019In one example, the p97 conjugate or p97 receptor ligand-conjugate comprises a therapeutic agent useful in treating a lysosomal storage disease. In one example, the therapeutic agent is an enzyme deficient in a patient having such a disorder. In one example the enzyme is iduronidase. Diclosed herein is a method of treating a lysosomal storage disease in a patient, the method comprising administering to the patient with a lysosomal storage disease a therapeutically effective amount of p97 conjugated to an enzyme with an activity which is deficient in the lysosomal storage disease. In one example, a modulator or LRP or LRP1B is co-administered to modulate the therapeutic or adverse effects of such a conjugate. In one example, the conjuate is a fusion protein comprising a p97 portion and an enzyme portion wherein the enzyme provides the enzymatic activity deficient in the lysosomal storage disease. In one example the enzyme is α-iduronidase. In one example, the p97 portion is a fragment of p97 sufficient for the endocytosis or transcytosis of the conjugate or fusion protein.

0020In one example, the p97 conjugate or p97 receptor modulator-conjugate comprises a therapeutic agent useful in treating a CNS tumor such as a glioblastoma. In one example, the therapeutic agents is cancer chemotherapeutic agent. Disclosed herein is a method of treating a patient with a brain or CNS tumor or glioblastoma by administering to the patient a therapeutically effective amount of p97 conjugated to the chemotherapeutic agent. In a preferred example, the conjugate binds to the LRP1B receptor. In one example, a modulator or LRP or LRP1B is co-administered to modulate the therapeutic or adverse effects of such a conjugate.

0021Disclosed herein is a method of modulating a melanotransferrin receptor ("MTf-R"), the method comprises contacting the MTf-R with a modulator identified using the above method.

0022Disclosed herein is a method for increasing the uptake of a melanotransferrin conjugated therapeutic agent into the brain of a patient, the method comprising administering a modulator of MTf-R biological activity and the melanotransferrin conjugated therapeutic agent. In one example, the modulator of MTf-R biological activity and the melanotransferrin conjugated therapeutic agent are administered contemporaneously. In another example, the modulator of MTf-R biological activity and the melanotransferrin conjugated therapeutic agent are administered sequentially.

0023Disclosed herein is a method of reducing the uptake of a melanotransferrin conjugated therapeutic agent into the brain of a patient, the method comprising admmistering a modulator of MTf-R biological activity with a melanotransterrin conjugated therapeutic agent, wherein they are administered either contemporaneously or sequentially. In certain examples, the modulator is first identified according to the above method.

0024Also disclosed herein are modulators of MTf-R biological activity, wherein the modulator is identified using the above method. Preferably, the modulator is useful for reducing a neurological side-effect of a therapeutic agent.

0025Disclosed herein is a method of identifying a compound that modulates melanotransferrin-mediated ("MTf-mediated) iron uptake, the method comprising: contacting a cell expressing MTf on its surface with the compound in the presence of MTf bound to iron ("holo-MTf") and in the absence of transferrin; and determining the amount of iron uptake into the cell. In certain examples, the compound increases the amount of iron uptake into the cell. In other examples, the compound decreases the amount of iron uptake into the cell.

0026In another aspect, described herein are pharmaceutical compositions comprising such modulators, conjugates, and ligands and methods of using such pharmaceutical compositions. Disclosed herein is a pharmaceutical composition comprising a p97 receptor ligands conjugate for delivering an active agent across the blood brain barrier or into an intracellular compartment. The conjugate can be administered in a pharmaceutically acceptable carrier or diluent.

0027The present invention refers to a pharmaceutical composition comprising (a) a conjugate comprising receptor associated protein (RAP) and a chemotherapeutic agent, and (b) a sterile pharmaceutically acceptable carrier, wherein the chemotherapeutic agent is selected from the group consisting of (i) an alkylating agent, (ii) an alkaloid, (iii) an antimetabolite, (iv) an anticellular proliferative agent selected from Hydroxyurea, Procarbazine, Dacarbazine, Cisplatin, Carboplatin, Asparaginase, Etoposide, Amsarcrine, Mitotane, and Mitoxantrone, and (v) an antibiotic, for use in the treatment of a neurological infection or neurological cancer by delivering the chemotherapeutic agent across the blood-brain barrier (BBB).

0028In other examples, a fusion protein comprising a peptide ligand of the LRP1 or LRP1B receptor may be used as the conjugate. The conjugate may therefore be a chimeric fusion protein combining a p97 peptide portion with a peptide active agent portion. The fusion protein active agent may be a substance having therapeutic activity such as a growth factor or lymphokine or peptide drug. The active agent may be an enzyme or other protein. In a preferred examples, the fusion protein comprises an active agent which is an enzyme that is deficient or has an activity deficient in a lysosomal storage disease. In particular, enzymes such as α-L-iduronidase or N-acetylgalactosamine 4-sulfatase are contemplated. Also described are examples where such a fusion protein is administered to a subject having a lysosomal storage disease. In other examples, the enzyme is an enzyme deficient in a human disease such as PKU (e.g., phenylase). In other examples, the enzyme is selected for its beneficial effect (e.g. heparinase I to limit the action of heparin). Such conjugates may alternatively be conjugated by synthetic chemistry reactions or joined by linger groups. One of ordinary skill in the art would understand how to make such fusion proteins. See, for instance, U.S. Patent Application No. <patcit id="pcit0006" dnum="US20010025026A1"><text>US 2001/0025026A1, published September 27, 2001</text></patcit>. In some examples, the fusion protein may be formed from expression of a nucleic acid encoding the full amino acid sequence of the chimeric protein. In other examples, the chimeric fusion protein may be formed by a synthetic reaction combining the two peptides portions via a peptide bond directly linking the two portions.

0029In other examples, the conjugate is alternatively formed by non-covalent bonds between the carrier and an antibody to which the active agent is attached.

0030Also described herein is a method of delivering an active agent across the blood brain barrier comprising administering a conjugate of p97 or another ligand of the LRP1B receptor which undergoes endocystosis or transcytosis. The compositions described herein may also be used for delivering an agent across the blood eye or blood placenta barrier or intracellular to a lysosome.

0031In some examples, the conjugate does not comprise p97, aprotinin, lactoferrin, or aprotinin, or or portions thereof with LRP1B or LRP binding activity. In some examples, the conjugate does not comprise PAI-1, plasminogen, pro-uPA, tissue factor inhibitor, tPA, activated α₂-macroglobulin, α₁-chymotrypsin, cathepsin G, lactoferrin, thyroglobulin, circumsporozite protein, saposin, gentamycin, polymixin B, pseudomonas exotoxin A, seminal vesicle secretory protein A, thrombospondin -1, β-VLDL, chylomicron remnants, IDL, Lp(a), VLDL (very low density lipoprotein), ApoB 100 (apolipoprotein B 100), and Apolipoprotein E (Apo E), or portions thereof with LRP1B or LRP binding activity.

0032In some examples, the conjugate comprises PAI-1, plasminogen, pro-uPA, tissue factor inhibitor, tPA, activated α₂-macroglobulin, α₁-chymotrypsin, cathepsin G, lactoferrin, thyroglobulin, circumsporozite protein, saposin, gentamycin, polymixin B, pseudomonas exotoxin A, seminal vesicle secretory protein A, thrombospondin -1, β-VLDL, chylomicron remnants, IDL, Lp(a), VLDL (very low density lipoprotein), ApoB 100 (apolipoprotein B 100), and Apolipoprotein E (Apo E), or portions thereof with LRP1B or LRP binding activity.

0033In preferred examples, the p97 or LRP1 or LRP1B ligand or modulator of human or mammalian origin. In other examples, the ligand is the native compound from the human or mammal. In other examples, the ligand is substantially homologous (at least 60% identical in amino acid sequence or atomic structure) to the native or endogenous ligand. In other examples, the p97 receptor is human. In other examples, the subject to which the conjugate, modulator, or ligand is to be administered is human.

BRIEF DESCRIPTION OF THE DRAWINGS

0034<ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001">Figure 1</figref>. Permeability coefficient (Pe) of sucrose with BBCECs. The permeability coefficient (Pe) for sucrose was calculated as previously described (Dehouck, <i>et al., J. Neurochem., 58</i>:1790-1797 (1992)) using filters coated or non-coated with BBCE cells co-cultured with astrocytes. Briefly, coated or non-coated filters with BBCE cells were transferred to 6-well plates containing 2 ml of Ringer-Hepes per well (basolateral compartment) for 2 hrs at 37°C. In each apical chamber, the culture medium was replaced by 1 ml Ringer-Hepes containing labeled [¹⁴C]-sucrose. At different times, inserts were placed into another well. At the end of the experiments, amounts of the radio tracers in the basolateral compartment were measured in a liquid scintillation counter. The results were plotted as the sucrose clearance (µl) as a function of time (min). The permeability of sucrose with non-coated filters with BBCE cells (PSf) and with coated filters with BBCE cells (PSt) were obtained. The results were plotted as the clearance of [¹⁴C]-sucrose (µl) as a function of time (min). PS = permeability x surface area of a filter of the coculture; PSt = permeability of the filter and endothelial monolayer; PSf= permeability of a filter coated with collagen and astrocytes plated on the bottom side of the filter. The permeability coefficient (Pe) of the endothelial monolayer was calculated as: <ol id="ol0001" compact="compact" ol-style=""><li>1) <maths id="math0001" num=""><math display="block"><mi>Clearance</mi><mfenced><mi mathvariant="normal">μl</mi></mfenced><mo>=</mo><mfrac><mrow><msub><mfenced open="[" close="]"><mi mathvariant="normal">C</mi></mfenced><mi mathvariant="normal">A</mi></msub><mo>×</mo><msub><mi mathvariant="normal">V</mi><mi mathvariant="normal">A</mi></msub></mrow><msub><mfenced open="[" close="]"><mi mathvariant="normal">C</mi></mfenced><mi mathvariant="normal">L</mi></msub></mfrac></math><img file="EP2147679B1_D0001.tif" /></maths><ul id="ul0002" list-style="none" compact="compact"><li>[C]_A = Abluminal tracer concentration</li><li>V_A = Volume of abluminal chamber</li><li>[C]_L = Luminal tracer concentration</li></ul></li><li>2) <maths id="math0002" num=""><math display="block"><mn>1</mn><mo>/</mo><mi>Pe</mi><mo>=</mo><mfenced separators=""><mn>1</mn><mo>/</mo><mi>PSt</mi><mo>-</mo><mn>1</mn><mo>/</mo><mi>PSf</mi></mfenced><mo>/</mo><mi>filter area</mi><mspace width="1em" /><mfenced separators=""><mn>4.2</mn><mspace width="1em" /><msup><mi>cm</mi><mn>2</mn></msup></mfenced></math><img file="EP2147679B1_D0002.tif" /></maths></li></ol></li><li><figref idref="f0002">Figure 2</figref>. Effect of p97 (1 µm) on sucrose permeability with BBCECs that were preincubated for 2 hrs at 37°C. The effect ofp97 at 1 µM on sucrose permeability of the <i>in vitro</i> BBB model was tested. The permeability coefficient (Pe) for sucrose was calculated as previously described (<nplcit id="ncit0019" npl-type="s"><text>Dehouck, et al., J. Neurochem, 58:1790-179 (1992</text></nplcit>)) using filters coated or non-coated with BBCE cells. Briefly, coated or non-coated filters with BBCE cells were transferred to 6-well plates containing 2 ml of Ringer/Hepes per well (basolateral compartment) for 2 hrs at 37°C. In each apical (<i>i.e.</i>, luminal) chamber, the culture medium was replaced by 1 ml Ringer/Hepes containing labeled [¹⁴C]-sucrose. At different times, inserts were placed into another well. At the end of the experiments, amounts of the radiotracers in the basolateral (<i>i.e.</i>, abluminal) compartment were measured in a liquid scintillation counter. The permeability coefficient (Pe) was calculated as described in <figref idref="f0001">Figure 1</figref>.</li><li><figref idref="f0003">Figure 3</figref>. Binding of p97 on bovine brain capillary endothelial cell monolayers. Binding p97 was performed with BBCECs that were pre-incubated 2 hrs in Ringer-Hepes to avoid any interference from the astrocytes. For the binding experiments, cells were incubated for 2 h at 4°C in Ringer/Hepes in the presence of [¹²⁵I]-p97 (25 nM) and 7.5 micromolar concentrations of cold-p97, transferrin or lactoferrin, respectively. At the end of the incubation, the filters were gently washed at 4°C three times with 4 ml of cold-PBS. Then the associated radioactivity of endothelial cells was determined by removing the membrane of the culture insert and counting it in a gamma counter.</li><li><figref idref="f0004">Figure 4</figref>. Binding of p97 on rat brain endothelial cells. Binding p97 was performed with RBE4 that were pre-incubated 2 hrs in Ringer-Hepes. For the binding experiments, cells in 24-well microplates were incubated for 2 h at 4°C in Ringer/Hepes in the presence of [¹²⁵I]-p97 (25 nM) and 10 micromolar concentrations of cold-p97, transferrin or lactoferrin, respectively. At the end of the incubation, the cells were gently washed at 4°C three times with 4 ml of cold-PBS. RBE4 cells were then lysed with Triton X-100 and the associated radioactivity of with cell lysates was determined.</li><li><figref idref="f0005">Figure 5</figref>. Accumulation of p97 in human brain capillaries. Accumulation of [¹²⁵I]-p97 was measured at 37°C for 1 h in isolated human brain capillaries (100 µg/assay). The incubation medium contained [¹²⁵I]-p97 and a final concentration of 100 nM p97 in Ringer/Hepes solution. The accumulation of [¹²⁵I]-p97 was performed in the presence or absence of 5 µM cold-p97, holo-transferrin or lactoferrin. After incubation, the accumulation was stopped by addition of 1 ml-cold stop solution (150 mM NaCl, 0.1% BSA and 5 mM Hepes, pH 7.5). The suspension was filtered under vacuum through a 0.45µM pore size Millipore filter. The filter was rinsed with 8 ml of stop solution, and the radioactivity was counted. Nonspecific binding of the radioactivity to the capillaries was determined by the addition of the ice-cold stop solution to the capillaries before adding the incubation medium. This value was subtracted from accumulation values obtained following an 1 h incubation. The results were expressed as ng of [¹²⁵I]-p97 accumulated per µg of brain capillaries.</li><li><figref idref="f0006">Figure 6</figref> is a repeat experiment, performed identically to the experiment of <figref idref="f0005">Figure 5</figref>, except this time with the additional competition assay for β-amyloid peptide. Evidently, the β -amyloid peptide 1-40 competes with p97 for receptor binding, along with lactoferrin, but not with transferrin.</li><li><figref idref="f0007">Figure 7</figref> illustrates the results of experiments where the ligands p97, Lf and Tfwere heated or not heated prior to the binding study. In all cases, binding experiments were conducted in the transwell apparatus as described previously, with the exception that binding was conducted at either 4°C or at 37°C. For the 37°C trial, a separate experiment was conducted where the ligand was boiled for 30 mins then rapidly cooled prior to administration on the transwell plates.</li><li><figref idref="f0008">Figure 8</figref> demonstrates that p97 transcytosis was significantly higher at 37°C than at 4°C. This result demonstrates that p97 is actively transported in an energy-dependent process across this blood-brain barrier model in a temperature-dependent fashion, presumably by receptor mediated uptake.</li><li><figref idref="f0009">Figure 9</figref> confirms that transcytosis of p97 is also a saturable phenomenon, thus further implicating a specific MTf-receptor protein in this model of the blood-brain barrier. These experiments were conducted as previously described. Measurements of the amount of transcytosis were made at the time points indicated.</li><li><figref idref="f0010 f0011">Figure 10</figref> assesses the ability of several agents to interfere with p97 transyctosis. <figref idref="f0010">Figure 10a</figref>, transcytosis of I125-P97 was compared in the presence of cold p97 (5 micromolar), Lf (5 micromolar), and Tf (5 micromolar). <figref idref="f0010">Figure 10b</figref>, β -amyloid protein (5 micromolar) also failed to slow or reduce transcytosis of labelled p97. <figref idref="f0011">Figure 10c</figref>, RAP, a known poly-peptide inhibitor of the LDL-Receptor family was applied to the cells (25 micrograms/ml). RAP significantly inhibited the transcytosis of p97, thus directly implicating the LDL-receptor family, especially LRP1 as the MTf-R.</li><li><figref idref="f0012">Figure 11</figref> examines the accumulation and transcytosis of p97. <figref idref="f0012">Figure 11a</figref>, of [¹²⁵I]-p97, [¹²⁵I]-BSA and [¹²⁵I]-holo-transferrin one hour after i.v. injection. The radioactivity in brain was compared between the three compounds. (n=3); <figref idref="f0012">Figure 11b</figref>, Accumulation of p97 and transferrin into the mouse brain parenchyma. In situ brain perfusion was performed with human [¹²⁵I]-p97 or [¹²⁵I]-holo-transferrin at a final concentration of 10 nM via a catheter inserted in the right common carotid artery. The volume of distribution (VD) of [¹²⁵I]-proteins were obtained in the whole brain homogenate (white bars), in brain capillaries (solid bars) and in brain parenchyma (hatched bars) after isolation of the right hemisphere and capillary depletion. Results were obtained for p97 (n=10) and for holo-transferrin (n=6); <figref idref="f0012">Figure 11c</figref>, Transcytosis of p97 across BBCEC monolayers. Transcytosis experiments were performed 37 °C (open circles) and 4 °C (closed circles) with BBCEC monolayers. [¹²⁵I]-p97 (1mg/ml) was added to the upper side of the cell-covered filter. One representative experiment is shown (n=4); <figref idref="f0012">Figure 11d</figref>, Preferential transport of p97 across the BBCEC monolayers. Apical-to-basal and basal-to-apical transport of [¹²⁵I]-p97 (25 nM) was measured for 2 hrs at 37°C. At the end of the experiment, [¹²⁵I]-p97was assessed in the lower or upper chambers of each well by TCA precipitation (n=4).</li><li><figref idref="f0013">Figure 12</figref> illustrates the Binding of p97 to BBCE cells. <figref idref="f0013">Figure 12a</figref>, p97 binding experiments were performed with BBCECs that were either pre-incubated in Ringer-Hepes solution or pre-treated with saponin. BBCECs were then incubated for 2 hrs at 4 °C with [¹²⁵I]-p97 (25 nM). At the end of the incubation, the filters were gently washed with cold PBS and then the radioactivity associated with the ECs was quantified; <figref idref="f0013">Figure 12b</figref>, The binding of [¹²⁵I]-p97 was also performed with increasing concentrations of unlabelled p97 following saponin treatment. The results were expressed as the percentage of the [¹²⁵I]-p97 binding measured in the absence of unlabelled p97; <figref idref="f0013">Figure 12c</figref>, The results were also transformed with a Scatchard plot and expressed as the ratio of bound p97/free p97 as a function of the bound p97 (n=5).</li><li><figref idref="f0014">Figure 13</figref> illustrates the transcytosis and accumulation of p97 and transferrin in BBCEC monolayers. <figref idref="f0014">Figure 13a</figref>, Transcytosis experiments were performed at 37 °C (solid bars) or 4 °C (white bars). [¹²⁵I]-p97 or bovine [¹²⁵I]-holo-transferrin (1 mg/ml) was added to the uper side of the cell-covered filter. At the end of the experiment, radiolabelled proteins were measured in the lower chamber of each well by TCA precipitation (n=4). Controls were also performed at 37 °C with denatured [¹²⁵I]-p97 or bovine [¹²⁵I]-holo-transferrin boiled for 30 min (grey bars) (n=2); <figref idref="f0014">Figure 13b</figref>, Biospecific interaction analysis was performed with native or boiled p97 for the indicated times. MAb L235 (5 µg) was immobilized on a sensor chip (SM5) using standard procedures incorporating NHS, EDC and ethanolamine. Native and boiled p97 (5 to 30 min) diluted at 1 mg/ml in Ringer/Hepes was cooled and injected into the BIAcore. The surface plasmon resonance response obtained for native p97 and boiled p97 was plotted (in relative units (RU)) as a function of time; <figref idref="f0014">Figure 13c</figref>, The accumulation of both proteins into BBEC cells were also measured. Briefly, after incubation at 37 °C (solid bars) or 4 °C (white bars) with either [¹²⁵I]-protein, cells were washed four times with cold PBS. Accumulation of both denatured proteins (grey bars) was also measured at 37 °C. Filters were then removed, and the radioactivity associated with the cells was quantified <i>(n</i> =3).</li><li><figref idref="f0015">Figure 14</figref> illustrates the stability of p97 and integrity of the BBCEC monolayers following p97 transcytosis. <figref idref="f0015">Figure 14a</figref>, Transcytosis experiments were performed at 37 °C and 4 °C by adding p97 (1 mg/ml) to the upper compartment. At the end of the experiment, 50 µl from each lower chamber was used for SDS-polyacrylamide gel electrophoresis. After electrophoresis, the gels were stained with Coomassie Blue. A standard curve was also made with known amounts of recombinant p97 (0-2 µg); <figref idref="f0015">Figure 14b</figref>, The gels were dried and scanned to quantify the amount of intact p97 that crossed the BBCEC monolayers at 37 °C and 4 °C. (n=3); <figref idref="f0015">Figure 14c</figref>, Effect of p97 on sucrose permeability of BBCE cell monolayers co-cultured with astrocytes. The passage of [₁₄C]-sucrose was measured with filters (à) or with filters coated with BBCE cells in the absence (open circles) or in the presence of p97 (up to 1 mg/ml (closed circles). One representative experiment is shown. The results were plotted as the sucrose clearance (µl) as a function of time (min); <figref idref="f0015">Figure 14d</figref>, The sucrose permeability coefficient (Pe) was determined in the presence (+p97) or in the absence (-p97) of p97, and was calculated as described in the Examples (n=3).</li><li><figref idref="f0016">Figure 15</figref> shows the inhibition of [¹²⁵I]-p97 transcytosis in BBCEC monolayers. <figref idref="f0016">Figure 15a</figref>, Transport of [¹²⁵I]-p97 (25 nM) from the apical to the basolateral side of ECs was measured in the absence (open circles) or in the presence (closed circles) of a 200-fold molar excess of unlabelled p97; <figref idref="f0016">Figure 15b</figref>, The effects of a 200-fold molar excess of either human or bovine transferrin (Tf) and p97 were also evaluated on [¹²⁵I]-p97 transcytosis across BBCEC monolayers (n=5); <figref idref="f0016">Figure 15c</figref>, Transcytosis of [¹²⁵I]-p97 (25 nM) was alsomeasured in the presence of mouse IgGs (open circles) or mAb OX-26 (closed circles) at a concentration of 5 µg/ml (n=3)</li><li><figref idref="f0017">Figure 16</figref> illustrates the identification of a p97 receptor according to ligand specificity. <figref idref="f0017">Figure 16a</figref>, Uptake of [¹²⁵I]-p97 into human brain capillaries. The uptake of [¹²⁵I]-p97 at 100 nM (control) into isolated human brain capillaries was measured for 1 h at 37 °C in the presence of a 50-fold molar excess of unlabelled p97, human holo-transferrin or human lactoferrin. (*<i>P</i> &lt; 0.5 Student's t-test (n=5); <figref idref="f0017">Figure 16b</figref>, Effect of RAP on [¹²⁵I]-p97 Transcytosis across BBCEC monolayers. Apical-to-basal transport of [¹²⁵I]-p97 was measured in the presence (closed circles) or absence (open circles) of RAP (25 µg/ml) (n=5); <figref idref="f0017">Figure 16c</figref>, Inhibition of bovine [¹²⁵I]-lactoferrin transport by p97. Transcytosis of bovine [¹²⁵I]-lactoferrin (50 nM) was measured in the presence (closed circles) or absence (open circles) of unlabelled p97 (5 µM) at 37 °C (n=3).</li><li><figref idref="f0018">Figure 17</figref>. Examination of the role of LRP on p97 transcytosis. <figref idref="f0018">Figure 17A</figref>. Apical-to-basal transport of [¹²⁵I]-p97 was measured for 2 h at 37°C in the absence (Control) or presence of RAP (0.6 µM), aprotinin (5µM) or BSA (5 µM). Results represent means ± SD (n=5 for RAP; n=3 for aprotinin and BSA). [¹²⁵I]-p97 was assessed in the lower chamber of each well by TCA precipitation. <figref idref="f0018">Figure 17B</figref>. Inhibition of bovine [¹²⁵I]-lactoferrin transport by p97. Transcytosis of bovine [¹²⁵I]-lactoferrin (50 nM) was measured in the presence or absence of unlabelled p97 (5 µM) at 37°C for 2 hrs. Bovine [¹²⁵I]-lactoferrin was assessed in the lower or upper chambers of each well by TCA precipitation. Results represent means ± SD (n=6). Some ligands for LRP and megalin are presented in <figref idref="f0018">Figure 17C</figref>.</li><li><figref idref="f0019">Figure 18</figref> illustrates the time-course of the uptake of p97 in BBCE and accumulation in early endosome.</li><li><figref idref="f0020">Figure 19</figref> is a schematic of how the p97 transport rate across the blood brain barrier of the model is determined.</li><li><figref idref="f0021">Figure 20</figref> presents the results of measuring the rate of internalization and transcytosis of p97 in the blood brain barrier model.</li><li><figref idref="f0022">Figure 21</figref> depicts the transcellular co-localization of p97 and clathrin turning transcytosis.</li><li><figref idref="f0023">Figure 22</figref> is a schematic drawing of two of several pathways possible following endocytosis of a p97 compound or conjugate: transcytosis and lysosomal delivery.</li><li><figref idref="f0024">Figure 23</figref> is a schematic drawing of the LRP receptor associated with a cell membrane and a number of the ligands of such LRP receptors.</li><li><figref idref="f0025">Figure 24</figref>. Uptake of p97 in rat astrocytes and astrocytoma cells. Cells were grown in monolayer in six-multiwell microplates at 37°C under 5% CO2. Uptake of [¹²⁵I]-p97 was measured at 37°C for 2 h in astrocytes and astrocytomas. The incubation medium contained [¹²⁵I]-p97 and a final concentration of 50 nM p97 in Ringer/Hepes solution. After incubation, the cell monolayer was washed three times with cold Ringer/Hepes solution. Triton X-100 0.1% was added and the [¹²⁵I]-p97 uptake was assessed in the Triton X-100 soluble fraction by TCA precipitation. Results represent means ± SD (n=3, Astro; n=6, CTX and RG2; n=9, C6).</li><li><figref idref="f0026">Figure 25</figref>. Immunodetection of LRP/LRP 1B. Cells lysates (25 (g) were subjected to SDS-PAGE under non-reducing conditions and electroblotted onto PVDF membranes. LRP/LRP1B was immunodetected using a rabbit polyclonal antibody raised against the low density lipoprotein receptor-related protein as previously described (<nplcit id="ncit0020" npl-type="s"><text>Bu et al., J. Biol. Chem. 17:13002-13009, 1993</text></nplcit>). (n=1)</li><li><figref idref="f0027">Figure 26</figref>. Immunodetection ofmegalin. Cells lysates were subjected to SDS-PAGE under non-reducing conditions and electroblotted onto PVDF membranes. Megalin was immunodetected using a mouse monoclonal antibody diluted 1/2500 in TBS-Tween 0.1 %, 3% BSA and 0.01% NaN3. (n=1)</li><li><figref idref="f0028">Figure 27</figref>. mRNA extraction and RT-PCR of LRP 1B. For RNA extraction, cells were grown in 75 cm² plastic tissue culture flasks at 37°C under 5% CO2 with optimal culture medium to 80-90% confluence. Total RNA from each cell line was extracted as described in the Examples. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized under ultraviolet light. (n=1).</li><li><figref idref="f0029">Figure 28</figref> presents some results of experiments correlating p97 uptake with LRP 1B expression.</li><li><figref idref="f0030">Figure 29</figref>. LRP/LRP1B migrates as a high molecular weight dissociable complex in presence of p97. U-87 cells were grown in monolayer in six-multiwell microplates at 37°C under 5% CO2. The cell monolayer were incubated at 4°C with p97 or RAP in Ringer/Hepes solution. After incubation, cell monolayer was washed 3 times with cold- Ringer/Hepes solution and proteins were cross-linked with 1mM DSS. After, cells were lysed and equal quantities of protein (25 µg) were subjected to SDS-PAGE under non-reducing conditons and electroblotted onto PVDF membranes. LRP/LRP1B was immunodetected as described in Examples using a mouse monoclonal antibody raised against human LRP α-subunit (clone 8G1 from Research Diagnostics inc.) at a 1/500 dilution in TBS-Tween 0.1%, 3% BSA and 0.01% NaN₃. (n=2).</li><li><figref idref="f0030">Figure 29</figref>. LRP/LRP1B migrates as a high molecular weight dissociable complex in presence of p97. U-87 cells were grown in monolayer in six-multiwell microplates at 37°C under 5% CO2. The cell monolayer were incubated at 4°C with p97 or RAP in Ringer/Hepes solution. After incubation, cell monolayer was washed 3 times with cold- Ringer/Hepes solution and proteins were cross-linked with 1mM DSS. After, cells were lysed and equal quantities of protein (25 µg) were subjected to SDS-PAGE under non-reducing conditons and electroblotted onto PVDF membranes. LRP/LRP1B was immunodetected as described in Examples using a mouse monoclonal antibody raised against human LRP α-subunit (clone 8G1 from Research Diagnostics inc.) at a 1/500 dilution in TBS-Tween 0.1%, 3% BSA and 0.01% NaN₃. (n=2).</li><li><figref idref="f0031">Figure 30</figref>. Reducing condition induces release of p97 from high molecular weigh complex. U-87 cells were grown in monolayer in six-multiwell microplates at 37°C under 5% CO2. The cell monolayer were incubated at 4°C with [¹²⁵I]-p97 in Ringer/Hepes solution. After incubation, cell monolayer was washed with cold-Ringer/Hepes solution and [¹²⁵I]-p97 cross-linked was performed with 1 mM DTSP according to the manufacturer's protocol. After, cells were lysed and proteins were subjected to SDS-PAGE under reducing conditions or not. Autoradiography of [¹²⁵I]-p97 was performed following gel fixation and drying. (n=2)</li><li><figref idref="f0032">Figure 31</figref>. p97 migrates as a high molecular weight protein complex. The ligand binding was performed as described in the Examples. Concerning human brain capillaries, the ligand binding was performed on 100µg capillaries with the same protocol as cell monolayer. After ligand binding, cells were lysed with lysis buffer at 4°C for 30 min. Equal quantities of protein were subjected to SDS-PAGE under non-reducing conditions. Autoradiography of[¹²⁵I]-p97 was performed following gel fixation and drying. (n=2)</li><li><figref idref="f0033">Figure 32</figref>. Expression of members of LDL receptor family (RT-PCR). RNA from isolated human capillaries and U87 were performed as described in the Examples. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized under ultraviolet light. (n=2)</li><li><figref idref="f0035">Figure 34</figref>. Effect of p97 and RAP treatments in U-87 cells. A. Cells(2 x 10⁵) were plated onto six-multiwell microplates and grown at 37°C under 5% CO₂ with optimal culture medium supplemented with serum for 4 days. Treatment was performed in serum free medium for 72 hours. RNA isolation and RT-PCR were performed as described in the Exampples. B. LRP/LRP1B was immunodetected in cells lysates as described in the Examples. Mouse monoclonal antibody raised against human LRP að-subunit (clone 8G1 from Research Diagnostics inc.) was used at a 1/500 dilution in TBS-Tween 0.1%, 3% BSA and 0.01% NaN3. (N=2 for p97 treatment), (n=1 for RAP treatment)</li><li><figref idref="f0036">Figure 35</figref>. Effect of p97 and RAP treatments in U-87 cells (RT-PCR). Cells (2 x 10⁵) were plated onto six-multiwell microplates and grown at 37°C under 5% CO2 with optimal culture medium supplemented with serum for 4 days. Treatment was performed in serum free medium for 72 hours. RNA isolation and RT-PCR were performed as described in the Examples. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized under ultraviolet light. (n=2 for p97), (n=1 for RAP)</li><li><figref idref="f0037">Figure 36</figref>. Effect of p97 and RAP treatments in U-87 cells (RT-PCR) on endogenous melanotransferrin. Cells (2 x 105) were plated onto six-multiwell microplates and grown at 37°C under 5% CO2 with optimal culture medium supplemented with serum for 4 days. Treatment with p97 and RAP was performed in serum free medium for 72 hours. RNA isolation and RT-PCR were performed as described in the Examples. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized under ultraviolet light. (n=1).</li><li><figref idref="f0038">Figure 37</figref>. Effect of p97 and RAP treatments in U-87 cells. <figref idref="f0038">Figure 37a</figref> Agarose gel electrophoresis of PCR products. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized under ultraviolet light. (n=1).</li><li><figref idref="f0039">Figure 38</figref>. Effect of p97 and RAP treatments in U-87 cells. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized under ultraviolet light. (n=1). Quantification was by densitometry of p97 and RAP treatments on the RNA levels of LRP1B, LRP, LDL-R, cubilin, endogenous p97 and megalin. Results are expressed as ratios of RNA levels between control and treated U-87 cells.</li><li><figref idref="f0040">Figure 39</figref> show the expression of members of the LDL receptor family in MG1391. RT-PCR. RT-PCR was performed for members of the LDL-R family: LRP, LRP1B, megalin, LDL, VLDL, LRP8, LR11, LRP5 and cubulin. DNA (cDNA) synthesis was performed with 1 µg of total RNA using a cDNA one step synthesis kit (Invitrogen,USA) following the manufacturer's protocol. (1x of reaction mix, RNA 1µg, 0.2µM of both primers, 1µl of RT/Platinum Tag mix). The cDNA generated was amplified using primers produced with MacVector 7.0 (Oxford molecular Ltd, Oxford, UK). All the subsequent assays were then performed under conditions that produced amplifications of cDNA within a linear range. RT inverse-transcription was performed at 50°C for 30 min. PCR amplification for 35 or 40 cycles for all was performed as follows: denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min. Finalisation stage was performed at 72°C for 5 min. Tubes containing all the ingredients except templates were included in all runs and served as negative controls. The amplified PCR products were electrophoresed on a 1% agarose gel in TAE (40 mM Tris, 360 mM acetic acid, 1 mM EDTA, 12.5 fM Ethidum bromide) and were visualized under ultraviolet light followed by densitrometric analysis.</li><li><figref idref="f0041">Figure 40</figref> shows the effect of p97 treatment on the expression of LRP1B, LRP, LDL-R, and LRP5 in MG1391 cells using the above methods.</li><li><figref idref="f0042">Figure 41</figref> shows the effect of p97 on the expression of LRP1B, LRP, LDL-R, and LRP5 in MG1391 cells using the above methods.</li><li><figref idref="f0043">Figure 42</figref> shows the expression of LDL-receptor family members in human endothelial cells.</li><li><figref idref="f0044">Figure 43</figref> shows the expression of LDL receptor family members in BBCE cells cultured in the presence and absence of astrocytes.</li></ul>

DETAILED DESCRIPTION OF THE INVENTION

A. <u>Overview</u>

0035The present invention is in its breadest sense defined by the appended claims.

0036Described herein are, <i>inter alia,</i> novel compounds, pharmaceutical compositions and methods for modulating the activity of the melanotransferrin receptor and for modulating the transcytosis, endocytosis, and blood brain barrier transport of active agents conjugated to melanotransferrin ("MTf" or "p97") or other ligands of LRP1, and more particularly, LRP1B. Also described are screening methods for identifying such compounds, compositions, and methods. The invention is based on the discovery, disclosed here for the first time, that melanotransferrin binding to brain capillary endothelial cells (BCECs) is competitively inhibited by lactoferrin (Lf), RAP, and β-amyloid protein, but not significantly by transferrin (Tf) or bovine serum albumin (BSA). This binding spectra establishes the p97 receptor is a member of the family of LRP family. This assignment is consistent with the characterization herein of the mechanism for p97 transcytosis and endocytosis.

0037The BCEC receptor for MTf is therefore not the transferrin receptor (Tf-R), as previously hypothesized, but an unrelated class of receptor (herein called the "MTf-R" or "p97 receptor") for which Lf is a competitive inhibitor of p97. This surprising discovery provides a new pathway for the uptake of MTf into the brain; and simultaneously identifies a new role for receptors of lactoferrin and/or β-amyloid proteins in BCECs, namely MTf binding and uptake.

0038Also disclosed herein is the unexpected discovery that LRP1B is a major p97 receptor involved in the transcytosis and endocytosis of p97. Applicants have, for instance, found that p97 binds to the LRP1B receptor to form a high molecule weight complex which is dissociable and subject to competitive inhibition. Applicants have also found that the trancystosis and uptake of p97 in cells is associated with their expression of the LRP1B gene and that LRP1B is induced by treatment with p97.

0039In addition, it has been discovered that, in contrast to several other tumors, the LRP1B gene is upregulated in astrocytomas. In a preferred embodiment, therefore, p97 and ligands binding LRP1B are conjugated to chemotherapeutic agents active against such cells.

0040MTf-R biological activity is modulated to , influence blood-brain barrier transport of compounds conjugated to p97, and MTf-R biological activity is modulated to influence the transport of melanotransferrin conjugated therapeutic agents (MTf-TA) into the brain.

0041Modulation of delivery across the blood-brain barrier is achieved by modulating the activity and/or expression of MTf-Rs on the serum face (i.e., the apical side, or the "inside" of the blood vessel) of brain capillary endothelial cells. Described are methods of modulating MTf-TA uptake into the brain, compositions useful for modulating MTf-TA uptake into the brain, and screening assays and methods for identifying modulators of MTf-TA uptake into the brain. The screening assays and compounds identified using such screening assays can also be used for modulating the expression and/or activity of Lf-Rs in general, and thus for treating the diseases and disorders associated with the expression and/or activity ofLf-Rs, such as those disclosed hereinbelow.

0042The blood brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of pharmacological agents to cerebral tissues, necessitating the use of vectors for their transit. We have discovered that recombinant human melanotransferrin is highly transported into the brain by using animal models and a well-defined in vitro model of the BBB. Transcytosis of p97 is at least 14-fold higher than that of holo-hansferrin with no apparent intraendothelial degradation. The transport of p97 is not due to changes in endothelial barrier integrity but to receptor-mediated endocytosis. We have also discovered a member of the low-density lipoprotein receptor protein family, likely LRP, is involved in p97 transendothelial transport. The brain accumulation, high rate of p97 transcytosis and its very low level in the blood (100,000-fold lower than transferrin) indicate that p97 is particularly and advantageous carrier as a delivery system to target drugs directly to the brain.

0043Blood-brain barrier (BBB) permeability is frequently a rate-limiting factor for the penetration of drugs or peptides into the central nervous system (CNS) (see <nplcit id="ncit0021" npl-type="s"><text>Pardridge, W.M. J. Neurovirol. 5: 556-569 (1999</text></nplcit>); <nplcit id="ncit0022" npl-type="s"><text>Bickel, U., Yoshikawa, T. &amp; Pardridge, W.M. Adv. Drug Deliv. Rev. 46:247-279 (2001</text></nplcit>)). The brain is shielded against potentially toxic substances by the BBB, which is formed by brain capillary endothelial cells that are closely sealed by tight junctions. In addition, brain capillaries possess few fenestrae and few endocytic vesicles, compared to the capillaries ofother organs (see <nplcit id="ncit0023" npl-type="s"><text>Pardridge, W.M. J. Neurovirol. 5: 556-569 (1999</text></nplcit>)). There is little transit across the BBB of large, hydrophilic molecules aside from some specific proteins such as transferrin, lactoferrin and low-density lipoproteins, which are taken up by receptor-mediated endocytosis (see <nplcit id="ncit0024" npl-type="s"><text>Pardridge, W.M. J. Neurovirol. 5: 556-569 (1999</text></nplcit>); <nplcit id="ncit0025" npl-type="s"><text>Tsuji, A. &amp; Tamai, I. Adv.Drug Deliv.Rev. 36: 277-290 (1999</text></nplcit>); <nplcit id="ncit0026" npl-type="s"><text>Kusuhara, H. &amp; Sugiyama, Y. Drug Discov. Today 6:150-156 (2001</text></nplcit>); <nplcit id="ncit0027" npl-type="s"><text>Dehouck, B. et al. J. Cell. Biol. 138: 877-889 (1997</text></nplcit>); and <nplcit id="ncit0028" npl-type="s"><text>Fillebeen, C. et al. J. Biol. Chem. 274: 7011-7017 (1999</text></nplcit>)).

0044Melanotransferrin is a glycosylated protein that was first named human melanoma antigen p97 when it was found at high levels in malignant melanoma cells (see <nplcit id="ncit0029" npl-type="s"><text>Brown, J.P., Woodbury, RG., Hart, C.E., Hellstrom, I. &amp; Hellstrom, K.E. Proc. Natl.Acad Sci. U.S.A. 78: 539-543 (1981</text></nplcit>); and <nplcit id="ncit0030" npl-type="s"><text>Brown, J.P. et al. Nature 296:171-173 (1982</text></nplcit>)). It was later renamed Mtf due to its high level of sequence homology (37-39%) with human serum transferrin, human lactoferrin and chicken transferrin. (See <nplcit id="ncit0031" npl-type="s"><text>Brown, J.P. et al. Nature 296:171-173 (1982</text></nplcit>), <nplcit id="ncit0032" npl-type="s"><text>Rose, T.M. et al. Proc. Natl. Acad. Sci. U.S.A. 83,1261-1265 (1986</text></nplcit>)). In contrast to transferrin and lactoferrin, no cellular receptor for p97 has been identified. It has also been shown that p97 reversibly binds iron and that it exists in two forms, one of which is bound to cell membranes by a glycosyl phosphatidylinositol anchor while the other form is both soluble and actively secreted (see <nplcit id="ncit0033" npl-type="s"><text>Baker, E.N. et al. FEBS Lett. 298: 215-218 (1992</text></nplcit>); <nplcit id="ncit0034" npl-type="s"><text>Alemany, R. et al. J. Cell Sci. 104: 1155-1162 (1993</text></nplcit>); and <nplcit id="ncit0035" npl-type="s"><text>Food M.R et al. J. Biol. Chem. 269: 3034-3040 (1994</text></nplcit>)). The exact physiological role of membrane-bound p97 remains to be clearly established while the function of secreted p97 is largely unexplored (see <nplcit id="ncit0036" npl-type="s"><text>Sekyere, E. and Richardson, D.R FEBS Lett. 483: 11-16, (2000</text></nplcit>)).

0045More recently, it was reported that p97 mRNA is also widespread in normal human tissues, with the highest levels in salivary glands (see <nplcit id="ncit0037" npl-type="s"><text>Richardson, D.R. Eur J Biochem. 267: 1290-1298 (2000</text></nplcit>)). In normal human brain, p97 is present in capillary endothelium whereas in brain from patients with Alzheimer's disease it is located in microglial cells associated with senile plaques (see<nplcit id="ncit0038" npl-type="s"><text> Rothenberger, S. et al. Brain Res. 712: 117-121 (1996</text></nplcit>); <nplcit id="ncit0039" npl-type="s"><text>Jefferies, W.A. et al. Brain Res. 712: 122-126 (1996</text></nplcit>); and <nplcit id="ncit0040" npl-type="s"><text>Yamada, T. et al. Brain Res. 845: 1-5 (1999</text></nplcit>)). Serum contains very low levels of p97 (<nplcit id="ncit0041" npl-type="s"><text>Brown, J.P., Woodbury, R.G., Hart, C.E., Hellstrom, I. &amp; Hellstrom, K.E. Proc. Natl.Acad. Sci. U.S.A. 78: 539-543 (1981</text></nplcit>). The fact that p97 levels are very low in serum while high p97 levels are reported in senile plaques shows that p97 crosses the BBB to a greater extent than do other proteins present in the serum.

0046To investigate this hypothesis we evaluated the uptake of p97 in brain following its administration in animals and compared it to those of holo-transferrin and bovine serum albumin (BSA). We further studied and characterized p97 transcytosis using a well-established model of the BBB, consisting of bovine brain endothelial cells (BBCECs) co-cultured with rat astrocytes. (see <nplcit id="ncit0042" npl-type="s"><text>Fillebeen, C. et al. J. Biol. Chem. 274: 7011-7017 (1999</text></nplcit>); (<nplcit id="ncit0043" npl-type="s"><text>Dehouck, M.P. et al. J. Neurochem. 58: 1790-1797 (1992</text></nplcit>)). We also used isolated human brain capillaries for measuring p97 uptake.

0047The results obtained with in vivo and in vitro models show a much greater passage of p97 across the BBB than holo-transferrin and futher indicate the low-density lipoprotein receptor-related protein (LRP) might be involved in its passage.

0048p97 transcytosis is 14 times higher than transferrin transcytosis. Transcytosis is mediated by a receptor as it is temperature sensitive, saturable, and p97 conformation-dependent. Transcytosis of p97 occurs without compromising the structural integrity of the blood brain barrier (BBB) and p97 is not substantially degraded.

B. Definitions

0049"Melanotransferrin," as used herein, is sometimes referred to as "MTf" or "p97". As used in this disclosure, MTf includes membrane bound p97 (<i>i.e.</i>, p97 attached to a GPI anchor or some other anchor), secreted p97, soluble p97, cleaved p97, analogs of p97 which are equivalents of p97 (having greater than 40% homology at the peptide sequence level, including allelic variants of p97), human, mouse, chicken and/or rabbit p97, and derivatives, portions, or fragments thereof. p97 can be in the form of acidic or basic salts, or in its neutral form. In addition, individual amino acid residues can be modified, such as by oxidation or reduction. Moreover, various substitutions, deletions, or additions can be made to the amino acid or nucleic acid sequences, the net effect of which is to retain or improve upon the desired biological activity of p97. Due to code degeneracy, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence.

0050"p97 fragment," as used herein, includes any portion of p97 or its biologically equivalent analogs that contains a sufficient portion of p97 to enable it to bind to the MTf-R and to be transported across the blood-brain barrier; or that otherwise retains or improves upon the desired biological activities of p97.

0051"Melanotransferrin conjugated therapeutic agent" or, alternatively, "MTf-TA," as used herein, refers to a composition comprising p97 or a p97 fragment covalently conjugated to another compound. The conjugation can be direct or indirect (<i>i.e.,</i> through an extended linker) so long as it is a chemical conjugation. Linkers may be as taught in U.S. Provisional Patent Application titled, "The Use of Isocyanate Linkers for the Synthesis of Hydrolyzable Active Agent Biopolymer Conjugates" with inventors Qingqi Chen, Damian Sowa, and Reinhard Gabathuler, filed on July 12, 2002 and assigned to the same assignee as the present application.

0052The general construct of the MTf-TA is as follows: <img file="EP2147679B1_D0003.tif" />

0053Melanotransferrin conjugated therapeutic agents (MTf-TAs) can be used to treat many diseases including, but not limited to, neurological diseases and conditions such as Alzheimer's Disease, Parkinson's Disease, schizophrenia, epilepsy and others; neurological cancers, such as primary brain tumors including glioma, meningioma, neurinoma, pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma, epidermoid, sarcoma and intracranial metastasis from other tumor sources; and neurological infections or inflammatory conditions. Further, MTf-TAs can be used to treat non-CNS (<i>i.e.</i>, non-BBB delimited) diseases, such as cancers, diseases and conditions of non-CNS organs. Detailed descriptions of MTf-TAs and their uses are set out in <patcit id="pcit0007" dnum="US226242P" dnum-type="L"><text>U.S. Patent Application Nos. 60/226,242</text></patcit> and <patcit id="pcit0008" dnum="US60226254P" dnum-type="L"><text>60/226,254</text></patcit>.

0054"Modulate," as used herein, refers to the ability to alter, by increase or decrease (e.g., to act as an antagonist or agonist).

0055"Melanotransferrin receptor" ("MTf-R"), as used herein, refers to any biological system that specifically or preferentially binds MTf. This term is intended to include those receptors which competitively bind Lf and/or β-amyloid protein, but excludes those receptors which are specific for Tf such as the transferrin receptor (Tf-R) (which is described at OMIM # *190010, and which is also known as TFR, TRFR and CD71). A receptor known to specifically or preferentially bind Lf is herein called a "lactotransferrin receptor" (Lf-R). Known Lf-Rs include, but are not limited to, the LDL-related receptors. A known LDL-related receptor is lipoprotein receptor-related protein/<i>alpha</i>2- macroglobulin receptor ("LRP1"). The term MTf-R specifically includes other receptors found on endothelial cells that specifically bind both MTf and Lf, but not Tf. As described, the MTf-R is the LRP1. As described, the MTf-R is LRP1B.

0056Members of the low density lipoprotein (LDL) receptor family include LDL-R (132 kDa); LRP/LRP1 and LRP1B (600 kDa); Megalin ((LRP2), 600 kDa); VLDL-R (130 kDa); ER-2 (LRP-8, 130 kDa); Mosaic LDL-R (LR11, 250 KDa); and other members such as LRP3, LRP6, and LRP-7. Characeristic features of the LDL-R family include cell- surface expression; extracellular ligand binding domain repeats (DxSDE); requirement of Ca++ for ligand binding; recognition of RAP and ApoE; EGF precursor homology domain repeats (YWTD); single membrane spaning region; internalization signal in the cytoplasmic domain (FDNPXY); and receptor mediated endocytosis of various ligands.

0057LRP refers to the low density lipoprotein receptor related protein and members of this receptor family. LRP is a large protein of 4525 amino acids (600 kDa) which is cleaved by furin to produce two subunits of 515-(α) and 85-(β) kDa that remain non-covalently bound. LRP is mainly expressed in the liver, kidney, neuron, CNS, BBB, SMC and various cultured cells.

0058LRP ligands. A number of molecules are known to bind LRP. These molecules include, for instance, lactoferrin, RAP, lipoprotein lipase, ApoE, Factor VIII, β-amyloid precursor, α2-macroglobulin, thrombospondin 2 MMP-2, MPP-9-TIMP-1; uPA:PAI-I:uPAR; and tPA:PAI-1:uPAR (see also <figref idref="f0024">Figure 23</figref>).

0059LRP 1B is a recently discovered member of the low density lipoprotein receptor family. 600 kDa multifunctional cell surface receptor. See<nplcit id="ncit0044" npl-type="s"><text> Liu et al., J. Biol. Chem. 276 (31):28889-28896 (2001</text></nplcit>). See also <nplcit id="ncit0045" npl-type="s"><text>Liu et al., Genomics 69, 271-274 (2000</text></nplcit>); and <nplcit id="ncit0046" npl-type="s"><text>Liu et al., Cancer Res. 60, 1961-1967 (2000</text></nplcit>). This receptor is more closely related to LRP than megalin and shares a 59% homology at cDNA level and a 52% homology at predicted amino acid level. the LRP 1B gene is expressed in the brain, thyroid and salivary gland. Known ligands for LRP 1B include RAP, tPA, PAI-1.

0060Mouse LRP1B is accessible through GenBank Accession Nos. XM 143023 XM 130241. Human LRP1B is accessible through GenBank Accession Nos. XM 015452.

0061"Lipoprotein receptor-related protein/<i>alpha</i>2-macroglobulin receptor" ("LRP1"), as used herein, refers to a multifunctional receptor. It is believed that the clustering of cysteine-rich type. A binding repeats, resembling those found in the LDL receptor, is the molecular principle for the ability to bind a variety of ligands that were previously thought to be unrelated: activated <i>alpha</i>-2-macroglobulin, apolipoprotein E, lipoprotein lipase, plasminogen activators and complexes with their inhibitor (PA and PA/PAI-1), lipoprotein(a), pseudomonas exotoxin A, human rhinovirus, Lf and the so-called receptor associated protein (RAP). <i>See,</i><nplcit id="ncit0047" npl-type="s"><text>Meilinger, et al., FEBSLet., 360:70-74 (1995</text></nplcit>).

0062LRPI is accessible through GenBank Accession No.: X 13916 and Swiss-Prot Primary Accession No.: Q07954.

0063Alternative names for the LRP1 gene/protein include: Low-density lipoprotein receptor-related protein 1 [precursor], LRP, Alpha-2-macroglobulin receptor, A2MR, Apolipoprotein E receptor, APOER, CD91, LRP1 or A2MR.

0064The screening assays can also employ homologs of human LRP1 (hLRP1) or LRP1B. Such homologs can be derived from other organisms, specifically eukaryotes and preferably from mammals.

0065Preferred homologs of human LRP include, but are not limited to, the following LRP proteins: <tables id="tabl0001" num="0001"><table frame="none"><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="31mm" /><colspec colnum="2" colname="col2" colwidth="115mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><tbody><row><entry><b><i>H. sapiens</i>:</b></entry><entry><u>SP:Q07954</u> - LRP1_HUMAN LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 1 PRECURSOR</entry><entry><b>100 % / 4543 aa</b></entry></row><row><entry><b><i>M. musculus:</i></b></entry><entry><u>PID:g49942</u> - AM2 receptor</entry><entry><b>97 % / 4543 aa</b></entry></row><row><entry><b><i>R. norvegicus</i>:</b></entry><entry><u>SP:P98158 -</u> LRP2 RAT LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 2 PRECURSOR</entry><entry><b>39 % / 4207 aa</b></entry></row><row><entry><b><i>D. melanogaster</i>:</b></entry><entry><u>SP:P98163 -</u> YL DROME PUTATIVE VITELLOGENIN RECEPTOR PRECURSOR</entry><entry><b>28 % / 1550 aa</b></entry></row><row><entry><b><i>C. elegans</i>:</b></entry><entry><u>PID:g3876533</u> - predicted using Genefinder</entry><entry><b>35 % / 4205 aa</b></entry></row><row><entry><b><i>S. cerevisiae</i>:</b></entry><entry><u>PID:g557822</u> - mal5, sta1, len: 1367, CAI: 0.3, AMYH YEAST P08640 GLUCOAMYLASE S1</entry><entry><b>24 % / 1221 aa</b></entry></row><row><entry><b><i>E. coli:</i></b></entry><entry><u>PID:g1787636</u> - putative membrane protein</entry><entry><b>25 % / 370 aa</b></entry></row></tbody></tgroup></table></tables>

0066Those skilled in the art can readily identify other homologs of LRP1 or more particularly, LRP1B, suitable for use in the present invention.

0067"Receptor for Advanced Glycation End products" ("RAGE"), as used herein, refers to a multiligand member of the immunoglobulin superfamily of cell surface molecules. RAGE was originally identified and characterized based on its ability to bind advanced glycation end products (AGEs), adducts formed by glycoxidation that accumulate in disorders such as diabetes and renal failure. Subsequent studies demonstrate that RAGE serves as a cell surface receptor for amyloid-β peptide (the cleavage product of β-amyloid precursor protein (β-APP), a major component of neuritic plaques of Alzheimer's Disease). Other ligands include amphoterin and s100/calgranulin-like molecules (<i>see</i>, <nplcit id="ncit0048" npl-type="s"><text>Hofmann, et al., Cell, 97(7):889-901 (1999</text></nplcit>)). RAGE is readily accessible through GenBank Accession No.: M91211 and Swiss-Prot Primary Accession No.: Q15109.

0068By "determining the functional effect" is meant assaying for a compound that modulates, <i>e.g.,</i> increases or decreases, a parameter that is indirectly or directly under the influence of the MTf-R, <i>e.g.,</i> functional, physical and chemical effects. In addition to the functional effects specifically described herein, it will be readily apparent to those of skill in the art that other functional effects can be measured by any means know to those skilled in the art, <i>e.g.,</i> changes in spectroscopic characteristics (<i>e.g.,</i> fluorescence, absorbance, refractive index), hydrodynamic (<i>e.g.,</i> shape), chromatographic, or solubility properties, changes in gene expression of MTf-Rs, and the like.

0069"Inhibitors," "activators" and "modulators" of MTf-Rs are used interchangeably to refer to inhibitory, activating or modulating compounds identified using in vitro and/or in vivo assays for MTF-R.

0070Samples or assays comprising MTf-Rs that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative MTf-R activity value of 100%. Inhibition of a MTf-R is achieved when the MTf-R activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation of a MTf-R is achieved when the MTf-R activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.

C. Screening Assays for Modulators of Melanotransferrin Receptors (MTf-Rs)

0071Disclosed herein is a screening assay employing an MTf-R, wherein compounds are tested for their ability to influence a measurable activity of the MTf-R. The MTf-R can be situated in a whole cell, a cell extract, semi-purified, purified or any other format that allows for measurement of its activity. The activity can be any activity in the expression, function or degradation of MTf-R including, for example, the amount or timing of such activities. Such activities include, for example, transcription, transcript processing, translation or transcript stability of the <i>MTf</i>-R gene sequence or mRNA transcript. Such activities include, for example, the synthesis of new MTf-R, the sub-cellular localization of MTf-R and activation of MTf-R biological activity. Such activities include, for example, the ability of MTf R to bind substances, adopt conformations, catalyze reactions, bind known ligands and the like. Such activities include, for example, the amount or stability of MTf-R, the processing and removal or degradation of MTf-R and the like. In preferred examples, the MTf-R receptor for use in screening is LRP1 or LRP1B.

0072In a preferred screening assay, compounds are tested to identify modulators of a biological activity of MTf-R such as: MTf-R interactions with MTf, MTf-R interactions with Lf or other putative ligands or uptake of iron/other metal; transport of MTf across the blood-brain barrier ("BBB"), <i>i.e.,</i> in a BBB model; transport of Lf, other ligands or metals across a BBB model; and/or measurement of the rate or amount of transcription, translation or expression levels of <i>MTf</i>-R genes or mRNA.

0073Described herein are a variety of different screening formats. Some designs are considered low throughput and test only one or a few compounds in series or in parallel. High throughput screening assays are suitable for screening tens of thousands or hundreds of thousands of compounds in a matter of weeks or months. "In silico" screening formats employ computer-aided rational design techniques to identify potential modulators of MTf-R biological activity.

0074The test compounds may be obtained from any source; but a preferred commercial embodiment employs commercially available compound libraries of hundreds of thousands of compounds, many of which are potential therapeutic agents. These compounds are tested in series or in parallel to identify modulating activity of MTf-R activity. Preferred test compounds can be identified by examining known and putative ligands of MTf-R for predicting chemical structure of inhibitors, <i>etc</i>. Other computer-aided design techniques can be employed to eliminate unsuitable candidates, such as those candidates thought to cause toxic side-effects. Those skilled in the art are familiar with the combinatorial and medicinal chemistry techniques that can be used to further select test compounds and the potential therapeutic agents,

0075The object of the screening assays is to identify modulators of MTf-R activity that are suitable for animal or human clinical trials and as therapeutic agents. As such, the screening assays identify stimulants, agonists or antagonists of MTf-R. The method of identifying stimulants, agonists or antagonists of MTf-R can comprise contacting a substance suspected of being a stimulant, agonist or antagonist with MTf and the MTf-R under conditions such that MTf is capable of binding to the MTf-R; measuring the amount of MTf bound to the MTf-R; and determining the effect of the substance by comparing the amount of MTf bound to MTf-R with an amount determined for a control. The MTf that can be used in this method includes MTf cleaved of GPI, soluble MTf, cleaved MTf or derivatives thereof, preferably recombinant MTf. In this method, the amount of MTf bound to MTf-R can be determined by measuring the amount of MTf bound to the MTf-R, the amount of unbound MTf or the amount of unbound MTf-R MTfbound to MTf-R can be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the measurement of MTf bound to MTf-R or of unbound MTf, or unbound MTf-R, antibodies against MTf or MTf-R can, for example, be utilized.

0076In one example, disclosed herein is a method for identifying a stimulant, agonist or antagonist of MTf-mediated iron uptake comprising: incubating a cell expressing MTf-R on its surface and a test substance (<i>e.g.,</i> compound) suspected of being a stimulant, agonist or antagonist of MTf-R in the presence of MTf bound to iron (holo-MTf) and in the absence of transferrin, measuring the amount of iron uptake into the cell and identifying a stimulant, agonist or antagonist of MTf-R mediated iron uptake by comparing the amount of iron uptake in the cell with the amount of iron uptake in a cell from a control incubation in the absence of the test substance. Iron uptake refers to the internalization of iron into the cell across the cell plasma membrane.

D. Modulating Uptake of Melanotransferrin Conjugated Therapeutic Agents (MTf-TAs) by Modulating MTf-R Activity

0077In another example, disclosed herein is a method of using compounds that modulate MTf-R biological activity to modulate the amount of uptake into the cells of melanotransferrin conjugated therapeutic agents (MTf-TAs). In particular, disclosed herein is a method of increasing the uptake of an MTf-TA into the brain, the method comprising administering a modulator of MTf-R biological activity either contemporaneously or sequentially with the MTf-TA. Alternatively, disclosed herein is a method to reduce uptake of an MTf-TA into the brain comprising administering a modulator of MTf-R biological activity either contemporaneously or sequentially with the MTf-TA. In a preferred embodiment, these methods employ modulators of MTf-R that are first identified using a screening assay as described hereinabove. In preferred examples, the MTf-R receptor for use in screening is LRP1 or LRP1B.

0078Those skilled in the art will appreciate that increasing MTf-TA uptake and delivery across the blood-brain barrier is useful and desirable in situations such as, but not limited to, where the MTf-TA is being used to treat a neurological condition and increased amounts of delivery provide therapeutic benefit. Those skilled in the art will appreciate that decreasing MTf-TA uptake and delivery across the blood-brain barrier is useful and desirable for a variety of reasons including, but not limited to, where the MTf-TA is being used for its cardio-protective effect or used in other (non-CNS) organs and side-effects of brain uptake are to be avoided.

0079Modulators of MTf-R activity can be readily identified using a modification of the transwell apparatus set out in Example I below. In the modified form, a compound is added to the luminal surface of the cells in the transwell apparatus in combination with MTf-TAs. The compound is then scored to determine if it increases or decreases the transport of the MTf-TA across the BBCECs to the abluminal side. A library of compounds can be readily screened or tested to identify pharmacologically superior modulators.

E. Diseases Treatable by Lf-R Modulators

0080As a result of the surprising discovery that certain MTf-Rs are Lf-Rs, it has now been discovered that modulators of Lf-R biological activity can have an impact on disease processes known to be linked to MTf biological activity. As such, the following uses for modulators of Lf-Rs identified, for example, by the screening assays described herein have now been identified.

00811) Rapidly proliferating cells, such as malignant cells, have an increased requirement for iron and must possess efficient mechanisms to obtain iron. Limiting the ability of malignant cells to acquire iron provides a method of killing tumor cells or of modulating their uncontrolled cell growth.

00822) These findings lead to the discovery that modulators of Lf-R can be used to modulate iron uptake in cells. Iron uptake in cells can be modulated by varying the amount of Lf-R on cell surfaces, or by inhibiting Lf-R binding to MTf. Accordingly, stimulants, agonists or antagonists of Lf-R can be useful in the treatment of conditions or diseases where there is a disturbance in iron metabolism. For example, such substances are useful in the treatment of conditions such as hemochromatosis, neurodegenerative diseases, ischemic tissue damage, including ischemic stroke or trauma, heart disease and tumors (<i>e.g.,</i> skin cancer).

00833) MTf plays a role in the binding and uptake of other metals such as zinc and aluminum (<nplcit id="ncit0049" npl-type="s"><text>Sekyere, E. and Richardson, D.R., FEBS Letters, 483:11-16 (2000</text></nplcit>) and, thus, modulation of Lf-R activity or expression plays a role in modulating or regulating Alzheimer's Disease or metaloproteins, such as zinc-dependent metaloproteins.

00844) Conditions that involve disturbances in iron metabolism that can be treated using the compounds and methods described herein (<i>i.e</i>., using stimulants, agonists and antagonists of MTf-mediated iron uptake) are, for example, those involving excessive iron absorption from the diet, defects in iron uptake into cells, excessive iron uptake into cells or those requiring regular treatment by blood transfusion (<i>e.g</i>., dyserythropoietic anemias, in particular thalassaemia disorders. Examples of such conditions include, but are not limited to, hemochromatosis, neurodegenerative diseases (<i>e.g</i>., Alzheimer's Disease, Huntington's Disease and Parkinson's Disease), ischemic tissue damage, heart disease and tumors, inflammation and infections (<i>see,</i><nplcit id="ncit0050" npl-type="b"><text>Pippard, "Clinical Use of Iron Chelation," in Iron in Immunity, Cancer and Inflammation (M. de Sousa and J. H. Brock (Eds.), 1989, John Wiley &amp; Sons</text></nplcit>),

00855) Diseases which can be treated using modulators of Lf-R activity or expression include neurological diseases and conditions such as Alzheimer's Disease, Parkinson's Disease, schizophrenia, epilepsy and others, neurological cancers, such as primary brain tumors including glioma, meningioma, neurinoma, pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma, epidermoid, sarcoma and intracranial metastasis from other tumor sources, and neurological infections or inflammatory conditions. modulators of Lf-R activity or expression can be used to treat non-CNS (<i>i.e</i>., non-BBB delimited) diseases, such as cancers, diseases and conditions of non-CNS organs.

0086Once a potential therapeutic agent has been identified from among the test compounds, those skilled in the art are familiar with the basic pharmacological steps required to develop an acceptable human or animal therapeutic agent. For example, therapeutic doses can be derived from animal studies and human clinical trials, as can suitable pharmacological carriers and excipients.

F. Therapeutic and Diagnostic Uses of MTf-R Proteins/Polypeptides

0087Also disclosed herein is a method for administering a MTf-R protein, or a fragment thereof, to an animal in need thereof, to effect a therapeutic or prophylactic result. <i>In vivo</i> an MTf-R protein or fragment thereof will bind to MTf and effectively reduce the circulating concentration of MTf. This treatment is useful in diseases where increased levels of MTf are know to be implicated in disease progression, particularly, Alzheimer's Disease (<i>see,</i><nplcit id="ncit0051" npl-type="s"><text>Kennard, et al., Nat. Med., 2:1230-1235 (1996</text></nplcit>); <patcit id="pcit0009" dnum="US5981194A"><text>U.S. Patent No. 5,981,194</text></patcit>). As a diagnostic tool, administration of an MTf-R protein or a fragment thereof, labeled with a detectable label, will identify the location or amount ofMTf in the subject. Such a tool would be particularly useful for finding tumor cells that are known to express higher levels of MTf. Presumably, labeling of MTf-R with a strong <i>beta</i> emitting particle, such as certain Iodine or Yttrium isotopes, would assist in killing tumor cells to which the MTf-R would bind.

0088Other therapeutical targets relate to HIV infection as a TAT receptor, activation of the NMDA receptor with respect to synaptic plasticity, Alzheimer's disease as LRP is a receptor for APoE, alpha2-macroglobulin, APP which have all been linked to this disase; and in the regulation of lipid degrading lysosomal enzymes (SAP-dependent).

G. MTf-R Antibodies

0089Antibodies that specifically recognize one or more epitopes of MTf-R, or epitopes of conserved variants of MTf-R, or peptide fragments of MTf-R are also disclosed herein.

0090Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

0091The antibodies can be used, for example, in conjunction with compound screening assays, as described above, for the evaluation of the effect of test compounds on expression and/or activity of the MTf-R gene product. Additionally, such antibodies can be used in conjunction with gene therapy techniques to, for example, evaluate the success of transfection of normal and/or engineered MTf-R-expression. Such antibodies can be used as antagonists of MTf-R biological activity, and can additionally be used as a method for the inhibition of abnormal MTf-R activity. Thus, such antibodies can be utilized as therapeutic agents,

0092The MTF-R antibodies can also be used in diagnostic techniques to identify the location, quantity, behavior and/or the like of MTf-R in an animal.

0093For the production of antibodies, various host animals may be immunized by injection with the MTf-R, a MTf-R protein or peptide (<i>e.g.,</i> one corresponding to a functional domain of the receptor), truncated MTf-R polypeptides (MTf-R in which one or more domains has been deleted), functional equivalents of the MTf-R or mutants of the MTf-R. Such host animals include, but are not limited to, rabbits, mice, and rats, to name but a few. Various adjuvants can be used to increase the immunological response, depending on the host species including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.

H. MTf-R Antisense Oligonucleotides and Ribozymes

0094Also disclosed herein is the use of antisense oligonucleotide sequences having sequences specific for hybridization to the MTf-R (e.g., LRP1 or LRP1B) mRNA or precursor RNA, <i>i.e</i>., sequences that specifically or selectively hybridize to MTf-R or mRNA or precursor RNA. Those skilled in the art can readily identify suitable antisense oligonucleotide sequences based on the known MTf-R mRNA sequence referenced above. Those skilled in the art are aware of modifications to the chemistry of oligonucleotides that improve their clinical utility, such as phosphorothioate modifications. MTf-R antisense oligonucleotides are an example of compounds that can be used to modulate MTf-R expression and, in particular, to down-regulate such expression. Those skilled in the art are also able to design ribozymes which achieve similar objectives. Methods of delivering such nucleic acids are described below.

I. MTf-R Gene Therapy

0095Certain examples disclosed herein employ the use of the <i>MTf</i>-<i>R</i> gene in a gene therapy method. The obj ect of such intervention is to deliver a functional copy of the MTf-R gene to a cell in need thereof in order to increase the transcription and subsequent translation and expression of the MTf-R protein in the cell. Those skilled in the art are familiar with viral, non-viral (<i>i.e</i>., lipid based), naked DNA and polymeric methods of delivering gene therapy vectors that include the <i>MTf</i>-<i>R</i> gene. Such methods can employ <i>in vivo</i> or <i>ex vivo</i> gene therapy techniques known to and used by those of skill in the art.

J. Diagnosis of Neurological Diseases

0096Also disclosed herein is the diagnosis of a neurological disease comprising detecting the amount or activity of MTf-R expressed at the BBB, in neural tissue or in any tissue associated with a non-CNS target organ, such as the lung, liver, kidney, spleen, <i>etc.</i> The diagnostic method employs a diagnostic agent comprising an agent specific for MTf-R (<i>i.e</i>., an MTf-R antibody, a ligand such as MTf or Lf, or another ligand which specifically binds MTf-R) and a detectable conjugate or label (<i>i.e</i>., radioisotopes of technetium or iodine). Those skilled in the art can identify diseases or conditions for which the determination of MTf-R amount or localization is relevant. Most preferred are diseases or conditions, such as Alzheimer's Disease, in which MTf-R is directly implicated.

K. Binding of different Ligands to LDL-R receptor family members.

0097A large number of ligands have been found to bind to members of the LDL-R receptor family. These ligands and their receptor are indicated in Tables 1-3. <tables id="tabl0002" num="0002"><table frame="all"><title>Table 1. Lipoproteins and apolipoproteins</title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="38mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="17mm" /><colspec colnum="5" colname="col5" colwidth="16mm" /><colspec colnum="6" colname="col6" colwidth="13mm" /><colspec colnum="7" colname="col7" colwidth="13mm" /><thead><row><entry valign="top">Ligands</entry><entry valign="top">LDL-R</entry><entry valign="top">LRP</entry><entry valign="top">Megalin</entry><entry valign="top">VLPL-R</entry><entry valign="top">ER-2</entry><entry valign="top">LR11</entry></row></thead><tbody><row><entry>β-VLDL</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry></row><row><entry>Chylomicron remnants</entry><entry>Yes</entry><entry>Yes</entry><entry /><entry>Yes</entry><entry /><entry /></row><row><entry>IDL</entry><entry>Yes</entry><entry /><entry /><entry>Yes</entry><entry /><entry /></row><row><entry>Lp(a)</entry><entry /><entry /><entry /><entry>Yes</entry><entry /><entry /></row><row><entry>YLDL</entry><entry>Yes</entry><entry /><entry /><entry>Yes</entry><entry>Yes</entry><entry /></row><row><entry>ApoB100</entry><entry>Yes</entry><entry /><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>ApoE</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry></row></tbody></tgroup></table></tables><tables id="tabl0003" num="0003"><table frame="all"><title>Table 2. Protease inhibitors and protease/inhibitor complexes.</title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="17mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><colspec colnum="6" colname="col6" colwidth="13mm" /><colspec colnum="7" colname="col7" colwidth="13mm" /><thead><row><entry valign="top">Ligands</entry><entry valign="top">LDL-R</entry><entry valign="top">LRP</entry><entry valign="top">Megalin</entry><entry valign="top">VLDL-R</entry><entry valign="top">ER-2</entry><entry valign="top">LR11</entry></row></thead><tbody><row><entry>PAI-1</entry><entry /><entry>Yes</entry><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>Plasminogen</entry><entry /><entry /><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>Pro-uPA</entry><entry /><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry /><entry /></row><row><entry>Tissue factor inhibitor</entry><entry /><entry>Yes</entry><entry /><entry /><entry /><entry /></row><row><entry>tPA</entry><entry /><entry>Yes</entry><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>activated α2-macroglobulin</entry><entry /><entry>Yes</entry><entry>No</entry><entry /><entry /><entry /></row><row><entry>α1-chymotrypsin/cathepsin G</entry><entry /><entry>No</entry><entry>Yes</entry><entry /><entry /><entry /></row></tbody></tgroup></table></tables><tables id="tabl0004" num="0004"><table frame="all"><title>Table 3. Other ligands.</title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="54mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="17mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><colspec colnum="6" colname="col6" colwidth="13mm" /><colspec colnum="7" colname="col7" colwidth="13mm" /><thead><row><entry valign="top">Ligands</entry><entry valign="top">LDL-R</entry><entry valign="top">LRP</entry><entry valign="top">Megalin</entry><entry valign="top">VLDL-R</entry><entry valign="top">ER-2</entry><entry valign="top">LR11</entry></row></thead><tbody><row><entry>Albumin</entry><entry /><entry /><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>ApoJ/clusterin</entry><entry /><entry /><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>ApoJ/β-amyloid</entry><entry /><entry>No</entry><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>β-APP</entry><entry /><entry>Yes</entry><entry /><entry>Yes</entry><entry /><entry /></row><row><entry>Lactoferrin</entry><entry /><entry>Yes</entry><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>RAP</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry>Yes</entry><entry /><entry /></row><row><entry>Thyroglobulin</entry><entry /><entry /><entry>Yes</entry><entry /><entry>Yes</entry><entry>Yes</entry></row><row><entry>Circumsporozite protein</entry><entry /><entry /><entry>Yes</entry><entry>Yes</entry><entry /><entry /></row><row><entry>Saposin</entry><entry /><entry>Yes</entry><entry /><entry /><entry /><entry /></row><row><entry>Gentamycin</entry><entry /><entry>Yes</entry><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>Polymixin B</entry><entry /><entry>Yes</entry><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>Pseudomonas Exotoxin A</entry><entry /><entry>Yes</entry><entry>No</entry><entry /><entry /><entry /></row><row><entry>Seminal Vesicle Secretory Protein A</entry><entry /><entry>Yes</entry><entry>Yes</entry><entry /><entry /><entry /></row><row><entry>Thrombospondin -1</entry><entry /><entry>Yes</entry><entry /><entry>Yes</entry><entry /><entry /></row></tbody></tgroup></table></tables>

L. Active agents

0098The chemotherapeutic agents used in the present invention are defined in the appended claims.

0099Active agents, disclosed herein include agents that affect any biological process. The term "drug" or "therapeutic agent" refers to an active agent that has a pharmacological activity or benefits health when administered in a therapeutically effective amount Examples of drugs or therapeutic agents include substances that are used in the prevention, diagnosis, alleviation, treatment or cure of a disease or condition.

0100The active agent conjugated to the p97 protein or fragment or LRP 1 modulator or LRP ligand may be any molecule, as well as any binding portion or fragment thereof, that is capable of modulating a biological process in a living host. Generally, the active agent may be of any size, but is preferably a small organic molecule that is capable of binding to the target of interest. A drug moiety of the conjugate, when a small molecule, generally has a molecular weight of at least about 50 D, usually at least about 100 D, where the molecular weight may be as high as 500 D or higher, but will usually not exceed about 2000 D.

0101The drug moiety is capable of interacting with a target in the host into which the conjugate is administered during practice of the subject methods. The target may be a number of different types of naturally occurring structures, where targets of interest include both intracellular and extracellular targets, where such targets may be proteins, phospholipids, nucleic acids and the like, where proteins are of particular interest. Specific proteinaceous targets of interest include, without limitation, enzymes, <i>e.g</i>., kinases, phosphatases, reductases, cyclooxygenases, proteases and the like, targets comprising domains involved in protein-protein interactions, such as the SH2, SH3, PTB and PDZ domains, structural proteins, <i>e.g</i>., actin, tubulin, <i>etc</i>., membrane receptors, immunoglobulins, <i>e.g..</i> IgE, cell adhesion receptors, such as integrins, <i>etc</i>, ion channels, transmembrane pumps, transcription factors, signaling proteins, and the like.

0102The active agent or drug has a hydroxy or an amino group for reacting with the isocyanate reagent or the active agent is chemically modified to introduce a hydroxy or an amino group for reacting with the isocyanate reagent

0103In some examples, the active agent or drug will also comprise a region that may be modified and/or participate in covalent linkage, preferably, without loss of the desired biological activity of the active agent The drug moieties often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Also of interest as drug moieties are structures found among biomolecules, proteins, enzymes, polysaccharides, and polynucleic acids, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof

0104The conjugate can comprise one or more active agents linked to the same biopolymer. For example, conjugation reactions may conjugate from 1 to 5, about 5, about 1-10, about 5 to 10, about 10-20, about 20-30, or 30 or more molecules of an active agent to the biopolymer. These formulations can be employed as mixtures, or they may be purified into specific (mol:mol) formulations. Those skilled in the art are able to determine which format and which mol:mol ratio is preferred. Further, more than one type of active agent may be linked to the biopolymer where delivery of more than one type of an agent to a target site or compartment is desired. A plurality of active agent species may be attached to the same biopolymers such as adriamycin-cisplatinum conjugate compositions where the biopolymer is a p97 related protein. Thus, the conjugates may consist of a range of mol:mol ratios and incorporate more than one type of active agent. These, too, may be separated into purified mixtures or they may be employed in aggregate. Active agents include those identified <patcit id="pcit0010" dnum="US6372712B"><text>U.S. Patent No. 6,372,712</text></patcit>;

0105Specific drugs of interest from which the drug moiety may be derived include, but are not limited to: psychopharmacological agents, such as (1) central nervous system depressants, <i>e.g</i>., general anesthetics (barbiturates, benzodiazepines, steroids, cyclohexanone derivatives, and miscellaneous agents), sedative-hypnotics (benzodiazepines, barbiturates, piperidinediones and triones, quinazoline derivatives, carbamates, aldehydes and derivatives, amides, acyclic ureides, benzazepines and related drugs, phenothiazines, <i>etc</i>.), central voluntary muscle tone modifying drugs (anticonvulsants, such as hydantoins, barbiturates, oxazolidinediones, succinimides, acylureides, glutarimides, benzodiazepines, secondary and tertiary alcohols, dibenzazepine derivatives, valproic acid and derivatives, GABA analogs, <i>etc</i>.), analgesics (morphine and derivatives, oripavine derivatives, morphinan derivatives, phenylpiperidines, 2,6-methane-3-benzazocaine derivatives, diphenylpropylamines and isosteres, salicylates, p-aminophenol derivatives, 5-pyrazolone derivatives, arylacetic acid derivatives, fenamates and isosteres, <i>etc.</i>) and antiemetics (anticholinergics, antihistamines, antidopaminergics, <i>etc.</i>), (2) central nervous system stimulants, <i>e</i>.<i>g</i>., analeptics (respiratory stimulants, convulsant stimulants, psychomotor stimulants), narcotic antagonists (morphine derivatives, oripavine derivatives, 2,6-methane-3-benzoxacine derivatives, morphinan derivatives) nootropics, (3) psychopharmacologicals; <i>e</i>.<i>g</i>., anxiolytic sedatives (benzodiazepines, propanediol carbamates) antipsychotics (phenothiazine derivatives, thioxanthine derivatives, other tricyclic compounds, butyrophenone derivatives and isosteres, diphenylbutylamine derivatives, substituted benzamides, arylpiperazine derivatives, indole derivatives, <i>etc.</i>), antidepressants (tricyclic compounds, MAO inhibitors, etc.), (4) respiratory tract drugs, <i>e.g.</i>, central antitussives (opium alkaloids and their derivatives); pharmacodynamic agents, such as (1) peripheral nervous system drugs, <i>e</i>.<i>g</i>., local anesthetics (ester derivatives, amide derivatives), (2) drugs acting at synaptic or neuroeffeetor junctional sites, <i>e</i>.<i>g</i>., cholinergic agents, cholinergic blocking agents, neuromuscular blocking agents, adrenergic agents, antiadrenergic agents, (3) smooth muscle active drugs, <i>e</i>.<i>g</i>., spasmolytics (anticholinergics, musculotropic spasmolytics), vasodilators, smooth muscle stimulants, (4) histamines and antihistamines, e.g., histamine and derivative thereof (betazole), antihistamines (H₁ -antagonists, H₂ -antagonists), histamine metabolism drugs, (5) cardiovascular drugs, <i>e</i>.<i>g</i>., cardiotonics (plant extracts, butenolides, pentadienolids, alkaloids from erythrophleum species, ionophores, adrenoceptor stimulants, <i>etc</i>), antiarrhythmic drugs, antihypertensive agents, antilipidemic agents (clofibric acid derivatives, nicotinic acid derivatives, hormones and analogs, antibiotics, salicylic acid and derivatives), antivaricose drugs, hemostyptics, (6) blood and hemopoietic system drugs, <i>e</i>.<i>g</i>., antianemia drugs, blood coagulation drugs (hemostatics, anticoagulants, antithrombotics, thrombolytics, blood proteins and their fractions), (7) gastrointestinal tract drugs, <i>e</i>.<i>g</i>., digestants (stomachics, choleretics), antiulcer drugs, antidiarrheal agents, (8) locally acting drugs; chemotherapeutic agents, such as (1) anti-infective agents, <i>e</i>.<i>g</i>., ectoparasiticides (chlorinated hydrocarbons, pyrethins, sulfurated compounds), anthelmintics, antiprotozoal agents, antimalarial agents, antiamebic agents, antileiscmanial drugs, antitrichomonal agents, antitrypanosomal agents, sulfonamides, antimycobacterial drugs, antiviral chemotherapeutics, <i>etc</i>., and (2) cytostatics, <i>i</i>.<i>e</i>., antineoplastic agents or cytotoxic drugs, such as alkylating agents, <i>e.g.,</i> Mechlorethamine hydrochloride (Nitrogen Mustard, Mustargen, HN2), Cyclophosphamide (Cytovan, Endoxana), Ifosfamide (IFEX), Chlorambucil (Leukeran), Melphalan (Phenylalanine Mustard, L-sarcolysin, Alkeran, L-PAM), Busulfan (Myleran), Thiotepa (Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine (CeeNU, CCNU), Streptozocin (Zanosar) and the like; plant alkaloids, <i>e.g.,</i> Vincristine (Oncovin), Vinblastine (Velban, Velbe), Paclitaxel (Taxol), and the like; antimetabolites, <i>e.g.,</i> Methotrexate (MTX), Mercaptopurine (Purinethol, 6-MP), Thioguanine (6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U, Ara-C), Azacitidine (Mylosar, 5-AZA) and the like; antibiotics, <i>e.g.,</i> Dactinomycin (Actinomycin D, Cosmegen), Doxorubicin (Adriamycin), Daunorubicin (duanomycin, Cerubidine), Idarubicin (Idamycin), Bleomycin (Blenoxane), Picamycin (Mithramycin, Mithracin), Mitomycin (Mutamycin) and the like, and other anticellular proliferative agents, <i>e</i>.<i>g</i>., Hydroxyurea (Hydrea), Procarbazine (Mutalane), Dacarbazine (DTIC-Dome), Cisplatin (Platinol) Carboplatin (Paraplatin), Asparaginase (Elspar) Etoposide (VePesid, VP-16-213), Amsarcrine (AMSA, m-AMSA), Mitotane (Lysodren), Mitoxantrone (Novatrone), and the like;

0106Antibiotics, such as: aminoglycosides, <i>e.g.,</i> amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin, gentamicin, isepamicin, kanamycin, micronomcin, neomycin, netilmicin, paromycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, trospectomycin; amphenicols, <i>e</i>.<i>g</i>., azidamfenicol, chloramphenicol, florfenicol, and theimaphenicol; ansamycins, <i>e.g</i>., rifamide, rifampin, rifamycin, rifapentine, rifaximin; beta.-Iactams, <i>e</i>.<i>g</i>., carbacephems, carbapenems, cephalosporins, cehpamycins, monobactams, oxaphems, penicillins; lincosamides, <i>e</i>.<i>g</i>., clinamycin, lincomycin; macrolides, <i>e</i>.<i>g</i>., clarithromycin, dirthromycin, erythromycin, <i>etc</i>.; polypeptides, <i>e.g.,</i> amphomycin, bacitracin, capreomycin, <i>etc</i>.; tetracyclines, <i>e</i>.<i>g</i>., apicycline, chlortetracycline, clomocycline, <i>etc</i>.; synthetic antibacterial agents, such as 2,4-diaminopyrimidines, nitrofurans, quinolones and analogs thereof, sulfonamides, sulfones;

0107Antifungal agents, such as: polyenes, <i>e</i>.<i>g</i>., amphotericin B, candicidin, dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin; synthetic antifungals, such as allylamines, <i>e.g.,</i> butenafine, naftifine, terbinafine; imidazoles, <i>e.g.,</i> bifonazole, butoconazole, chlordantoin, chlormidazole, <i>etc</i>., thiocarbamates, <i>e.g.,</i> tolciclate, triazoles, <i>e.g.,</i> fluconazole, itraconazole, terconazole;

0108Anthelmintics, such as: arecoline, aspidin, aspidinol, dichlorophene, embelin, kosin, napthalene, niclosamide, pelletierine, quinacrine, alantolactone, amocarzine, amoscanate, ascaridole, bephenium, bitoscanate, carbon tetrachloride, carvacrol, cyclobendazole, diethylcarbamazine, etc.;

0109Antimalarials, such as: acedapsone, amodiaquin, arteether, artemether, artemisinin, artesunate, atovaquone, bebeerine, berberine, chirata, chlorguanide, chloroquine, chlorprogaunil, cinchona, cinchonidine, cinchonine, cycloguanil, gentiopicrin, halofantrine, hydroxychloroquine, mefloquine hydrochloride, 3-methylarsacetin, pamaquine, plasmocid, primaquine, pyrimethamine, quinacrine, quinidine, quinine, quinocide, quinoline, dibasic sodium arsenate;

0110Antiprotozoan agents, such as: acranil, tinidazole, ipronidazole, ethylstibamine, pentamidine, acetarsone, aminitrozole, anisomycin, nifuratel, tinidazole, benzidazole, suramin, and the like.

0111Drug compounds of interest from which drug moieties may be derived are also listed in: <nplcit id="ncit0052" npl-type="b"><text>Goodman &amp; Gilman's, The Pharmacological Basis of Therapeutics (9th Ed) (Goodman et al. eds) (McGraw-Hill) (1996</text></nplcit>); and 1999 Physician's Desk Reference (1998).

0112Specific compounds of interest also include, but are not limited to: <ul id="ul0003" list-style="none" compact="compact"><li>antineoplastic agents, as disclosed in <patcit id="pcit0011" dnum="US5880161A"><text>U.S. Pat. Nos. 5,880,161</text></patcit>, <patcit id="pcit0012" dnum="US5877206A"><text>5,877,206</text></patcit>, <patcit id="pcit0013" dnum="US5786344A"><text>5,786,344,</text></patcit><patcit id="pcit0014" dnum="US5760041A"><text>5,760,041</text></patcit>, <patcit id="pcit0015" dnum="US5753668A"><text>5,753,668</text></patcit>,<patcit id="pcit0016" dnum="US5698529A"><text> 5,698,529</text></patcit>, <patcit id="pcit0017" dnum="US5684004A"><text>5,684,004</text></patcit>, <patcit id="pcit0018" dnum="US5665715A"><text>5,665,715</text></patcit>, <patcit id="pcit0019" dnum="US5654484A"><text>5,654,484</text></patcit>, <patcit id="pcit0020" dnum="US5624924A"><text>5,624,924</text></patcit>, <patcit id="pcit0021" dnum="US5618813A"><text>5,618,813</text></patcit>,<patcit id="pcit0022" dnum="US5610292A"><text> 5,610,292</text></patcit>, <patcit id="pcit0023" dnum="US5597831A"><text>5,597,831</text></patcit>,<patcit id="pcit0024" dnum="US5530026A"><text> 5,530,026</text></patcit>,<patcit id="pcit0025" dnum="US5525633A"><text> 5,525,633</text></patcit>,<patcit id="pcit0026" dnum="US5525606A"><text> 5,525,606</text></patcit>, <patcit id="pcit0027" dnum="US5512678A"><text>5,512,678</text></patcit>, <patcit id="pcit0028" dnum="US5508277A"><text>5,508,277</text></patcit>, <patcit id="pcit0029" dnum="US5463181A"><text>5,463,181</text></patcit>, <patcit id="pcit0030" dnum="US5409893A"><text>5,409,893</text></patcit>, <patcit id="pcit0031" dnum="US5358952A"><text>5,358,952</text></patcit>,<patcit id="pcit0032" dnum="US5318965A"><text> 5,318,965</text></patcit> ,<patcit id="pcit0033" dnum="US5223503A"><text>5,223,503</text></patcit>,<patcit id="pcit0034" dnum="US5214068A"><text> 5,214,068</text></patcit>,<patcit id="pcit0035" dnum="US5196424A"><text> 5,196,424</text></patcit>, <patcit id="pcit0036" dnum="US5109024A"><text>5,109,024</text></patcit>, <patcit id="pcit0037" dnum="US5106996A"><text>5,106,996</text></patcit>, <patcit id="pcit0038" dnum="US5101072A"><text>5,101,072</text></patcit>, <patcit id="pcit0039" dnum="US5077404A"><text>5,077,404</text></patcit>, <patcit id="pcit0040" dnum="US5071848A"><text>5,071,848</text></patcit>,<patcit id="pcit0041" dnum="US5066493A"><text> 5,066,493</text></patcit>,<patcit id="pcit0042" dnum="US5019390A"><text> 5,019,390</text></patcit>, <patcit id="pcit0043" dnum="US4996229A"><text>4,996,229</text></patcit>, <patcit id="pcit0044" dnum="US4996206A"><text>4,996,206</text></patcit>, <patcit id="pcit0045" dnum="US4970318A"><text>4,970,318</text></patcit>, <patcit id="pcit0046" dnum="US4968800A"><text>4,968,800</text></patcit>, <patcit id="pcit0047" dnum="US4962114A"><text>4,962,114</text></patcit>, <patcit id="pcit0048" dnum="US4927828A"><text>4,927,828</text></patcit>, <patcit id="pcit0049" dnum="US4892887A"><text>4,892,887</text></patcit>, <patcit id="pcit0050" dnum="US4889859A"><text>4,889,859</text></patcit>, <patcit id="pcit0051" dnum="US4886790A"><text>4,886,790</text></patcit>, <patcit id="pcit0052" dnum="US4882334A"><text>4,882,334</text></patcit>,<patcit id="pcit0053" dnum="US4882333A"><text> 4,882,333</text></patcit>, <patcit id="pcit0054" dnum="US4871746A"><text>4,871,746</text></patcit>, <patcit id="pcit0055" dnum="US4863955A"><text>4,863,955</text></patcit>, <patcit id="pcit0056" dnum="US4849563A"><text>4,849,563</text></patcit>, <patcit id="pcit0057" dnum="US4845216A"><text>4,845,216</text></patcit>, <patcit id="pcit0058" dnum="US4833145A"><text>4,833,145</text></patcit>,<patcit id="pcit0059" dnum="US4824955A"><text> 4,824,955</text></patcit>, <patcit id="pcit0060" dnum="US4785085A"><text>4,785,085</text></patcit>, <patcit id="pcit0061" dnum="US4684747A"><text>4,684,747</text></patcit>, <patcit id="pcit0062" dnum="US4618685A"><text>4,618,685</text></patcit>, <patcit id="pcit0063" dnum="US4611066A"><text>4,611,066</text></patcit>, <patcit id="pcit0064" dnum="US4550187A"><text>4,550,187</text></patcit>, <patcit id="pcit0065" dnum="US4550186A"><text>4,550,186</text></patcit>,<patcit id="pcit0066" dnum="US4544501A"><text> 4,544,501</text></patcit>, <patcit id="pcit0067" dnum="US4541956A"><text>4,541,956</text></patcit>,<patcit id="pcit0068" dnum="US4532327A"><text> 4,532,327</text></patcit>, <patcit id="pcit0069" dnum="US4490540A"><text>4,490,540</text></patcit>, <patcit id="pcit0070" dnum="US4399283A"><text>4,399,283</text></patcit>, <patcit id="pcit0071" dnum="US4391982A"><text>4,391,982</text></patcit>, <patcit id="pcit0072" dnum="US4383994A"><text>4,383,994</text></patcit>, <patcit id="pcit0073" dnum="US4294763A"><text>4,294,763</text></patcit>, <patcit id="pcit0074" dnum="US4283394A"><text>4,283,394</text></patcit>, <patcit id="pcit0075" dnum="US4246411A"><text>4,246,411</text></patcit>, <patcit id="pcit0076" dnum="US4214089A"><text>4,214,089</text></patcit>,<patcit id="pcit0077" dnum="US4150231A"><text> 4,150,231</text></patcit>, <patcit id="pcit0078" dnum="US4147798A"><text>4,147,798</text></patcit>,<patcit id="pcit0079" dnum="US4056673A"><text> 4,056,673</text></patcit>, <patcit id="pcit0080" dnum="US4029661A"><text>4,029,661</text></patcit>, <patcit id="pcit0081" dnum="US4012448A"><text>4,012,448</text></patcit>;</li><li>psychopharmacological/psychotropic agents, as disclosed in <patcit id="pcit0082" dnum="US5192799A"><text>U.S. Pat. Nos. 5,192,799</text></patcit>, <patcit id="pcit0083" dnum="US5036070A"><text>5,036,070</text></patcit>, <patcit id="pcit0084" dnum="US4778800A"><text>4,778,800</text></patcit>, <patcit id="pcit0085" dnum="US4753951A"><text>4,753,951</text></patcit>, <patcit id="pcit0086" dnum="US4590180A"><text>4,590,180</text></patcit>, <patcit id="pcit0087" dnum="US4690930A"><text>4,690,930</text></patcit>, <patcit id="pcit0088" dnum="US4645773A"><text>4,645,773</text></patcit>, <patcit id="pcit0089" dnum="US4427694A"><text>4,427,694</text></patcit>, <patcit id="pcit0090" dnum="US4424202A"><text>4,424,202</text></patcit>, <patcit id="pcit0091" dnum="US4440781A"><text>4,440,781</text></patcit>, <patcit id="pcit0092" dnum="US5686482A"><text>5,686,482</text></patcit>, <patcit id="pcit0093" dnum="US5478828A"><text>5,478,828</text></patcit>, <patcit id="pcit0094" dnum="US5461062A"><text>5,461,062</text></patcit>, <patcit id="pcit0095" dnum="US5387593A"><text>5,387,593</text></patcit>, <patcit id="pcit0096" dnum="US5387586A"><text>5,387,586</text></patcit>, <patcit id="pcit0097" dnum="US5256664A"><text>5,256,664</text></patcit>, <patcit id="pcit0098" dnum="US5192799A"><text>5,192,799</text></patcit>, <patcit id="pcit0099" dnum="US5120733A"><text>5,120,733</text></patcit>, <patcit id="pcit0100" dnum="US5036070A"><text>5,036,070</text></patcit>,<patcit id="pcit0101" dnum="US4977167A"><text> 4,977,167</text></patcit>, <patcit id="pcit0102" dnum="US4904663A"><text>4,904,663</text></patcit>, <patcit id="pcit0103" dnum="US4788188A"><text>4,788,188</text></patcit>, <patcit id="pcit0104" dnum="US4778800A"><text>4,778,800</text></patcit>, <patcit id="pcit0105" dnum="US4753951A"><text>4,753,951</text></patcit>, <patcit id="pcit0106" dnum="US4690930A"><text>4,690,930</text></patcit>, <patcit id="pcit0107" dnum="US4645773A"><text>4,645,773</text></patcit>, <patcit id="pcit0108" dnum="US4631285A"><text>4,631,285</text></patcit>, <patcit id="pcit0109" dnum="US4617314A"><text>4,617,314</text></patcit>, <patcit id="pcit0110" dnum="US4613600A"><text>4,613,600</text></patcit>,<patcit id="pcit0111" dnum="US4590180A"><text> 4,590,180</text></patcit>, <patcit id="pcit0112" dnum="US4560684A"><text>4,560,684</text></patcit>, <patcit id="pcit0113" dnum="US4548938A"><text>4,548,938</text></patcit>, <patcit id="pcit0114" dnum="US4529727A"><text>4,529,727</text></patcit>, <patcit id="pcit0115" dnum="US4459306A"><text>4,459,306</text></patcit>, <patcit id="pcit0116" dnum="US4443451A"><text>4,443,451</text></patcit>, <patcit id="pcit0117" dnum="US4440781A"><text>4,440,781</text></patcit>, <patcit id="pcit0118" dnum="US4427694A"><text>4,427,694</text></patcit>,<patcit id="pcit0119" dnum="US4424202A"><text> 4,424,202</text></patcit>, <patcit id="pcit0120" dnum="US4397853A"><text>4,397,853</text></patcit>, <patcit id="pcit0121" dnum="US4358451A"><text>4,358,451</text></patcit>, <patcit id="pcit0122" dnum="US4324787A"><text>4,324,787</text></patcit>, <patcit id="pcit0123" dnum="US4314081A"><text>4,314,081</text></patcit>, <patcit id="pcit0124" dnum="US4313896A"><text>4,313,896</text></patcit>, <patcit id="pcit0125" dnum="US4294828A"><text>4,294,828</text></patcit>,<patcit id="pcit0126" dnum="US4277476A"><text> 4,277,476</text></patcit>, <patcit id="pcit0127" dnum="US4267328A"><text>4,267,328</text></patcit>, <patcit id="pcit0128" dnum="US4264499A"><text>4,264,499</text></patcit>, <patcit id="pcit0129" dnum="US4231930A"><text>4,231,930</text></patcit>, <patcit id="pcit0130" dnum="US4194009A"><text>4,194,009</text></patcit>, <patcit id="pcit0131" dnum="US4188388A"><text>4,188,388</text></patcit>,<patcit id="pcit0132" dnum="US4148796A"><text> 4,148,796</text></patcit>, <patcit id="pcit0133" dnum="US4128717A"><text>4,128,717</text></patcit>, <patcit id="pcit0134" dnum="US4062858A"><text>4,062,858</text></patcit>,<patcit id="pcit0135" dnum="US4031226A"><text> 4,031,226</text></patcit>,<patcit id="pcit0136" dnum="US4020072A"><text> 4,020,072</text></patcit>,<patcit id="pcit0137" dnum="US4018895A"><text> 4,018,895</text></patcit>, <patcit id="pcit0138" dnum="US4018779A"><text>4,018,779</text></patcit>, <patcit id="pcit0139" dnum="US4013672A"><text>4,013,672</text></patcit>, <patcit id="pcit0140" dnum="US3994898A"><text>3,994,898</text></patcit>, <patcit id="pcit0141" dnum="US3968125A"><text>3,968,125</text></patcit>, <patcit id="pcit0142" dnum="US3939152A"><text>3,939,152,</text></patcit><patcit id="pcit0143" dnum="US3928356A"><text> 3,928,356</text></patcit>, <patcit id="pcit0144" dnum="US3880834A"><text>3,880,834</text></patcit><patcit id="pcit0145" dnum="US3668210A"><text>, 3,668,210</text></patcit>;</li><li>cardiovascular agents, as disclosed in <patcit id="pcit0146" dnum="US4966967A"><text>U.S. Pat. Nos. 4,966,967</text></patcit>,<patcit id="pcit0147" dnum="US5661129A"><text> 5,661,129</text></patcit>, <patcit id="pcit0148" dnum="US5552411A"><text>5,552,411</text></patcit>, <patcit id="pcit0149" dnum="US5332737A"><text>5,332,737</text></patcit>,<patcit id="pcit0150" dnum="US5389675A"><text> 5,389,675</text></patcit>, <patcit id="pcit0151" dnum="US5198449A"><text>5,198,449</text></patcit>, <patcit id="pcit0152" dnum="US5079247A"><text>5,079,247</text></patcit>, <patcit id="pcit0153" dnum="US4966967A"><text>4,966,967</text></patcit>, <patcit id="pcit0154" dnum="US4874760A"><text>4,874,760</text></patcit>, <patcit id="pcit0155" dnum="US4954526A"><text>4,954,526</text></patcit>, <patcit id="pcit0156" dnum="US5051423A"><text>5,051,423</text></patcit>, <patcit id="pcit0157" dnum="US4888335A"><text>4,888,335</text></patcit>, <patcit id="pcit0158" dnum="US4853391A"><text>4,853,391</text></patcit>, <patcit id="pcit0159" dnum="US4906634A"><text>4,906,634</text></patcit>, <patcit id="pcit0160" dnum="US4775757A"><text>4,775,757</text></patcit>, <patcit id="pcit0161" dnum="US4727072A"><text>4,727,072</text></patcit>,<patcit id="pcit0162" dnum="US4542160A"><text> 4,542,160</text></patcit>, <patcit id="pcit0163" dnum="US4522949A"><text>4,522,949</text></patcit>, <patcit id="pcit0164" dnum="US4524151A"><text>4,524,151</text></patcit>, <patcit id="pcit0165" dnum="US4525479A"><text>4,525,479</text></patcit>, <patcit id="pcit0166" dnum="US4474804A"><text>4,474,804</text></patcit>, <patcit id="pcit0167" dnum="US4520026A"><text>4,520,026</text></patcit>, <patcit id="pcit0168" dnum="US4520026A"><text>4,520,026</text></patcit>, <patcit id="pcit0169" dnum="US5869478A"><text>5,869,478</text></patcit>, <patcit id="pcit0170" dnum="US5859239A"><text>5,859,239</text></patcit>, <patcit id="pcit0171" dnum="US5837702A"><text>5,837,702</text></patcit>,<patcit id="pcit0172" dnum="US5807889A"><text> 5,807,889,</text></patcit><patcit id="pcit0173" dnum="US5731322A"><text>5,731,322</text></patcit>, <patcit id="pcit0174" dnum="US5726171A"><text>5,726,171</text></patcit>, <patcit id="pcit0175" dnum="US5723457A"><text>5,723,457</text></patcit>,<patcit id="pcit0176" dnum="US5705523A"><text> 5,705,523</text></patcit>, <patcit id="pcit0177" dnum="US5696111A"><text>5,696,111</text></patcit>, <patcit id="pcit0178" dnum="US5691332A"><text>5,691,332</text></patcit>, <patcit id="pcit0179" dnum="US5679672A"><text>5,679,672</text></patcit>, <patcit id="pcit0180" dnum="US5661129A"><text>5,661,129</text></patcit>, <patcit id="pcit0181" dnum="US5654294A"><text>5,654,294</text></patcit>, <patcit id="pcit0182" dnum="US5646276A"><text>5,646,276</text></patcit>, <patcit id="pcit0183" dnum="US5637586A"><text>5,637,586</text></patcit>, <patcit id="pcit0184" dnum="US5631251A"><text>5,631,251</text></patcit>, <patcit id="pcit0185" dnum="US5612370A"><text>5,612,370</text></patcit>,<patcit id="pcit0186" dnum="US5612323A"><text> 5,612,323,</text></patcit><patcit id="pcit0187" dnum="US5574037A"><text>5,574,037</text></patcit>, <patcit id="pcit0188" dnum="US5563170A"><text>5,563,170</text></patcit>, <patcit id="pcit0189" dnum="US5552411A"><text>5,552,411</text></patcit>,<patcit id="pcit0190" dnum="US5552397A"><text> 5,552,397</text></patcit>, <patcit id="pcit0191" dnum="US5547966A"><text>5,547,966</text></patcit>, <patcit id="pcit0192" dnum="US5482925A"><text>5,482,925</text></patcit>, <patcit id="pcit0193" dnum="US5457118A"><text>5,457,118</text></patcit>, <patcit id="pcit0194" dnum="US5414017A"><text>5,414,017</text></patcit>, <patcit id="pcit0195" dnum="US5414013A"><text>5,414,013</text></patcit>, <patcit id="pcit0196" dnum="US5401758A"><text>5,401,758</text></patcit>, <patcit id="pcit0197" dnum="US5393771A"><text>5,393,771</text></patcit>,<patcit id="pcit0198" dnum="US5362902A"><text> 5,362,902</text></patcit>, <patcit id="pcit0199" dnum="US5332737A"><text>5,332,737</text></patcit>, <patcit id="pcit0200" dnum="US5310731A"><text>5,310,731</text></patcit>, <patcit id="pcit0201" dnum="US5260444A"><text>5,260,444</text></patcit>, <patcit id="pcit0202" dnum="US5223516A"><text>5,223,516</text></patcit>, <patcit id="pcit0203" dnum="US5217958A"><text>5,217,958</text></patcit>, <patcit id="pcit0204" dnum="US5208245A"><text>5,208,245</text></patcit>, <patcit id="pcit0205" dnum="US5202330A"><text>5,202,330</text></patcit>,<patcit id="pcit0206" dnum="US5198449A"><text> 5,198,449</text></patcit>, <patcit id="pcit0207" dnum="US5189036A"><text>5,189,036</text></patcit>, <patcit id="pcit0208" dnum="US5185362A"><text>5,185,362</text></patcit>, <patcit id="pcit0209" dnum="US5140031A"><text>5,140,031</text></patcit>, <patcit id="pcit0210" dnum="US5128349A"><text>5,128,349</text></patcit>, <patcit id="pcit0211" dnum="US5116861A"><text>5,116,861</text></patcit>, <patcit id="pcit0212" dnum="US5079247A"><text>5,079,247,</text></patcit><patcit id="pcit0213" dnum="US5070099A"><text>5,070,099</text></patcit>, <patcit id="pcit0214" dnum="US5061813A"><text>5,061,813</text></patcit>, <patcit id="pcit0215" dnum="US5055466A"><text>5,055,466</text></patcit>, <patcit id="pcit0216" dnum="US5051423A"><text>5,051,423</text></patcit>, <patcit id="pcit0217" dnum="US5036065A"><text>5,036,065</text></patcit>, <patcit id="pcit0218" dnum="US5026712A"><text>5,026,712</text></patcit>, <patcit id="pcit0219" dnum="US5011931A"><text>5,011,931</text></patcit>,<patcit id="pcit0220" dnum="US5006542A"><text> 5,006,542</text></patcit>, <patcit id="pcit0221" dnum="US4981843A"><text>4,981,843</text></patcit>, <patcit id="pcit0222" dnum="US4977144A"><text>4,977,144</text></patcit>, <patcit id="pcit0223" dnum="US4971984A"><text>4,971,984</text></patcit>, <patcit id="pcit0224" dnum="US4966967A"><text>4,966,967</text></patcit>, <patcit id="pcit0225" dnum="US4959383A"><text>4,959,383</text></patcit>, <patcit id="pcit0226" dnum="US4954526A"><text>4,954,526</text></patcit>, <patcit id="pcit0227" dnum="US4952692A"><text>4,952,692</text></patcit>, <patcit id="pcit0228" dnum="US4939137A"><text>4,939,137</text></patcit>, <patcit id="pcit0229" dnum="US4906634A"><text>4,906,634</text></patcit>, <patcit id="pcit0230" dnum="US4889866A"><text>4,889,866</text></patcit>,<patcit id="pcit0231" dnum="US4888335A"><text> 4,888,335</text></patcit>, <patcit id="pcit0232" dnum="US4883872A"><text>4,883,872</text></patcit>, <patcit id="pcit0233" dnum="US4883811A"><text>4,883,811</text></patcit>, <patcit id="pcit0234" dnum="US4847379A"><text>4,847,379</text></patcit>, <patcit id="pcit0235" dnum="US4835157A"><text>4,835,157</text></patcit>, <patcit id="pcit0236" dnum="US4824831A"><text>4,824,831</text></patcit>, <patcit id="pcit0237" dnum="US4780538A"><text>4,780,538</text></patcit>, <patcit id="pcit0238" dnum="US4775757A"><text>4,775,757</text></patcit>, <patcit id="pcit0239" dnum="US4774239A"><text>4,774,239</text></patcit>, <patcit id="pcit0240" dnum="US4771047A"><text>4,771,047</text></patcit>, <patcit id="pcit0241" dnum="US4769371A"><text>4,769,371</text></patcit>, <patcit id="pcit0242" dnum="US4767756A"><text>4,767,756</text></patcit>, <patcit id="pcit0243" dnum="US4762837A"><text>4,762,837</text></patcit>,<patcit id="pcit0244" dnum="US4753946A"><text> 4,753,946</text></patcit>, <patcit id="pcit0245" dnum="US4752616A"><text>4,752,616</text></patcit>,<patcit id="pcit0246" dnum="US4749715A"><text> 4,749,715</text></patcit>, <patcit id="pcit0247" dnum="US4738978A"><text>4,738,978</text></patcit>, <patcit id="pcit0248" dnum="US4735962A"><text>4,735,962</text></patcit>, <patcit id="pcit0249" dnum="US4734426A"><text>4,734,426</text></patcit>, <patcit id="pcit0250" dnum="US4734425A"><text>4,734,425</text></patcit>, <patcit id="pcit0251" dnum="US4734424A"><text>4,734,424</text></patcit>, <patcit id="pcit0252" dnum="US4730052A"><text>4,730,052</text></patcit>, <patcit id="pcit0253" dnum="US4727072A"><text>4,727,072</text></patcit>,<patcit id="pcit0254" dnum="US4721796A"><text>4,721,796</text></patcit>,<patcit id="pcit0255" dnum="US4707550A"><text>4,707,550</text></patcit>,<patcit id="pcit0256" dnum="US470438A"><text>4,704,38</text></patcit><patcit id="pcit0257" dnum="US24703120B"><text>2,4,703,120</text></patcit><patcit id="pcit0258" dnum="US4681970A"><text>,4,681,970</text></patcit>,<patcit id="pcit0259" dnum="US4681882A"><text>4,681,882</text></patcit>,<patcit id="pcit0260" dnum="US4670560A"><text>4,670,560</text></patcit>, <patcit id="pcit0261" dnum="US4670453A"><text>4,670,453</text></patcit>, <patcit id="pcit0262" dnum="US4668787A"><text>4,668,787</text></patcit>, <patcit id="pcit0263" dnum="US4663337A"><text>4,663,337</text></patcit>, <patcit id="pcit0264" dnum="US4663336A"><text>4,663,336</text></patcit>, <patcit id="pcit0265" dnum="US4661506A"><text>4,661,506</text></patcit>, <patcit id="pcit0266" dnum="US4656267A"><text>4,656,267</text></patcit>, <patcit id="pcit0267" dnum="US4656185A"><text>4,656,185</text></patcit>, <patcit id="pcit0268" dnum="US4654357A"><text>4,654,357</text></patcit>, <patcit id="pcit0269" dnum="US4654356A"><text>4,654,356</text></patcit>, <patcit id="pcit0270" dnum="US4654355A"><text>4,654,355</text></patcit>, <patcit id="pcit0271" dnum="US4654335A"><text>4,654,335</text></patcit>, <patcit id="pcit0272" dnum="US4652578A"><text>4,652,578</text></patcit>,<patcit id="pcit0273" dnum="US4652576A"><text> 4,652,576</text></patcit>, <patcit id="pcit0274" dnum="US4650874A"><text>4,650,874</text></patcit>, <patcit id="pcit0275" dnum="US4650797A"><text>4,650,797</text></patcit>, <patcit id="pcit0276" dnum="US4649139A"><text>4,649,139</text></patcit>, <patcit id="pcit0277" dnum="US4647585A"><text>4,647,585</text></patcit>, <patcit id="pcit0278" dnum="US4647573A"><text>4,647,573</text></patcit>, <patcit id="pcit0279" dnum="US4647565A"><text>4,647,565</text></patcit>, <patcit id="pcit0280" dnum="US4647561A"><text>4,647,561</text></patcit>, <patcit id="pcit0281" dnum="US4645836A"><text>4,645,836</text></patcit>, <patcit id="pcit0282" dnum="US4639461A"><text>4,639,461</text></patcit>, <patcit id="pcit0283" dnum="US4638012A"><text>4,638,012</text></patcit>, <patcit id="pcit0284" dnum="US4638011A"><text>4,638,011</text></patcit>, <patcit id="pcit0285" dnum="US4632931A"><text>4,632,931</text></patcit>, <patcit id="pcit0286" dnum="US4631283A"><text>4,631,283</text></patcit>, <patcit id="pcit0287" dnum="US4628095A"><text>4,628,095</text></patcit>, <patcit id="pcit0288" dnum="US4626548A"><text>4,626,548</text></patcit>, <patcit id="pcit0289" dnum="US4614825A"><text>4,614,825</text></patcit>, <patcit id="pcit0290" dnum="US4611007A"><text>4,611,007</text></patcit>, <patcit id="pcit0291" dnum="US4611006A"><text>4,611,006</text></patcit>,<patcit id="pcit0292" dnum="US4611005A"><text> 4,611,005</text></patcit>, <patcit id="pcit0293" dnum="US4609671A"><text>4,609,671</text></patcit>, <patcit id="pcit0294" dnum="US4608386A"><text>4,608,386</text></patcit>, <patcit id="pcit0295" dnum="US4607049A"><text>4,607,049</text></patcit>, <patcit id="pcit0296" dnum="US4607048A"><text>4,607,048</text></patcit>, <patcit id="pcit0297" dnum="US4595692A"><text>4,595,692</text></patcit>,<patcit id="pcit0298" dnum="US4593042A"><text> 4,593,042</text></patcit>, <patcit id="pcit0299" dnum="US4593029A"><text>4,593,029</text></patcit>, <patcit id="pcit0300" dnum="US4591603A"><text>4,591,603</text></patcit>, <patcit id="pcit0301" dnum="US4588743A"><text>4,588,743</text></patcit>, <patcit id="pcit0302" dnum="US4588742A"><text>4,588,742</text></patcit>,<patcit id="pcit0303" dnum="US4588741A"><text> 4,588,741</text></patcit>, <patcit id="pcit0304" dnum="US4582854A"><text>4,582,854</text></patcit>, <patcit id="pcit0305" dnum="US4575512A"><text>4,575,512</text></patcit>, <patcit id="pcit0306" dnum="US4568762A"><text>4,568,762</text></patcit>, <patcit id="pcit0307" dnum="US4560698A"><text>4,560,698</text></patcit>, <patcit id="pcit0308" dnum="US4556739A"><text>4,556,739</text></patcit>, <patcit id="pcit0309" dnum="US4556675A"><text>4,556,675</text></patcit>, <patcit id="pcit0310" dnum="US4555571A"><text>4,555,571</text></patcit>, <patcit id="pcit0311" dnum="US4555570A"><text>4,555,570</text></patcit>, <patcit id="pcit0312" dnum="US4555523A"><text>4,555,523</text></patcit>, <patcit id="pcit0313" dnum="US4550120A"><text>4,550,120</text></patcit>, <patcit id="pcit0314" dnum="US4542160A"><text>4,542,160</text></patcit>, <patcit id="pcit0315" dnum="US4542157A"><text>4,542,157</text></patcit>, <patcit id="pcit0316" dnum="US4542156A"><text>4,542,156</text></patcit>, <patcit id="pcit0317" dnum="US4542155A"><text>4,542,155</text></patcit>, <patcit id="pcit0318" dnum="US4542151A"><text>4,542,151</text></patcit>, <patcit id="pcit0319" dnum="US4537981A"><text>4,537,981</text></patcit>, <patcit id="pcit0320" dnum="US4537904A"><text>4,537,904</text></patcit>, <patcit id="pcit0321" dnum="US4536514A"><text>4,536,514</text></patcit>, <patcit id="pcit0322" dnum="US4536513A"><text>4,536,513</text></patcit>, <patcit id="pcit0323" dnum="US4533673A"><text>4,533,673</text></patcit>, <patcit id="pcit0324" dnum="US4526901A"><text>4,526,901</text></patcit>, <patcit id="pcit0325" dnum="US4526900A"><text>4,526,900</text></patcit>, <patcit id="pcit0326" dnum="US4525479A"><text>4,525,479</text></patcit>, <patcit id="pcit0327" dnum="US4524151A"><text>4,524,151</text></patcit>, <patcit id="pcit0328" dnum="US4522949A"><text>4,522,949</text></patcit>, <patcit id="pcit0329" dnum="US4521539A"><text>4,521,539</text></patcit>, <patcit id="pcit0330" dnum="US4520026A"><text>4,520,026</text></patcit>, <patcit id="pcit0331" dnum="US4517188A"><text>4,517,188</text></patcit>, <patcit id="pcit0332" dnum="US4482562A"><text>4,482,562</text></patcit>, <patcit id="pcit0333" dnum="US4474804A"><text>4,474,804</text></patcit>, <patcit id="pcit0334" dnum="US4474803A"><text>4,474,803</text></patcit>, <patcit id="pcit0335" dnum="US4472411A"><text>4,472,411</text></patcit>, <patcit id="pcit0336" dnum="US4466979A"><text>4,466,979</text></patcit>, <patcit id="pcit0337" dnum="US4463015A"><text>4,463,015</text></patcit>, <patcit id="pcit0338" dnum="US4456617A"><text>4,456,617</text></patcit>, <patcit id="pcit0339" dnum="US4456616A"><text>4,456,616</text></patcit>, <patcit id="pcit0340" dnum="US4456615A"><text>4,456,615</text></patcit>, <patcit id="pcit0341" dnum="US4418076A"><text>4,418,076</text></patcit>, <patcit id="pcit0342" dnum="US4416896A"><text>4,416,896</text></patcit>, <patcit id="pcit0343" dnum="US4252815A"><text>4,252,815,</text></patcit><patcit id="pcit0344" dnum="US4220594A"><text>4,220,594</text></patcit>,<patcit id="pcit0345" dnum="US4190587A"><text> 4,190,587</text></patcit>, <patcit id="pcit0346" dnum="US4177280A"><text>4,177,280</text></patcit>, <patcit id="pcit0347" dnum="US4164586A"><text>4,164,586</text></patcit>, <patcit id="pcit0348" dnum="US4151297A"><text>4,151,297</text></patcit>, <patcit id="pcit0349" dnum="US4145443A"><text>4,145,443</text></patcit>, <patcit id="pcit0350" dnum="US4143054A"><text>4,143,054</text></patcit>,<patcit id="pcit0351" dnum="US4123550A"><text> 4,123,550</text></patcit>, <patcit id="pcit0352" dnum="US4083968A"><text>4,083,968</text></patcit>, <patcit id="pcit0353" dnum="US4076834A"><text>4,076,834</text></patcit>, <patcit id="pcit0354" dnum="US4064259A"><text>4,064,259</text></patcit>, <patcit id="pcit0355" dnum="US4064258A"><text>4,064,258,</text></patcit><patcit id="pcit0356" dnum="US4064257A"><text>4,064,257</text></patcit>, <patcit id="pcit0357" dnum="US4058620A"><text>4,058,620</text></patcit>, <patcit id="pcit0358" dnum="US4001421A"><text>4,001,421</text></patcit>, <patcit id="pcit0359" dnum="US3993639A"><text>3,993,639</text></patcit>, <patcit id="pcit0360" dnum="US3991057A"><text>3,991,057</text></patcit>, <patcit id="pcit0361" dnum="US3982010A"><text>3,982,010</text></patcit>, <patcit id="pcit0362" dnum="US3980652A"><text>3,980,652</text></patcit>,<patcit id="pcit0363" dnum="US3968117A"><text> 3,968,117</text></patcit>, <patcit id="pcit0364" dnum="US3959296A"><text>3,959,296</text></patcit>, <patcit id="pcit0365" dnum="US3951950A"><text> 3,951,950</text></patcit>, <patcit id="pcit0366" dnum="US3933834A"><text>3,933,834</text></patcit>, <patcit id="pcit0367" dnum="US3925369A"><text>3,925,369</text></patcit>, <patcit id="pcit0368" dnum="US3923818A"><text>3,923,818</text></patcit>, <patcit id="pcit0369" dnum="US3898210A"><text>3,898,210</text></patcit>, <patcit id="pcit0370" dnum="US3897442A"><text>3,897,442</text></patcit>, <patcit id="pcit0371" dnum="US3897441A"><text>3,897,441</text></patcit>, <patcit id="pcit0372" dnum="US3886157A"><text>3,886,157</text></patcit>, <patcit id="pcit0373" dnum="US3883540A"><text>3,883,540</text></patcit>, <patcit id="pcit0374" dnum="US3873715A"><text>3,873,715</text></patcit>, <patcit id="pcit0375" dnum="US3867383A"><text>3,867,383</text></patcit>, <patcit id="pcit0376" dnum="US3873715A"><text>3,873,715</text></patcit>, <patcit id="pcit0377" dnum="US3867383A"><text>3,867,383</text></patcit>,<patcit id="pcit0378" dnum="US3691216A"><text> 3,691,216</text></patcit>, <patcit id="pcit0379" dnum="US3624126A"><text>3,624,126</text></patcit>;</li><li>antimicrobial agents as disclosed in <patcit id="pcit0380" dnum="US5902594A"><text>U.S. Pat. Nos. 5,902,594</text></patcit>, <patcit id="pcit0381" dnum="US5874476A"><text>5,874,476</text></patcit>, <patcit id="pcit0382" dnum="US5874436A"><text>5,874,436</text></patcit>, <patcit id="pcit0383" dnum="US5859027A"><text>5,859,027</text></patcit>, <patcit id="pcit0384" dnum="US5856320A"><text>5,856,320</text></patcit>, <patcit id="pcit0385" dnum="US5854242A"><text>5,854,242</text></patcit>, <patcit id="pcit0386" dnum="US5811091A"><text>5,811,091</text></patcit>, <patcit id="pcit0387" dnum="US5786350A"><text>5,786,350</text></patcit>,<patcit id="pcit0388" dnum="US5783177A"><text> 5,783,177</text></patcit>, <patcit id="pcit0389" dnum="US5773469A"><text>5,773,469</text></patcit>, <patcit id="pcit0390" dnum="US5762919A"><text>5,762,919</text></patcit>, <patcit id="pcit0391" dnum="US5753715A"><text>5,753,715</text></patcit>, <patcit id="pcit0392" dnum="US5741526A"><text>5,741,526</text></patcit>, <patcit id="pcit0393" dnum="US5709870A"><text>5,709,870</text></patcit>, <patcit id="pcit0394" dnum="US5707990A"><text>5,707,990</text></patcit>, <patcit id="pcit0395" dnum="US5696117A"><text>5,696,117</text></patcit>, <patcit id="pcit0396" dnum="US5684042A"><text>5,684,042</text></patcit>, <patcit id="pcit0397" dnum="US5683709A"><text>5,683,709</text></patcit>, <patcit id="pcit0398" dnum="US5656591A"><text>5,656,591</text></patcit>, <patcit id="pcit0399" dnum="US5643971A"><text>5,643,971</text></patcit>, <patcit id="pcit0400" dnum="US5643950A"><text>5,643,950</text></patcit>, <patcit id="pcit0401" dnum="US5610196A"><text>5,610,196</text></patcit>, <patcit id="pcit0402" dnum="US5608056A"><text>5,608,056</text></patcit>, <patcit id="pcit0403" dnum="US5604262A"><text>5,604,262</text></patcit>, <patcit id="pcit0404" dnum="US5595742A"><text>5,595,742</text></patcit>, <patcit id="pcit0405" dnum="US5576341A"><text>5,576,341</text></patcit>, <patcit id="pcit0406" dnum="US5554373A"><text>5,554,373</text></patcit>, <patcit id="pcit0407" dnum="US5541233A"><text>5,541,233</text></patcit>, <patcit id="pcit0408" dnum="US5534546A"><text>5,534,546</text></patcit>, <patcit id="pcit0409" dnum="US5534508A"><text>5,534,508</text></patcit>, <patcit id="pcit0410" dnum="US5514715A"><text>5,514,715,</text></patcit><patcit id="pcit0411" dnum="US5508417A"><text>5,508,417</text></patcit>, <patcit id="pcit0412" dnum="US5464832A"><text>5,464,832</text></patcit>, <patcit id="pcit0413" dnum="US5428073A"><text>5,428,073,</text></patcit><patcit id="pcit0414" dnum="US5428016A"><text>5,428,016</text></patcit>, <patcit id="pcit0415" dnum="US5424396A"><text>5,424,396</text></patcit>, <patcit id="pcit0416" dnum="US5399553A"><text>5,399,553</text></patcit>, <patcit id="pcit0417" dnum="US5391544A"><text>5,391,544</text></patcit>, <patcit id="pcit0418" dnum="US5385902A"><text>5,385,902</text></patcit>, <patcit id="pcit0419" dnum="US5359066A"><text>5,359,066</text></patcit>, <patcit id="pcit0420" dnum="US5356803A"><text>5,356,803</text></patcit>, <patcit id="pcit0421" dnum="US5354862A"><text>5,354,862</text></patcit>, <patcit id="pcit0422" dnum="US5346913A"><text>5,346,913</text></patcit>, <patcit id="pcit0423" dnum="US5302592A"><text>5,302,592</text></patcit>, <patcit id="pcit0424" dnum="US5288693A"><text>5,288,693</text></patcit>, <patcit id="pcit0425" dnum="US5266567A"><text>5,266,567</text></patcit>,<patcit id="pcit0426" dnum="US5254685A"><text> 5,254,685</text></patcit>, <patcit id="pcit0427" dnum="US5252745A"><text>5,252,745</text></patcit>,<patcit id="pcit0428" dnum="US5209930A"><text> 5,209,930,</text></patcit><patcit id="pcit0429" dnum="US5196441A"><text>5,196,441</text></patcit>, <patcit id="pcit0430" dnum="US5190961A"><text>5,190,96 1</text></patcit>, <patcit id="pcit0431" dnum="US5175160A"><text>5,175,160</text></patcit>, <patcit id="pcit0432" dnum="US5157051A"><text>5,157,051</text></patcit>, <patcit id="pcit0433" dnum="US5096700A"><text>5,096,700</text></patcit>, <patcit id="pcit0434" dnum="US5093342A"><text>5,093,342</text></patcit>, <patcit id="pcit0435" dnum="US5089251A"><text>5,089,251</text></patcit>, <patcit id="pcit0436" dnum="US5073570A"><text>5,073,570</text></patcit>, <patcit id="pcit0437" dnum="US5061702A"><text>5,061,702</text></patcit>, <patcit id="pcit0438" dnum="US5037809A"><text>5,037,809</text></patcit>, <patcit id="pcit0439" dnum="US5036077A"><text>5,036,077,</text></patcit><patcit id="pcit0440" dnum="US5010109A"><text>5,010,109,</text></patcit><patcit id="pcit0441" dnum="US4970226A"><text>4,970,226</text></patcit>, <patcit id="pcit0442" dnum="US4916156A"><text>4,916,156</text></patcit>, <patcit id="pcit0443" dnum="US4888434A"><text>4,888,434</text></patcit>, <patcit id="pcit0444" dnum="US4870093A"><text>4,870,093</text></patcit>, <patcit id="pcit0445" dnum="US4855318A"><text>4,855,318</text></patcit>, <patcit id="pcit0446" dnum="US4784991A"><text>4,784,991</text></patcit>, <patcit id="pcit0447" dnum="US4746504A"><text>4,746,504</text></patcit>,<patcit id="pcit0448" dnum="US4686221A"><text>4,686,221</text></patcit>,<patcit id="pcit0449" dnum="US4599228A"><text>4,599,228</text></patcit>,<patcit id="pcit0450" dnum="US4552882A"><text>4,552,882</text></patcit>,<patcit id="pcit0451" dnum="US4492700A"><text>4,492,700</text></patcit><patcit id="pcit0452" dnum="US4489098A"><text>,4,489,098</text></patcit><patcit id="pcit0453" dnum="US4489085A"><text>,4,489,085</text></patcit>,<patcit id="pcit0454" dnum="US4487776A"><text>4,487,776</text></patcit>, <patcit id="pcit0455" dnum="US4479953A"><text>4,479,953</text></patcit>, <patcit id="pcit0456" dnum="US4477448A"><text>4,477,448</text></patcit>, <patcit id="pcit0457" dnum="US4474807A"><text>4,474,807</text></patcit>, <patcit id="pcit0458" dnum="US4470994A"><text>4,470,994</text></patcit>, <patcit id="pcit0459" dnum="US4370484A"><text>4,370,484</text></patcit>, <patcit id="pcit0460" dnum="US4337199A"><text>4,337,199</text></patcit>, <patcit id="pcit0461" dnum="US4311709A"><text>4,311,709</text></patcit>, <patcit id="pcit0462" dnum="US4308283A"><text>4,308,283</text></patcit>, <patcit id="pcit0463" dnum="US4304910A"><text>4,304,910</text></patcit>, <patcit id="pcit0464" dnum="US4260634A"><text>4,260,634</text></patcit>, <patcit id="pcit0465" dnum="US4233311A"><text>4,233,311</text></patcit>, <patcit id="pcit0466" dnum="US4215131A"><text>4,215,131</text></patcit>, <patcit id="pcit0467" dnum="US4166122A"><text>4,166,122</text></patcit>, <patcit id="pcit0468" dnum="US4141981A"><text>4,141,981</text></patcit>, <patcit id="pcit0469" dnum="US4130664A"><text>4,130,664</text></patcit>, <patcit id="pcit0470" dnum="US4089977A"><text>4,089,977</text></patcit>, <patcit id="pcit0471" dnum="US4089900A"><text>4,089,900,</text></patcit><patcit id="pcit0472" dnum="US4069341A"><text>4,069,341</text></patcit>, <patcit id="pcit0473" dnum="US4055655A"><text>4,055,655</text></patcit>, <patcit id="pcit0474" dnum="US4049665A"><text>4,049,665</text></patcit>, <patcit id="pcit0475" dnum="US4044139A"><text>4,044,139</text></patcit>, <patcit id="pcit0476" dnum="US4002775A"><text>4,002,775</text></patcit>, <patcit id="pcit0477" dnum="US3991201A"><text>3,991,201</text></patcit>, <patcit id="pcit0478" dnum="US3966968A"><text>3,966,968</text></patcit>, <patcit id="pcit0479" dnum="US3954868A"><text>3,954,868</text></patcit>, <patcit id="pcit0480" dnum="US3936393A"><text>3,936,393</text></patcit>, <patcit id="pcit0481" dnum="US3917476A"><text>3,917,476</text></patcit>, <patcit id="pcit0482" dnum="US3915889A"><text>3,915,889</text></patcit>, <patcit id="pcit0483" dnum="US3867548A"><text>3,867,548</text></patcit>, <patcit id="pcit0484" dnum="US3865748A"><text>3,865,748</text></patcit>, <patcit id="pcit0485" dnum="US3867548A"><text>3,867,548</text></patcit>, <patcit id="pcit0486" dnum="US3865748A"><text>3,865,748</text></patcit>, <patcit id="pcit0487" dnum="US3783160A"><text>3,783,160</text></patcit>, <patcit id="pcit0488" dnum="US3764676A"><text>3,764,676</text></patcit>,<patcit id="pcit0489" dnum="US3764677A"><text> 3,764,677</text></patcit>;</li><li>anti-inflammatory agents as disclosed in <patcit id="pcit0490" dnum="US5872109A"><text>U.S. Pat. Nos. 5,872,109</text></patcit>,<patcit id="pcit0491" dnum="US5837735A"><text> 5,837,735</text></patcit>, <patcit id="pcit0492" dnum="US5827837A"><text>5,827,837</text></patcit>, <patcit id="pcit0493" dnum="US5821250A"><text>5,821,250</text></patcit>, <patcit id="pcit0494" dnum="US5814648A"><text>5,814,648</text></patcit>,<patcit id="pcit0495" dnum="US5780026A"><text> 5,780,026,</text></patcit><patcit id="pcit0496" dnum="US5776946A"><text>5,776,946</text></patcit>,<patcit id="pcit0497" dnum="US5760002A"><text> 5,760,002</text></patcit>, <patcit id="pcit0498" dnum="US5750543A"><text>5,750,543</text></patcit>, <patcit id="pcit0499" dnum="US5741798A"><text>5,741,798</text></patcit>, <patcit id="pcit0500" dnum="US5739279A"><text>5,739,279</text></patcit>, <patcit id="pcit0501" dnum="US5733939A"><text>5,733,939</text></patcit>, <patcit id="pcit0502" dnum="US5723481A"><text>5,723,481</text></patcit>, <patcit id="pcit0503" dnum="US5716967A"><text>5,716,967</text></patcit>, <patcit id="pcit0504" dnum="US5688949A"><text>5,688,949</text></patcit>, <patcit id="pcit0505" dnum="US5686488A"><text>5,686,488</text></patcit>, <patcit id="pcit0506" dnum="US5686471A"><text>5,686,471</text></patcit>, <patcit id="pcit0507" dnum="US5686434A"><text>5,686,434</text></patcit>, <patcit id="pcit0508" dnum="US5684204A"><text>5,684,204</text></patcit>, <patcit id="pcit0509" dnum="US5684041A"><text>5,684,041</text></patcit>, <patcit id="pcit0510" dnum="US5684031A"><text>5,684,031</text></patcit>, <patcit id="pcit0511" dnum="US5684002A"><text>5,684,002</text></patcit>, <patcit id="pcit0512" dnum="US5677318A"><text>5,677,318</text></patcit>, <patcit id="pcit0513" dnum="US5674891A"><text>5,674,891</text></patcit>, <patcit id="pcit0514" dnum="US5672620A"><text>5,672,620</text></patcit>, <patcit id="pcit0515" dnum="US5665752A"><text>5,665,752</text></patcit>, <patcit id="pcit0516" dnum="US5656661A"><text>5,656,661</text></patcit>, <patcit id="pcit0517" dnum="US5635516A"><text>5,635,516</text></patcit>, <patcit id="pcit0518" dnum="US5631283A"><text>5,631,283</text></patcit>, <patcit id="pcit0519" dnum="US5622948A"><text>5,622,948</text></patcit>, <patcit id="pcit0520" dnum="US5618835A"><text>5,618,835</text></patcit>, <patcit id="pcit0521" dnum="US5607959A"><text>5,607,959</text></patcit>, <patcit id="pcit0522" dnum="US5593980A"><text>5,593,980</text></patcit>, <patcit id="pcit0523" dnum="US5593960A"><text>5,593,960</text></patcit>, <patcit id="pcit0524" dnum="US5580888A"><text>5,580,888</text></patcit>, <patcit id="pcit0525" dnum="US5552424A"><text>5,552,424</text></patcit>, <patcit id="pcit0526" dnum="US5552422A"><text>5,552,422</text></patcit>,<patcit id="pcit0527" dnum="US5516764A"><text> 5,516,764</text></patcit>, <patcit id="pcit0528" dnum="US5510361A"><text>5,510,361</text></patcit>, <patcit id="pcit0529" dnum="US5508026A"><text>5,508,026</text></patcit>, <patcit id="pcit0530" dnum="US5500417A"><text>5,500,417</text></patcit>, 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<patcit id="pcit0719" dnum="US4145444A"><text>4,145,444</text></patcit>, <patcit id="pcit0720" dnum="US4128664A"><text>4,128,664</text></patcit>, <patcit id="pcit0721" dnum="US4125612A"><text>4,125,612</text></patcit>, <patcit id="pcit0722" dnum="US4124726A"><text>4,124,726</text></patcit>,<patcit id="pcit0723" dnum="US4124707A"><text> 4,124,707</text></patcit>, <patcit id="pcit0724" dnum="US4117135A"><text>4,117,135</text></patcit>, <patcit id="pcit0725" dnum="US4027031A"><text>4,027,031</text></patcit>, <patcit id="pcit0726" dnum="US4024284A"><text>4,024,284</text></patcit>, <patcit id="pcit0727" dnum="US4021553A"><text>4,021,553</text></patcit>, <patcit id="pcit0728" dnum="US4021550A"><text>4,021,550</text></patcit>, <patcit id="pcit0729" dnum="US4018923A"><text>4,018,923</text></patcit>, <patcit id="pcit0730" dnum="US4012527A"><text>4,012,527</text></patcit>, <patcit id="pcit0731" dnum="US4011326A"><text>4,011,326</text></patcit>, <patcit id="pcit0732" dnum="US3998970A"><text>3,998,970</text></patcit>, <patcit id="pcit0733" dnum="US3998954A"><text>3,998,954</text></patcit>, <patcit id="pcit0734" dnum="US3993763A"><text>3,993,763</text></patcit>, <patcit id="pcit0735" dnum="US3991212A"><text>3,991,212</text></patcit>,<patcit id="pcit0736" dnum="US3984405A"><text> 3,984,405</text></patcit>, <patcit id="pcit0737" dnum="US3978227A"><text>3,978,227</text></patcit>, <patcit id="pcit0738" dnum="US3978219A"><text>3,978,219</text></patcit>, <patcit id="pcit0739" dnum="US3978202A"><text>3,978,202</text></patcit>, <patcit id="pcit0740" dnum="US3975543A"><text>3,975,543</text></patcit>, <patcit id="pcit0741" dnum="US3968224A"><text>3,968,224</text></patcit>, <patcit id="pcit0742" dnum="US3959368A"><text>3,959,368</text></patcit>, <patcit id="pcit0743" dnum="US3949082A"><text>3,949,082</text></patcit>, <patcit id="pcit0744" dnum="US3949081A"><text>3,949,081</text></patcit>, <patcit id="pcit0745" dnum="US3947475A"><text>3,947,475</text></patcit>, <patcit id="pcit0746" dnum="US3936450A"><text>3,936,450</text></patcit>, <patcit id="pcit0747" dnum="US3934018A"><text>3,934,018</text></patcit>, <patcit id="pcit0748" dnum="US3930005A"><text>3,930,005</text></patcit>, <patcit id="pcit0749" dnum="US3857955A"><text>3,857,955</text></patcit>, <patcit id="pcit0750" dnum="US3856962A"><text>3,856,962</text></patcit>, <patcit id="pcit0751" dnum="US3821377A"><text>3,821,377</text></patcit>, <patcit id="pcit0752" dnum="US3821401A"><text>3,821,401</text></patcit>, <patcit id="pcit0753" dnum="US3789121A"><text>3,789,121</text></patcit>, <patcit id="pcit0754" dnum="US3789123A"><text>3,789,123</text></patcit>, <patcit id="pcit0755" dnum="US3726978A"><text>3,726,978</text></patcit>, <patcit id="pcit0756" dnum="US3694471A"><text>3,694,471</text></patcit>, <patcit id="pcit0757" dnum="US3691214A"><text>3,691,214</text></patcit>, <patcit id="pcit0758" dnum="US3678169A"><text>3,678,169</text></patcit>, <patcit id="pcit0759" dnum="US3624216A"><text>3,624,216</text></patcit>;</li><li>immunosuppressive agents, as disclosed in <patcit id="pcit0760" dnum="US4450159A"><text>U.S. Pat Nos. 4,450,159</text></patcit>, <patcit id="pcit0761" dnum="US4450159A"><text>4,450,159</text></patcit>, <patcit id="pcit0762" dnum="US5905085A"><text>5,905,085</text></patcit>, <patcit id="pcit0763" dnum="US5883119A"><text>5,883,119</text></patcit>, <patcit id="pcit0764" dnum="US5880280A"><text>5,880,280,</text></patcit><patcit id="pcit0765" dnum="US5877184A"><text>5,877,184</text></patcit>, <patcit id="pcit0766" dnum="US5874594A"><text>5,874,594</text></patcit>, <patcit id="pcit0767" dnum="US5843452A"><text>5,843,452</text></patcit>, <patcit id="pcit0768" dnum="US5817672A"><text>5,817,672</text></patcit>,<patcit id="pcit0769" dnum="US5817661A"><text> 5,817,661</text></patcit>, <patcit id="pcit0770" dnum="US5817660A"><text>5,817,660</text></patcit>, <patcit id="pcit0771" dnum="US5801193A"><text>5,801,193</text></patcit>,<patcit id="pcit0772" dnum="US5776974A"><text> 5,776,974</text></patcit>, <patcit id="pcit0773" dnum="US5763478A"><text>5,763,478</text></patcit>, <patcit id="pcit0774" dnum="US5739169A"><text>5,739,169</text></patcit>,<patcit id="pcit0775" dnum="US5723466A"><text> 5,723,466</text></patcit>, <patcit id="pcit0776" dnum="US5719176A"><text>5,719,176</text></patcit>, <patcit id="pcit0777" dnum="US5696156A"><text>5,696,156</text></patcit>, <patcit id="pcit0778" dnum="US5695753A"><text>5,695,753</text></patcit>, <patcit id="pcit0779" dnum="US5693648A"><text>5,693,648</text></patcit>,<patcit id="pcit0780" dnum="US5693645A"><text> 5,693,645</text></patcit>, <patcit id="pcit0781" dnum="US5691346A"><text>5,691,346</text></patcit>, <patcit id="pcit0782" dnum="US5686469A"><text>5,686,469</text></patcit>, <patcit id="pcit0783" dnum="US5686424A"><text>5,686,424</text></patcit>, <patcit id="pcit0784" dnum="US5679705A"><text>5,679,705</text></patcit>, <patcit id="pcit0785" dnum="US5679640A"><text>5,679,640</text></patcit>, <patcit id="pcit0786" dnum="US5670504A"><text>5,670,504</text></patcit>, <patcit id="pcit0787" dnum="US5665774A"><text>5,665,774</text></patcit>, <patcit id="pcit0788" dnum="US5665772A"><text>5,665,772</text></patcit>, <patcit id="pcit0789" dnum="US5648376A"><text>5,648,376</text></patcit>, <patcit id="pcit0790" dnum="US5639455A"><text>5,639,455</text></patcit>, <patcit id="pcit0791" dnum="US5633277A"><text>5,633,277</text></patcit>, <patcit id="pcit0792" dnum="US5624930A"><text>5,624,930</text></patcit>, <patcit id="pcit0793" dnum="US5622970A"><text>5,622,970</text></patcit>, <patcit id="pcit0794" dnum="US5605903A"><text>5,605,903</text></patcit>, <patcit id="pcit0795" dnum="US5604229A"><text>5,604,229</text></patcit>,<patcit id="pcit0796" dnum="US5574041A"><text> 5,574,041</text></patcit>, <patcit id="pcit0797" dnum="US5565560A"><text>5,565,560</text></patcit>, <patcit id="pcit0798" dnum="US5550233A"><text>5,550,233</text></patcit>, <patcit id="pcit0799" dnum="US5545734A"><text>5,545,734</text></patcit>, <patcit id="pcit0800" dnum="US5540931A"><text>5,540,931</text></patcit>,<patcit id="pcit0801" dnum="US5532248A"><text> 5,532,248</text></patcit>, <patcit id="pcit0802" dnum="US5527820A"><text>5,527,820</text></patcit>,<patcit id="pcit0803" dnum="US5516797A"><text> 5,516,797</text></patcit>, <patcit id="pcit0804" dnum="US5514688A"><text>5,514,688</text></patcit>, <patcit id="pcit0805" dnum="US5512687A"><text>5,512,687</text></patcit>, <patcit id="pcit0806" dnum="US5506233A"><text>5,506,233</text></patcit>, <patcit id="pcit0807" dnum="US5506228A"><text>5,506,228</text></patcit>, <patcit id="pcit0808" dnum="US5494895A"><text>5,494,895</text></patcit>, <patcit id="pcit0809" dnum="US5484788A"><text>5,484,788</text></patcit>, <patcit id="pcit0810" dnum="US5470857A"><text>5,470,857</text></patcit>, <patcit id="pcit0811" dnum="US5464615A"><text>5,464,615</text></patcit>, <patcit id="pcit0812" dnum="US5432183A"><text>5,432,183</text></patcit>,<patcit id="pcit0813" dnum="US5431896A"><text> 5,431,896</text></patcit>, <patcit id="pcit0814" dnum="US5385918A"><text>5,385,918</text></patcit>, <patcit id="pcit0815" dnum="US5349061A"><text>5,349,061</text></patcit>, <patcit id="pcit0816" dnum="US5344925A"><text>5,344,925</text></patcit>, <patcit id="pcit0817" dnum="US5330993A"><text>5,330,993</text></patcit>, <patcit id="pcit0818" dnum="US5308837A"><text>5,308,837</text></patcit>, <patcit id="pcit0819" dnum="US5290783A"><text>5,290,783</text></patcit>, <patcit id="pcit0820" dnum="US5290772A"><text>5,290,772</text></patcit>, <patcit id="pcit0821" dnum="US5284877A"><text>5,284,877</text></patcit>, <patcit id="pcit0822" dnum="US5284840A"><text>5,284,840</text></patcit>, <patcit id="pcit0823" dnum="US5273979A"><text>5,273,979</text></patcit>, <patcit id="pcit0824" dnum="US5262533A"><text>5,262,533</text></patcit>, <patcit id="pcit0825" dnum="US5260300A"><text>5,260,300</text></patcit>, <patcit id="pcit0826" dnum="US5252732A"><text>5,252,732</text></patcit>, <patcit id="pcit0827" dnum="US5250678A"><text>5,250,678</text></patcit>,<patcit id="pcit0828" dnum="US5247076A"><text> 5,247,076</text></patcit>, <patcit id="pcit0829" dnum="US5244896A"><text>5,244,896</text></patcit>,<patcit id="pcit0830" dnum="US5238689A"><text> 5,238,689</text></patcit>, <patcit id="pcit0831" dnum="US5219884A"><text>5,219,884</text></patcit>, <patcit id="pcit0832" dnum="US5208241A"><text>5,208,241</text></patcit>, <patcit id="pcit0833" dnum="US5208228A"><text>5,208,228</text></patcit>, <patcit id="pcit0834" dnum="US5202332A"><text>5,202,332</text></patcit>,<patcit id="pcit0835" dnum="US5192773A"><text> 5,192,773</text></patcit>, <patcit id="pcit0836" dnum="US5189042A"><text>5,189,042</text></patcit>, <patcit id="pcit0837" dnum="US5169851A"><text>5,169,851</text></patcit>, <patcit id="pcit0838" dnum="US5162334A"><text>5,162,334</text></patcit>, <patcit id="pcit0839" dnum="US5151413A"><text>5,151,413</text></patcit>, <patcit id="pcit0840" dnum="US5149701A"><text>5,149,701</text></patcit>, <patcit id="pcit0841" dnum="US5147877A"><text>5,147,877</text></patcit>, <patcit id="pcit0842" dnum="US5143918A"><text>5,143,918</text></patcit>, <patcit id="pcit0843" dnum="US5138051A"><text>5,138,051</text></patcit>, <patcit id="pcit0844" dnum="US5093338A"><text>5,093,338</text></patcit>, <patcit id="pcit0845" dnum="US5091389A"><text>5,091,389</text></patcit>, <patcit id="pcit0846" dnum="US5068323A"><text>5,068,323</text></patcit>, <patcit id="pcit0847" dnum="US5068247A"><text>5,068,247</text></patcit>, <patcit id="pcit0848" dnum="US5064835A"><text>5,064,835</text></patcit>, <patcit id="pcit0849" dnum="US5061728A"><text>5,061,728</text></patcit>, <patcit id="pcit0850" dnum="US5055290A"><text>5,055,290</text></patcit>, <patcit id="pcit0851" dnum="US4981792A"><text>4,981,792</text></patcit>, <patcit id="pcit0852" dnum="US4810692A"><text>4,810,692</text></patcit>, <patcit id="pcit0853" dnum="US4410696A"><text>4,410,696</text></patcit>, <patcit id="pcit0854" dnum="US4346096A"><text>4,346,096</text></patcit>, <patcit id="pcit0855" dnum="US4342769A"><text>4,342,769</text></patcit>, <patcit id="pcit0856" dnum="US4317825A"><text>4,317,825</text></patcit>, <patcit id="pcit0857" dnum="US4256766A"><text>4,256,766</text></patcit>, <patcit id="pcit0858" dnum="US4180588A"><text>4,180,588</text></patcit>, <patcit id="pcit0859" dnum="US4000275A"><text>4,000,275</text></patcit>, <patcit id="pcit0860" dnum="US3759921A"><text>3,759,921</text></patcit>;</li><li>analgesic agents, as disclosed in <patcit id="pcit0861" dnum="US5292736A"><text>U.S. Pat. Nos. 5,292,736</text></patcit>, <patcit id="pcit0862" dnum="US5688825A"><text>5,688,825,</text></patcit><patcit id="pcit0863" dnum="US5554789A"><text>5,554,789</text></patcit>, <patcit id="pcit0864" dnum="US5455230A"><text>5,455,230</text></patcit>, <patcit id="pcit0865" dnum="US5292736A"><text>5,292,736</text></patcit>, <patcit id="pcit0866" dnum="US5298522A"><text>5,298,522</text></patcit>, <patcit id="pcit0867" dnum="US5216165A"><text>5,216,165,</text></patcit><patcit id="pcit0868" dnum="US5438064A"><text>5,438,064</text></patcit>, <patcit id="pcit0869" dnum="US5204365A"><text>5,204,365</text></patcit>, <patcit id="pcit0870" dnum="US5017578A"><text>5,017,578</text></patcit>, <patcit id="pcit0871" dnum="US4906655A"><text>4,906,655</text></patcit>, <patcit id="pcit0872" dnum="US4906655A"><text>4,906,655</text></patcit>, <patcit id="pcit0873" dnum="US4994450A"><text>4,994,450,</text></patcit><patcit id="pcit0874" dnum="US4749792A"><text>4,749,792</text></patcit>, <patcit id="pcit0875" dnum="US4980365A"><text>4,980,365</text></patcit>, <patcit id="pcit0876" dnum="US4794110A"><text>4,794,110</text></patcit>, <patcit id="pcit0877" dnum="US4670541A"><text>4,670,541</text></patcit>, <patcit id="pcit0878" dnum="US4737493A"><text>4,737,493</text></patcit>, <patcit id="pcit0879" dnum="US4622326A"><text>4,622,326</text></patcit>, <patcit id="pcit0880" dnum="US4536512A"><text>4,536,512</text></patcit>, <patcit id="pcit0881" dnum="US4719231A"><text>4,719,231</text></patcit>, <patcit id="pcit0882" dnum="US4533671A"><text>4,533,671</text></patcit>, <patcit id="pcit0883" dnum="US4552866A"><text>4,552,866</text></patcit>, <patcit id="pcit0884" dnum="US4539312A"><text>4,539,312</text></patcit>, <patcit id="pcit0885" dnum="US4569942A"><text>4,569,942</text></patcit>, <patcit id="pcit0886" dnum="US4681879A"><text>4,681,879</text></patcit>, <patcit id="pcit0887" dnum="US4511724A"><text>4,511,724</text></patcit>, <patcit id="pcit0888" dnum="US4556672A"><text>4,556,672</text></patcit>, <patcit id="pcit0889" dnum="US4721712A"><text>4,721,712</text></patcit>, <patcit id="pcit0890" dnum="US4474806A"><text>4,474,806</text></patcit>, <patcit id="pcit0891" dnum="US4595686A"><text>4,595,686</text></patcit>, <patcit id="pcit0892" dnum="US4440779A"><text>4,440,779</text></patcit>, <patcit id="pcit0893" dnum="US4434175A"><text>4,434,175</text></patcit>, <patcit id="pcit0894" dnum="US4608374A"><text>4,608,374</text></patcit>, <patcit id="pcit0895" dnum="US4395402A"><text>4,395,402</text></patcit>, <patcit id="pcit0896" dnum="US4400534A"><text>4,400,534</text></patcit>, <patcit id="pcit0897" dnum="US4374139A"><text>4,374,139</text></patcit>, <patcit id="pcit0898" dnum="US4361583A"><text>4,361,583</text></patcit>, <patcit id="pcit0899" dnum="US4252816A"><text>4,252,816</text></patcit>, <patcit id="pcit0900" dnum="US4251530A"><text>4,251,530</text></patcit>, <patcit id="pcit0901" dnum="US5874459A"><text>5,874,459</text></patcit>, <patcit id="pcit0902" dnum="US5688825A"><text>5,688,825</text></patcit>, <patcit id="pcit0903" dnum="US5554789A"><text>5,554,789</text></patcit>, <patcit id="pcit0904" dnum="US5455230A"><text>5,455,230</text></patcit>, <patcit id="pcit0905" dnum="US5438064A"><text>5,438,064</text></patcit>, <patcit id="pcit0906" dnum="US5298522A"><text>5,298,522</text></patcit>, <patcit id="pcit0907" dnum="US5216165A"><text>5,216,165</text></patcit>, <patcit id="pcit0908" dnum="US5204365A"><text>5,204,365</text></patcit>, <patcit id="pcit0909" dnum="US5030639A"><text>5,030,639</text></patcit>, <patcit id="pcit0910" dnum="US5017578A"><text>5,017,578</text></patcit>, <patcit id="pcit0911" dnum="US5008264A"><text>5,008,264</text></patcit>, <patcit id="pcit0912" dnum="US4994450A"><text>4,994,450</text></patcit>, <patcit id="pcit0913" dnum="US4980365A"><text>4,980,365</text></patcit>, <patcit id="pcit0914" dnum="US4906655A"><text>4,906,655</text></patcit>, <patcit id="pcit0915" dnum="US4847290A"><text>4,847,290</text></patcit>, <patcit id="pcit0916" dnum="US4844907A"><text>4,844,907</text></patcit>, <patcit id="pcit0917" dnum="US4794110A"><text>4,794,110</text></patcit>, <patcit id="pcit0918" dnum="US4791129A"><text>4,791,129</text></patcit>, <patcit id="pcit0919" dnum="US4774256A"><text>4,774,256</text></patcit>, <patcit id="pcit0920" dnum="US4749792A"><text>4,749,792</text></patcit>, <patcit id="pcit0921" dnum="US4737493A"><text>4,737,493</text></patcit>, <patcit id="pcit0922" dnum="US4721712A"><text>4,721,712</text></patcit>, <patcit id="pcit0923" dnum="US4719231A"><text>4,719,231</text></patcit>, <patcit id="pcit0924" dnum="US4681879A"><text>4,681,879</text></patcit>, <patcit id="pcit0925" dnum="US4670541A"><text>4,670,541</text></patcit>, <patcit id="pcit0926" dnum="US4667039A"><text>4,667,039</text></patcit>, <patcit id="pcit0927" dnum="US4658037A"><text>4,658,037</text></patcit>, <patcit id="pcit0928" dnum="US4634708A"><text>4,634,708</text></patcit>, <patcit id="pcit0929" dnum="US4623648A"><text>4,623,648</text></patcit>, <patcit id="pcit0930" dnum="US4622326A"><text>4,622,326</text></patcit>, <patcit id="pcit0931" dnum="US4608374A"><text>4,608,374</text></patcit>, <patcit id="pcit0932" dnum="US4595686A"><text>4,595,686</text></patcit>, <patcit id="pcit0933" dnum="US4594188A"><text>4,594,188</text></patcit>, <patcit id="pcit0934" dnum="US4569942A"><text>4,569,942</text></patcit>, <patcit id="pcit0935" dnum="US4556672A"><text>4,556,672</text></patcit>, <patcit id="pcit0936" dnum="US4552866A"><text>4,552,866</text></patcit>, <patcit id="pcit0937" dnum="US4539312A"><text>4,539,312</text></patcit>, <patcit id="pcit0938" dnum="US4536512A"><text>4,536,512</text></patcit>, <patcit id="pcit0939" dnum="US4533671A"><text>4,533,671</text></patcit>, <patcit id="pcit0940" dnum="US4511724A"><text>4,511,724</text></patcit>, <patcit id="pcit0941" dnum="US4440779A"><text>4,440,779,</text></patcit><patcit id="pcit0942" dnum="US4434175A"><text>4,434,175</text></patcit>, <patcit id="pcit0943" dnum="US4400534A"><text>4,400,534</text></patcit>, <patcit id="pcit0944" dnum="US4395402A"><text>4,395,402</text></patcit>, <patcit id="pcit0945" dnum="US4391827A"><text>4,391,827</text></patcit>, <patcit id="pcit0946" dnum="US4374139A"><text>4,374,139</text></patcit>, <patcit id="pcit0947" dnum="US4361583A"><text>4,361,583</text></patcit>, <patcit id="pcit0948" dnum="US4322420A"><text>4,322,420</text></patcit>, <patcit id="pcit0949" dnum="US4306097A"><text>4,306,097</text></patcit>, <patcit id="pcit0950" dnum="US4252816A"><text>4,252,816</text></patcit>, <patcit id="pcit0951" dnum="US4251530A"><text>4,251,530</text></patcit>, <patcit id="pcit0952" dnum="US4244955A"><text>4,244,955</text></patcit>, <patcit id="pcit0953" dnum="US4232018A"><text>4,232,018</text></patcit>, <patcit id="pcit0954" dnum="US4209520A"><text>4,209,520</text></patcit>, <patcit id="pcit0955" dnum="US4164514A"><text>4,164,514</text></patcit>, <patcit id="pcit0956" dnum="US4147872A"><text>4,147,872</text></patcit>,<patcit id="pcit0957" dnum="US4133819A"><text> 4,133,819</text></patcit>, <patcit id="pcit0958" dnum="US4124713A"><text>4,124,713</text></patcit>, <patcit id="pcit0959" dnum="US4117012A"><text>4,117,012</text></patcit>, <patcit id="pcit0960" dnum="US4064272A"><text>4,064,272</text></patcit>, <patcit id="pcit0961" dnum="US4022836A"><text>4,022,836</text></patcit>,<patcit id="pcit0962" dnum="US3966944A"><text> 3,966,944</text></patcit>;</li><li>cholinergic agents, as disclosed in <patcit id="pcit0963" dnum="US5219872A"><text>U.S. Pat. Nos. 5,219,872</text></patcit>,<patcit id="pcit0964" dnum="US5219873A"><text>5,219,873</text></patcit>,<patcit id="pcit0965" dnum="US5073560A"><text>5,073,560</text></patcit>, <patcit id="pcit0966" dnum="US5073560A"><text>5,073,560</text></patcit>, <patcit id="pcit0967" dnum="US5346911A"><text>5,346,911</text></patcit>, <patcit id="pcit0968" dnum="US5424301A"><text>5,424,301</text></patcit>, <patcit id="pcit0969" dnum="US5073560A"><text>5,073,560</text></patcit>, <patcit id="pcit0970" dnum="US5219872A"><text>5,219,872</text></patcit>, <patcit id="pcit0971" dnum="US4900748A"><text>4,900,748</text></patcit>, <patcit id="pcit0972" dnum="US4786648A"><text>4,786,648</text></patcit>, <patcit id="pcit0973" dnum="US4798841A"><text>4,798,841</text></patcit>, <patcit id="pcit0974" dnum="US4782071A"><text>4,782,071</text></patcit>, <patcit id="pcit0975" dnum="US4710508A"><text>4,710,508</text></patcit>, <patcit id="pcit0976" dnum="US5482938A"><text>5,482,938</text></patcit>,<patcit id="pcit0977" dnum="US5464842A"><text> 5,464,842</text></patcit>, <patcit id="pcit0978" dnum="US5378723A"><text>5,378,723</text></patcit>, <patcit id="pcit0979" dnum="US5346911A"><text>5,346,911</text></patcit>, <patcit id="pcit0980" dnum="US5318978A"><text>5,318,978</text></patcit>, <patcit id="pcit0981" dnum="US5219873A"><text>5,219,873</text></patcit>, <patcit id="pcit0982" dnum="US5219872A"><text>5,219,872</text></patcit>, <patcit id="pcit0983" dnum="US5084281A"><text>5,084,281</text></patcit>, <patcit id="pcit0984" dnum="US5073560A"><text>5,073,560</text></patcit>, <patcit id="pcit0985" dnum="US5002955A"><text>5,002,955</text></patcit>, <patcit id="pcit0986" dnum="US4988710A"><text>4,988,710</text></patcit>, <patcit id="pcit0987" dnum="US4900748A"><text>4,900,748</text></patcit>, <patcit id="pcit0988" dnum="US4798841A"><text>4,798,841</text></patcit>, <patcit id="pcit0989" dnum="US4786648A"><text>4,786,648</text></patcit>, <patcit id="pcit0990" dnum="US4782071A"><text>4,782,071</text></patcit>, <patcit id="pcit0991" dnum="US4745123A"><text>4,745,123</text></patcit>, <patcit id="pcit0992" dnum="US4710508A"><text>4,710,508</text></patcit>;</li><li>adrenergic agents, as disclosed in <patcit id="pcit0993" dnum="US5091528A"><text>U.S. Pat. Nos. 5,091,528</text></patcit>, <patcit id="pcit0994" dnum="US5091528A"><text>5,091,528</text></patcit>,<patcit id="pcit0995" dnum="US4835157A"><text>4,835,157</text></patcit>, <patcit id="pcit0996" dnum="US5708015A"><text>5,708,015</text></patcit>, <patcit id="pcit0997" dnum="US5594027A"><text>5,594,027</text></patcit>, <patcit id="pcit0998" dnum="US5580892A"><text>5,580,892</text></patcit>,<patcit id="pcit0999" dnum="US5576332A"><text> 5,576,332</text></patcit>,<patcit id="pcit1000" dnum="US5510376A"><text> 5,510,376</text></patcit>, <patcit id="pcit1001" dnum="US5482961A"><text>5,482,961</text></patcit>, <patcit id="pcit1002" dnum="US5334601A"><text>5,334,601</text></patcit>, <patcit id="pcit1003" dnum="US5202347A"><text>5,202,347</text></patcit>, <patcit id="pcit1004" dnum="US5135926A"><text>5,135,926</text></patcit>, <patcit id="pcit1005" dnum="US5116867A"><text>5,116,867</text></patcit>, <patcit id="pcit1006" dnum="US5091528A"><text>5,091,528</text></patcit>, <patcit id="pcit1007" dnum="US5017618A"><text>5,017,618</text></patcit>, <patcit id="pcit1008" dnum="US4835157A"><text>4,835,157</text></patcit>, <patcit id="pcit1009" dnum="US4829086A"><text>4,829,086</text></patcit>, <patcit id="pcit1010" dnum="US4579867A"><text>4,579,867</text></patcit>,<patcit id="pcit1011" dnum="US4568679A"><text> 4,568,679</text></patcit>, <patcit id="pcit1012" dnum="US4469690A"><text>4,469,690</text></patcit>, <patcit id="pcit1013" dnum="US4395559A"><text>4,395,559</text></patcit>, <patcit id="pcit1014" dnum="US4381309A"><text>4,381,309</text></patcit>, <patcit id="pcit1015" dnum="US4363808A"><text>4,363,808</text></patcit>, <patcit id="pcit1016" dnum="US4343800A"><text>4,343,800</text></patcit>, <patcit id="pcit1017" dnum="US4329289A"><text>4,329,289</text></patcit>, <patcit id="pcit1018" dnum="US4314943A"><text>4,314,943</text></patcit>,<patcit id="pcit1019" dnum="US4311708A"><text> 4,311,708</text></patcit>, <patcit id="pcit1020" dnum="US4304721A"><text>4,304,721</text></patcit>, <patcit id="pcit1021" dnum="US4296117A"><text>4,296,117</text></patcit>, <patcit id="pcit1022" dnum="US4285873A"><text>4,285,873</text></patcit>, <patcit id="pcit1023" dnum="US4281189A"><text>4,281,189</text></patcit>, <patcit id="pcit1024" dnum="US4278608A"><text>4,278,608</text></patcit>, <patcit id="pcit1025" dnum="US4247710A"><text>4,247,710</text></patcit>, <patcit id="pcit1026" dnum="US4145550A"><text>4,145,550</text></patcit>, <patcit id="pcit1027" dnum="US4145425A"><text>4,145,425</text></patcit>, <patcit id="pcit1028" dnum="US4139535A"><text>4,139,535</text></patcit>, <patcit id="pcit1029" dnum="US4082843A"><text>4,082,843</text></patcit>, <patcit id="pcit1030" dnum="US4011321A"><text>4,011,321</text></patcit>, <patcit id="pcit1031" dnum="US4001421A"><text>4,001,421</text></patcit>, <patcit id="pcit1032" dnum="US3982010A"><text>3,982,010</text></patcit>, <patcit id="pcit1033" dnum="US3940407A"><text>3,940,407</text></patcit>, <patcit id="pcit1034" dnum="US3852468A"><text>3,852,468</text></patcit>, <patcit id="pcit1035" dnum="US3832470A"><text>3,832,470</text></patcit>;</li><li>antihistamine agents, as disclosed in <patcit id="pcit1036" dnum="US5874479A"><text>U.S. Pat. Nos. 5,874,479</text></patcit>, <patcit id="pcit1037" dnum="US5863938A"><text>5,863,938</text></patcit>, <patcit id="pcit1038" dnum="US5856364A"><text>5,856,364</text></patcit>, <patcit id="pcit1039" dnum="US5770612A"><text>5,770,612</text></patcit>, <patcit id="pcit1040" dnum="US5702688A"><text>5,702,688</text></patcit>, <patcit id="pcit1041" dnum="US5674912A"><text>5,674,912</text></patcit>, <patcit id="pcit1042" dnum="US5663208A"><text>5,663,208</text></patcit>, <patcit id="pcit1043" dnum="US5658957A"><text>5,658,957</text></patcit>, <patcit id="pcit1044" dnum="US5652274A"><text>5,652,274</text></patcit>, <patcit id="pcit1045" dnum="US5648380A"><text>5,648,380</text></patcit>, <patcit id="pcit1046" dnum="US5646190A"><text>5,646,190</text></patcit>, <patcit id="pcit1047" dnum="US5641814A"><text>5,641,814</text></patcit>, <patcit id="pcit1048" dnum="US5633285A"><text>5,633,285</text></patcit>, <patcit id="pcit1049" dnum="US5614561A"><text>5,614,561</text></patcit>, <patcit id="pcit1050" dnum="US5602183A"><text>5,602,183</text></patcit>, <patcit id="pcit1051" dnum="US4923892A"><text>4,923,892</text></patcit>, <patcit id="pcit1052" dnum="US4782058A"><text>4,782,058</text></patcit>, <patcit id="pcit1053" dnum="US4393210A"><text>4,393,210</text></patcit>, <patcit id="pcit1054" dnum="US4180583A"><text>4,180,583</text></patcit>, <patcit id="pcit1055" dnum="US3965257A"><text>3,965,257</text></patcit>, <patcit id="pcit1056" dnum="US3946022A"><text>3,946,022</text></patcit>, <patcit id="pcit1057" dnum="US3931197A"><text>3,931,197</text></patcit>;</li><li>steroidal agents, as disclosed in <patcit id="pcit1058" dnum="US5863538A"><text>U.S. Pat. Nos. 5,863,538</text></patcit>, <patcit id="pcit1059" dnum="US5855907A"><text>5,855,907</text></patcit>, <patcit id="pcit1060" dnum="US5855866A"><text>5,855,866</text></patcit>, <patcit id="pcit1061" dnum="US5780592A"><text>5,780,592</text></patcit>, <patcit id="pcit1062" dnum="US5776427A"><text>5,776,427</text></patcit>,<patcit id="pcit1063" dnum="US5651987A"><text> 5,651,987</text></patcit>, <patcit id="pcit1064" dnum="US5346887A"><text>5,346,887</text></patcit>, <patcit id="pcit1065" dnum="US5256408A"><text>5,256,408</text></patcit>, <patcit id="pcit1066" dnum="US5252319A"><text>5,252,319</text></patcit>, <patcit id="pcit1067" dnum="US5209926A"><text>5,209,926,</text></patcit><patcit id="pcit1068" dnum="US4996335A"><text>4,996,335</text></patcit>, <patcit id="pcit1069" dnum="US4927807A"><text>4,927,807</text></patcit>,<patcit id="pcit1070" dnum="US4910192A"><text> 4,910,192</text></patcit>, <patcit id="pcit1071" dnum="US4710495A"><text>4,710,495</text></patcit>, <patcit id="pcit1072" dnum="US4049805A"><text>4,049,805</text></patcit>, <patcit id="pcit1073" dnum="US4004005A"><text>4,004,005</text></patcit>, <patcit id="pcit1074" dnum="US3670079A"><text>3,670,079</text></patcit>,<patcit id="pcit1075" dnum="US3608076A"><text> 3,608,076</text></patcit>, <patcit id="pcit1076" dnum="US5892028A"><text>5,892,028</text></patcit>, <patcit id="pcit1077" dnum="US5888995A"><text>5,888,995</text></patcit>, <patcit id="pcit1078" dnum="US5883087A"><text>5,883,087</text></patcit>, <patcit id="pcit1079" dnum="US5880115A"><text>5,880,115</text></patcit>, <patcit id="pcit1080" dnum="US5869475A"><text>5,869,475</text></patcit>, <patcit id="pcit1081" dnum="US5866558A"><text>5,866,558</text></patcit>, <patcit id="pcit1082" dnum="US5861390A"><text>5,861,390</text></patcit>, <patcit id="pcit1083" dnum="US5861388A"><text>5,861,388</text></patcit>, <patcit id="pcit1084" dnum="US5854235A"><text>5,854,235</text></patcit>, <patcit id="pcit1085" dnum="US5837698A"><text>5,837,698</text></patcit>,<patcit id="pcit1086" dnum="US5834452A"><text> 5,834,452</text></patcit>, <patcit id="pcit1087" dnum="US5830886A"><text>5,830,886</text></patcit>, <patcit id="pcit1088" dnum="US5792758A"><text>5,792,758</text></patcit>, <patcit id="pcit1089" dnum="US5792757A"><text>5,792,757</text></patcit>, <patcit id="pcit1090" dnum="US5763361A"><text>5,763,361</text></patcit>, <patcit id="pcit1091" dnum="US5744462A"><text>5,744,462</text></patcit>,<patcit id="pcit1092" dnum="US5741787A"><text> 5,741,787</text></patcit>, <patcit id="pcit1093" dnum="US5741786A"><text>5,741,786</text></patcit>,<patcit id="pcit1094" dnum="US5733899A"><text> 5,733,899</text></patcit>, <patcit id="pcit1095" dnum="US5731345A"><text>5,731,345</text></patcit>, <patcit id="pcit1096" dnum="US5723638A"><text>5,723,638</text></patcit>, <patcit id="pcit1097" dnum="US5721226A"><text>5,721,226</text></patcit>, <patcit id="pcit1098" dnum="US5712264A"><text>5,712,264</text></patcit>, <patcit id="pcit1099" dnum="US5712263A"><text>5,712,263</text></patcit>, <patcit id="pcit1100" dnum="US5710144A"><text>5,710,144</text></patcit>, <patcit id="pcit1101" dnum="US5707984A"><text>5,707,984</text></patcit>, <patcit id="pcit1102" dnum="US5705494A"><text>5,705,494</text></patcit>, <patcit id="pcit1103" dnum="US5700793A"><text>5,700,793</text></patcit>, <patcit id="pcit1104" dnum="US5698720A"><text>5,698,720</text></patcit>, <patcit id="pcit1105" dnum="US5698545A"><text>5,698,545</text></patcit>,<patcit id="pcit1106" dnum="US5696106A"><text> 5,696,106</text></patcit>, <patcit id="pcit1107" dnum="US5677293A"><text>5,677,293</text></patcit>, <patcit id="pcit1108" dnum="US5674861A"><text>5,674,861</text></patcit>, <patcit id="pcit1109" dnum="US5661141A"><text>5,661,141</text></patcit>, <patcit id="pcit1110" dnum="US5656621A"><text>5,656,621</text></patcit>, <patcit id="pcit1111" dnum="US5646136A"><text>5,646,136</text></patcit>, <patcit id="pcit1112" dnum="US5637691A"><text>5,637,691</text></patcit>, <patcit id="pcit1113" dnum="US5616574A"><text>5,616,574</text></patcit>,<patcit id="pcit1114" dnum="US5614514A"><text> 5,614,514</text></patcit>, <patcit id="pcit1115" dnum="US5604215A"><text>5,604,215</text></patcit>, <patcit id="pcit1116" dnum="US5604213A"><text>5,604,213</text></patcit>, <patcit id="pcit1117" dnum="US5599807A"><text>5,599,807</text></patcit>, <patcit id="pcit1118" dnum="US5585482A"><text>5,585,482</text></patcit>, <patcit id="pcit1119" dnum="US5565588A"><text>5,565,588</text></patcit>, <patcit id="pcit1120" dnum="US5563259A"><text>5,563,259</text></patcit>, <patcit id="pcit1121" dnum="US5563131A"><text>5,563,131</text></patcit>, <patcit id="pcit1122" dnum="US5561124A"><text>5,561,124</text></patcit>, <patcit id="pcit1123" dnum="US5556845A"><text>5,556,845</text></patcit>, <patcit id="pcit1124" dnum="US5547949A"><text>5,547,949</text></patcit>, <patcit id="pcit1125" dnum="US5536714A"><text>5,536,714</text></patcit>, <patcit id="pcit1126" dnum="US5527806A"><text>5,527,806</text></patcit>, <patcit id="pcit1127" dnum="US5506354A"><text>5,506,354</text></patcit>, <patcit id="pcit1128" dnum="US5506221A"><text>5,506,221</text></patcit>, <patcit id="pcit1129" dnum="US5494907A"><text>5,494,907</text></patcit>, <patcit id="pcit1130" dnum="US5491136A"><text>5,491,136</text></patcit>, <patcit id="pcit1131" dnum="US5478956A"><text>5,478,956</text></patcit>, <patcit id="pcit1132" dnum="US5426179A"><text>5,426,179</text></patcit>, <patcit id="pcit1133" dnum="US5422262A"><text>5,422,262</text></patcit>, <patcit id="pcit1134" dnum="US5391776A"><text>5,391,776</text></patcit>, <patcit id="pcit1135" dnum="US5382661A"><text>5,382,661</text></patcit>, <patcit id="pcit1136" dnum="US5380841A"><text>5,380,841</text></patcit>,<patcit id="pcit1137" dnum="US5380840A"><text> 5,380,840</text></patcit>, <patcit id="pcit1138" dnum="US5380839A"><text>5,380,839</text></patcit>, <patcit id="pcit1139" dnum="US5373095A"><text>5,373,095</text></patcit>, <patcit id="pcit1140" dnum="US5371078A"><text>5,371,078</text></patcit>, <patcit id="pcit1141" dnum="US5352809A"><text>5,352,809</text></patcit>, <patcit id="pcit1142" dnum="US5344827A"><text>5,344,827</text></patcit>, <patcit id="pcit1143" dnum="US5344826A"><text>5,344,826</text></patcit>, <patcit id="pcit1144" dnum="US5338837A"><text>5338,837</text></patcit>, <patcit id="pcit1145" dnum="US5336686A"><text>5,336,686</text></patcit>, <patcit id="pcit1146" dnum="US5292906A"><text>5,292,906</text></patcit>, <patcit id="pcit1147" dnum="US5292878A"><text>5,292,878</text></patcit>, <patcit id="pcit1148" dnum="US5281587A"><text>5,281,587</text></patcit>, <patcit id="pcit1149" dnum="US5272140A"><text>5,272,140</text></patcit>,<patcit id="pcit1150" dnum="US5244886A"><text> 5,244,886</text></patcit>, <patcit id="pcit1151" dnum="US5236912A"><text>5,236,912</text></patcit>,<patcit id="pcit1152" dnum="US5232915A"><text> 5,232,915</text></patcit>, <patcit id="pcit1153" dnum="US5219879A"><text>5,219,879</text></patcit>, <patcit id="pcit1154" dnum="US5218109A"><text>5,218,109</text></patcit>, <patcit id="pcit1155" dnum="US5215972A"><text>5,215,972</text></patcit>, <patcit id="pcit1156" dnum="US5212166A"><text>5,212,166</text></patcit>, <patcit id="pcit1157" dnum="US5206415A"><text>5,206,415</text></patcit>, <patcit id="pcit1158" dnum="US5194602A"><text>5,194,602</text></patcit>, <patcit id="pcit1159" dnum="US5166201A"><text>5,166,201</text></patcit>,<patcit id="pcit1160" dnum="US5166055A"><text> 5,166,055</text></patcit>, <patcit id="pcit1161" dnum="US5126488A"><text>5,126,488</text></patcit>, <patcit id="pcit1162" dnum="US5116829A"><text>5,116,829</text></patcit>, <patcit id="pcit1163" dnum="US5108996A"><text>5,108,996</text></patcit>, <patcit id="pcit1164" dnum="US5099037A"><text>5,099,037</text></patcit>, <patcit id="pcit1165" dnum="US5096892A"><text>5,096,892</text></patcit>, <patcit id="pcit1166" dnum="US5093502A"><text>5,093,502</text></patcit>, <patcit id="pcit1167" dnum="US5086047A"><text>5,086,047</text></patcit>, <patcit id="pcit1168" dnum="US5084450A"><text>5,084,450</text></patcit>, <patcit id="pcit1169" dnum="US5082835A"><text>5,082,835</text></patcit>, <patcit id="pcit1170" dnum="US5081114A"><text>5,081,114</text></patcit>, <patcit id="pcit1171" dnum="US5053404A"><text>5,053,404</text></patcit>, <patcit id="pcit1172" dnum="US5041433A"><text>5,041,433</text></patcit>, <patcit id="pcit1173" dnum="US5041432A"><text>5,041,432</text></patcit>,<patcit id="pcit1174" dnum="US5034548A"><text> 5,034,548</text></patcit>,<patcit id="pcit1175" dnum="US5032586A"><text> 5,032,586</text></patcit>, <patcit id="pcit1176" dnum="US5026882A"><text>5,026,882</text></patcit>, <patcit id="pcit1177" dnum="US4996335A"><text>4,996,335</text></patcit>, <patcit id="pcit1178" dnum="US4975537A"><text>4,975,537</text></patcit>, <patcit id="pcit1179" dnum="US4970205A"><text>4,970,205</text></patcit>, <patcit id="pcit1180" dnum="US4954446A"><text>4,954,446</text></patcit>, <patcit id="pcit1181" dnum="US4950428A"><text>4,950,428</text></patcit>, <patcit id="pcit1182" dnum="US4946834A"><text>4,946,834</text></patcit>, <patcit id="pcit1183" dnum="US4937237A"><text>4,937,237</text></patcit>, <patcit id="pcit1184" dnum="US4921846A"><text>4,921,846</text></patcit>, <patcit id="pcit1185" dnum="US4920099A"><text>4,920,099</text></patcit>,<patcit id="pcit1186" dnum="US4910226A"><text> 4,910,226</text></patcit>, <patcit id="pcit1187" dnum="US4900725A"><text>4,900,725</text></patcit>, <patcit id="pcit1188" dnum="US4892867A"><text>4,892,867</text></patcit>, <patcit id="pcit1189" dnum="US4888336A"><text>4,888,336</text></patcit>, <patcit id="pcit1190" dnum="US4885280A"><text>4,885,280</text></patcit>, <patcit id="pcit1191" dnum="US4882322A"><text>4,882,322</text></patcit>, <patcit id="pcit1192" dnum="US4882319A"><text>4,882,319</text></patcit>, <patcit id="pcit1193" dnum="US4882315A"><text>4,882,315</text></patcit>, <patcit id="pcit1194" dnum="US4874855A"><text>4,874,855</text></patcit>, <patcit id="pcit1195" dnum="US4868167A"><text>4,868,167</text></patcit>, <patcit id="pcit1196" dnum="US4865767A"><text>4,865,767</text></patcit>, <patcit id="pcit1197" dnum="US4861875A"><text>4,861,875</text></patcit>,<patcit id="pcit1198" dnum="US4861765A"><text> 4,861,765</text></patcit>, <patcit id="pcit1199" dnum="US4861763A"><text>4,861,763</text></patcit>, <patcit id="pcit1200" dnum="US4847014A"><text>4,847,014</text></patcit>, <patcit id="pcit1201" dnum="US4774236A"><text>4,774,236</text></patcit>, <patcit id="pcit1202" dnum="US4753932A"><text>4,753,932</text></patcit>, <patcit id="pcit1203" dnum="US4711856A"><text>4,711,856</text></patcit>, <patcit id="pcit1204" dnum="US4710495A"><text>4,710,495</text></patcit>, <patcit id="pcit1205" dnum="US4701450A"><text>4,701,450</text></patcit>, <patcit id="pcit1206" dnum="US4701449A"><text>4,701,449</text></patcit>, <patcit id="pcit1207" dnum="US4689410A"><text>4,689,410</text></patcit>, <patcit id="pcit1208" dnum="US4680290A"><text>4,680,290</text></patcit>, <patcit id="pcit1209" dnum="US4670551A"><text>4,670,551</text></patcit>, <patcit id="pcit1210" dnum="US4664850A"><text>4,664,850</text></patcit>, <patcit id="pcit1211" dnum="US4659516A"><text>4,659,516</text></patcit>,<patcit id="pcit1212" dnum="US4647410A"><text> 4,647,410</text></patcit>, <patcit id="pcit1213" dnum="US4634695A"><text>4,634,695</text></patcit>, <patcit id="pcit1214" dnum="US4634693A"><text>4,634,693</text></patcit>,<patcit id="pcit1215" dnum="US4588530A"><text> 4,588,530</text></patcit>, <patcit id="pcit1216" dnum="US4567000A"><text>4,567,000</text></patcit>, <patcit id="pcit1217" dnum="US4560557A"><text>4,560,557</text></patcit>, <patcit id="pcit1218" dnum="US4558041A"><text>4,558,041</text></patcit>, <patcit id="pcit1219" dnum="US455287IA"><text>4,552,87i</text></patcit>,<patcit id="pcit1220" dnum="US4552868A"><text> 4,552,868</text></patcit>, <patcit id="pcit1221" dnum="US4541956A"><text>4,541,956</text></patcit>, <patcit id="pcit1222" dnum="US4519946A"><text>4,519,946</text></patcit>, <patcit id="pcit1223" dnum="US4515787A"><text>4,515;787</text></patcit>, <patcit id="pcit1224" dnum="US4512986A"><text>4,512,986</text></patcit>, <patcit id="pcit1225" dnum="US4502989A"><text>4,502,989</text></patcit>, <patcit id="pcit1226" dnum="US4495102A"><text>4,495,102</text></patcit>;</li></ul>

0113The drug moiety of the conjugate may be the whole compound or a binding fragment or portion thereof that retains its affinity and specificity for the target of interest while having a linkage site for covalent bonding to the presenter protein ligand or linker. The conjugates of such drugs may be used for the same disorders, diseases, and indications as the drugs themselves.

M. P97 and modulators

0114In one example, the active agent is conjugated to p97 or a a modulator or ligand of the LRP receptor family (e.g. LRP1; LRP1B), or is an antibody which is capable of specifically binding to p97 or the modulator, such as an antibody to p97. In a further example, the agent may be a substance having therapeutic activity such as a growth factor or lymphokine, enzyme or drug. Also disclosed herein is a method of delivering an active agent across the blood brain barrier comprising administering such a conjugate.

0115In one example, the p97 protein is soluble. p97 proteins as taught in <patcit id="pcit1227" dnum="US5981194A"><text>U.S. Patent No. 5,981,194</text></patcit> are particularly preferred. The p97 may be a human p97 protein or fragment thereof; the p97 may be from a mammal such as a mouse. Murine p97 is disclosed in <patcit id="pcit1228" dnum="WO0159549A2"><text>WO 01/59549 A2</text></patcit>:

0116"p97" as used in the compositions disclosed herein , includes membrane bound p97 (<i>i.e</i>., p97 linked to GPI or other lipids), soluble p97, cleaved p97, analogs of p97 which are equivalents of p97 (having greater than 40%, 60%, 80%, or 90% homology at the peptide sequence level, including allelic variants of p97), human, mouse, chicken and/or rabbit p97, and derivatives, portions, or fragments thereof. p97 may be in the form of acidic or basic salts, or in neutral form. In addition, individual amino acid residues may be modified, such as by oxidation or reduction. Various substitutions, deletions, or additions may be made to the amino acid or DNA nucleic acid sequences, the net effect of which is to retain or improve upon the desired biological activity of p97. Due to code degeneracy, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence. As used herein, p97 also includes fragments of p97, including any portion of p97 or its biologically equivalent analogs that contain a sufficient portion of p97 and homology to the corresponding native p97 amino acid sequence to enable it to retain or improve upon the desired biological activities of p97. In other aspects, described herein are p97 conjugates which have only minor substitutions in the amino acid sequence which do not substantially affect its receptor binding or transcytosis properties.

0117Preferred chemotherapeutic agents for use in p97-chemotherapeutic agent conjugates include all drugs which may be useful for treating brain tumours or other neoplasia in or around the brain, either in the free form, or, if not so useful in the free form, then useful when linked to p97. Such chemotherapeutic agents include adriamycin (also known as doxorubicin), cisplatin, paclitaxel, analogs thereof, and other chemotherapeutic agents which demonstrate activity against tumours <i>ex vivo</i> and <i>in vivo</i>. Such chemotherapeutic agents also include alkylating agents, antimetabolites, natural products (such as vinca alkaloids, epidophyllotoxins, antibiotics, enzymes and biological response modifiers), topoisomerase inhibitors, microtubule inhibitors, spindle poisons, hormones and antagonists, and miscellaneous agents such as platinum coordination complexes, anthracendioncs, substituted ureas, <i>etc</i>. those of skill in the art will know of other chemotherapeutic agents.

0118p97-chemotherapeutic agents can comprise one or more compound moieties linked to p97. For example, conjugation reactions may conjugate from 1 to 10 or more molecules of adriamycin to a single p97 molecule. Several atoms of gold or iodine can be conjugated to a single p97 polypeptide. These formulations can be employed as mixtures, or they may be purified into specific p97:compound (mol:mol) formulations. Those skilled in the art are able to determine which format and which mol:mol ratio is preferred. Further, mixtures of compounds may be linked to p97, such as the p97-adriamycin-cisplatinum composition set out in the examples. These p97-chemotherapeutic agents may consist of a range of mol:mol ratios. These, too, may be separated into purified mixtures or they may be employed in aggregate.

0119The compositions disclosed herein may also be used for delivering an agent across the blood eye barrier or blood placenta barrier

0120The compositions may also comprise a transcytosing or endocytosing ligand of the LRP1 or LRP1B receptor conjugated to an active agent

N. Labels

0121In some embodiments, the conjugate or modulator or LRP ligand is labeled to facilitate its detection. A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, labels suitable for use in the present invention include, for example, radioactive labels (<i>e.g.,</i>³²P), fluorophores (<i>e.g</i>., fluorescein), electron-dense reagents, enzymes (<i>e.g</i>., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, <i>e.g</i>., by incorporating a radiolabel into the hapten or peptide, or used to detect antibodies specifically reactive with the hapten or peptide.

0122As noted above, depending on the screening assay employed, the drug, the linker or the p97 or modulator or ligand portion of a conjugate may be labeled. The particular label or detectable group used is not a critical aspect, as long as it does not significantly interfere with the biological activity of the conjugate. The detectable group can be any material having a detectable physical or chemical property. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

0123Examples of labels suitable for use include, but are not limited to, fluorescent dyes (<i>e.g</i>., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (<i>e.g</i>., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (<i>e.g</i>., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (<i>e.g</i>., polystyrene, polypropylene, latex, <i>etc</i>.).

0124The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. Preferably, the label in one embodiment is covalently bound to the biopolymer using an isocyanate reagent for conjugating an active agent according to the invention. In one aspect, the bifunctional isocyanate reagents can be used to conjugate a label to a biopolymer to form a label biopolymer conjugate without an active agent attached thereto. The label biopolymer conjugate may be used as an intermediate for the synthesis of a labeled conjugate or may be used to detect the biopolymer conjugate. As indicated above, a wide variety of labels can be used, with the choice of label depending on sensitivity required, ease of conjugation with the desired component of the assay, stability requirements, available instrumentation, and disposal provisions. Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (<i>e.g</i>., biotin) is covalently bound to the molecule. The ligand then binds to another molecule (<i>e.g</i>., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.

0125The conjugates can also be conjugated directly to signal generating compounds, <i>e.g</i>., by conjugation with an enzyme or fluorophore. Enzymes suitable for use as labels include, but are not limited to, hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases. Fluorescent compounds, <i>i.e</i>., fluorophores, suitable for use as labels include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, <i>etc</i>. Further examples of suitable fluorophores include, but are not limited to, eosin, TRITC-amine, quinine, fluorescein W, acridine yellow, lissamine rhodamine, B sulfonyl chloride erythmscein, ruthenium (tris, bipyridinium), Texas Red, nicotinamide adenine dinucleotide, flavin adenine dinucleotide, <i>etc</i>. Chemiluminescent compounds suitable for use as labels include, but are not limited to, luciferin and 2,3-dihydrophthalazinediones, <i>e.g</i>., luminol. For a review of various labeling or signal producing systems that can be used in the methods described herein, <i>see</i><patcit id="pcit1229" dnum="US4391904A"><text>U.S. Patent No. 4,391,904</text></patcit>.

0126Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels may be detected simply by observing the color associated with the label. Other labeling and detection systems suitable for use in the methods will be readily apparent to those of skill in the art. Such labeled modulators and ligands may be used in the diagnosis of a disease or health condition.

O. Methods of Using, Pharmaceutical Compositions, and their Administration

0127The term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, buffers and excipients, including phosphate-buffered saline solution, water, and emulsions (such as an oil/water or water/oil emulsion), and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, 19th ed. 1995). Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent Typical modes of administration are described below.

0128The term "effective amount" means a dosage sufficient to produce a desired result on a health condition, pathology, disease of a subject or for a diagnositic purpose. The desired result may comprise a subjective or objective improvement in the recipient of the dosage.

0129A "prophylactic treatment" is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of a disease, wherein treatment is administered for the purpose of decreasing the risk of developing a pathology. The conjugate compounds may be given as a prophylactic treatment.

0130A "therapeutic treatment" is a treatment administered to a subject who exhibits signs of pathology, wherein treatment is administered for the purpose of diminishing or eliminating those pathological signs. The conjugate compounds, modulators, and ligands _ may be given as a prophylactic treatment or for diagnosis.

0131The term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions encompass any composition made by admixing a conjugate compound and a pharmaceutically acceptable carrier. The term "pharmaceutical composition" indicates a composition suitable for pharmaceutical use in a subject, including an animal or human. A pharmaceutical composition generally comprises an effective amount of a conjugate, modnlator, or LRP ligand or LRP1B ligand and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention are defined in the appended claims.

0132The conjugates, modulators, and LRP or LRP1B ligands may be administered by a variety of routes. For oral preparations, the conjugates can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

0133The conjugates, modulators, and LRP or LRP1B ligands can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

0134The conjugates, modulators, and LRP or LRP1B ligands can be utilized in aerosol formulation to be administered via inhalation. The compounds can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

0135Furthermore, the conjugates, modulators, and LRP or LRP1B ligands can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

0136Unit dosage forms of the conjugate, modulator, and LRP or LRP1B ligand for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing active agent. Similarly, unit dosage forms for injection or intravenous administration may comprise the conjugate in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms depend on the particular conjugate employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

0137In practical use, the conjugate, modulator, and LRP or LRP1B ligand can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, <i>e.g</i>., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

0138With respect to transdermal routes of administration, methods for transdermal administration of drugs are disclosed in <nplcit id="ncit0053" npl-type="b"><text>Remington's Pharmaceutical Sciences, 17th Edition, (Gennaro et al. Eds., Mack Publishing Co., 1985</text></nplcit>). Dermal or skin patches are a preferred means for transdermal delivery of the conjugates, modulators, and LRP or LRP1B ligands. Patches preferably provide an absorption enhancer such as DMSO to increase the absorption of the compounds. Other methods for transdermal drug delivery are disclosed in <patcit id="pcit1230" dnum="US5962012A"><text>U.S. Patents No. 5,962,012</text></patcit>, <patcit id="pcit1231" dnum="US6261595B"><text>6,261,595</text></patcit>, and <patcit id="pcit1232" dnum="US6261595B"><text>6,261,595</text></patcit>.

0139The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are commercially available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available.

0140Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

0141In each of these aspects, the compositions include, but are not limited to, compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend in part on the nature and severity of the conditions being treated and on the nature of the active ingredient. Exemplary routes of administration are the oral and intravenous routes. The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.

0142In practical use, the compounds (e.g., LRP or LRP 1B ligand conjugates, LRP or LRP1B modulator conjugates, LRP or LRP1B ligand-fusion proteins, LRP or LRP1B modulators, and LRP or LRP1B ligands) can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, <i>e</i>.<i>g</i>., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

0143Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. The percentage of an active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit

0144The conjugates, modulators, and ligands described herein are useful for therapeutic, prophylactic and diagnostic intervention in animals, and in particular in humans. As described herein, the conjugates show preferential accumulation and/or release of the active agent in any target organ, compartment, or site depending upon the biopolymer used.

0145Compositions described herein may be administered encapsulated in or attached to viral envelopes or vesicles, or incorporated into cells. Vesicles are micellular particles which are usually spherical and which are frequently lipidic. Liposomes are vesicles formed from a bilayer membrane. Suitable vesicles include, but are not limited to, unilamellar vesicles and multilamellar lipid vesicles or liposomes. Such vesicles and liposomes may be made from a wide range of lipid or phospholipid compounds, such as phosphatidylcholine, phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, glycolipids, gangliosides, <i>etc.</i> using standard techniques, such as those described in, <i>e.g.</i>, <patcit id="pcit1233" dnum="US4394448A"><text>U.S. Patent No. 4,394,448</text></patcit>. Such vesicles or liposomes may be used to administer compounds intracellularly and to deliver compounds to the target organs. Controlled release of a p97-composition of interest may also be achieved using encapsulation (<i>see</i>, <i>e.g.</i>, <patcit id="pcit1234" dnum="US5186941A"><text>U.S. Patent No. 5,186,941</text></patcit>).

0146Any route of administration which dilutes the conjugates, modulators, and LRP or LRP1B ligands composition into the blood stream, or at least outside of the blood-brain barrier, may be used. Preferably, the composition is administered peripherally, most preferably intravenously or by cardiac catheter. Intra-jugular and intra-carotid injections are also useful. Compositions may be administered locally or regionally, such as intra-peritoneally. In one aspect, compositions are administered with a suitable pharmaceutical diluent or carrier.

0147Dosages to be administered will depend on individual needs, on the desired effect, the active agent used, the biopolymer and on the chosen route of administration. Preferred dosages of a conjugate range from about 0.2 pmol/kg to about 25 nmol/kg, and particularly preferred dosages range from 2-250 pmol/kg; alternatively, preferred doses of the conjugate may be in the range of 0.02 to 2000 mg/kg. These dosages will be influenced by the number of active agent or drug moieties associated with the biopolymer. Alternatively, dosages may be calculated based on the active agent administered.

0148As described the conjugate comprises p97. For instance, doses of p97-adriamycin comprising from 0.005 to 100 mg/kg of adriamycin are also useful <i>in vivo.</i> Particularly preferred is a dosage of p97-adriamycin comprising from 0.05 mg/kg to 20 mg/kg of adriamycin. Those skilled in the art can determine suitable doses for other compounds linked to p97 based on the recommended dosage used for the free form of the compound. p97 generally reduces the amount of drug needed to obtain the same effect.

0149The p97-conjugates, modulators, and LRP1 or LRP1B ligands are useful for therapeutic, prophylactic and diagnostic intervention in animals, and in particular in humans. As described herein, p97-compounds show preferential accumulation in the lung, liver, kidney and spleen, and that they significantly reduce delivery of the compounds to the heart. Preferred medical indications for diagnostic uses include, for example, any condition associated with a target organ of interest (<i>e</i>.<i>g</i>., lung, liver, kidney, spleen) or any condition that requires a cardiotoxic compound that would benefit by reducing its cardiotoxicity.

0150The methods find use in the treatment of a variety of different disease conditions. In certain examples, of particular interest is the use of the methods in disease conditions where an active agent or drug having desired activity has been previously identified, but in which the active agent or drug is not targeted to the target site, area or compartment. With such active agents or drugs, the subject methods can be used to enhance the therapeutic efficacy and therapeutic index of active agent or drug.

0151The specific disease conditions treatable by with the conjugates are as varied as the types of drug moieties that can be present in the conjugate. Thus, disease conditions include cellular proliferative diseases, such as neoplastic diseases, autoimmune diseases, cardiovascular diseases, hormonal abnormality diseases, degenerative diseases, diseases of aging, diseases of the central nervous system (<i>e</i>.<i>g</i>., Alzheimer's disease, epilepsy, hyperlipidemias), psychiatric diseases and conditions( <i>e.g</i>., schizophrenia, mood disorders such as depression and anxiety), infectious diseases, and the like.

0152Treatment is meant to encompass any beneficial outcome to a subject associated with administration of a conjugate including a reduced liklihood of acquiring a disease, prevention of a disease, slowing, stopping or reversing, the progression of a disease or an amelioration of the symptoms associated with the disease condition afflicting the host, where amelioration or benefit is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, <i>e</i>.<i>g</i>., symptom, associated with the pathological condition being treated, such as inflammation and pain associated therewith. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, <i>e.g.,</i> prevented from happening, or stopped, <i>e.g.,</i> terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.

0153A variety of hosts or subjects are treatable. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (<i>e.g</i>., dogs and cats), rodentia <i>(e.g.,</i> mice, guinea pigs, and rats), and primates (<i>e.g</i>., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.

P. Combinatorial chemical libraries

0154Recently, attention has focused on the use of combinatorial chemical libraries to assist in the generation of new chemical compound leads. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, one commentator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (<nplcit id="ncit0054" npl-type="s"><text>Gallop et al. J. Med. Chem. 37(9):1233(1994</text></nplcit>)).

0155Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, benzodiazepines (<patcit id="pcit1235" dnum="US5288514A"><text>U.S. Pat. No. 5,288,514</text></patcit>), diversomers such as hydantoins, benzodiazepines and dipeptides (<nplcit id="ncit0055" npl-type="s"><text>Hobbs et aL PNAS USA 90: 6909(1993</text></nplcit>)), analogous organic syntheses of small compound libraries (<nplcit id="ncit0056" npl-type="s"><text>Chen et al.) J. Amer. Chem. Soc. 116: 2661(1994</text></nplcit>), oligocarbamates (<nplcit id="ncit0057" npl-type="s"><text>Cho, et al., Science 261: 1303(1993</text></nplcit>)), and/or peptidyl phosphonates (<nplcit id="ncit0058" npl-type="s"><text>Campbell et al., J. Org. Chem. 59: 658(1994</text></nplcit>)), and small organic molecule libraries (see, e.g., benzodiazepines (<nplcit id="ncit0059" npl-type="s"><text>Baum C&amp;EN, Jan 18, page 33(1993</text></nplcit>)), thiazolidinones and metathiazanones (<patcit id="pcit1236" dnum="US5549974A"><text>U.S. Patent 5,549,974</text></patcit>), pyrrolidines (<patcit id="pcit1237" dnum="US5525735A"><text>U.S. Patents 5,525,735</text></patcit> and <patcit id="pcit1238" dnum="US5519134A"><text>5,519,134</text></patcit>), benzodiazepines (<patcit id="pcit1239" dnum="US5288514A"><text>U.S. Patent 5,288,514</text></patcit>), and the like.

0156Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).

0157A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, HewlettPackard, Palo Alto, CA) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use as described herein. The nature and implementation of modifications to these devices so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd., Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

0158In a preferred embodiment, the combinatorial chemistry provides variants of the known native or endogenous human ligands of the LRP and LRP1B receptors as candidate ligands, modulators, and conjugates for use as described herein.

Q. High throughput assays of chemical libraries

0159The assays for compounds described herein are amenable to high throughput screening. Preferred assays thus detect activation of transcription (i.e., activation ofmRNA production) by the test compound(s), activation of protein expression by the test compound(s), or binding to the gene product (e.g., expressed protein) by the test compound(s). The BiaCore method is one such means for rapidly screening compounds for binding activity.

0160High throughput assays for the presence, absence, or quantification of particular protein products or binding assays are well known to those of skill in the art. Thus, for example, <patcit id="pcit1240" dnum="US5559410A"><text>U.S. Patent 5,559,410</text></patcit> discloses high throughput screening methods for proteins, and <patcit id="pcit1241" dnum="US5576220A"><text>U.S. Patents 5,576,220</text></patcit> and <patcit id="pcit1242" dnum="US5541061A"><text>5,541,061</text></patcit> disclose high throughput methods of screening for ligand/antibody binding.

0161In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

R. Conjugation and labeling

0162One of ordinary skill in the art would know how to conjugate and active agent to a protein or peptide. Methods of conjugating active agents and labels to proteins are well known in the art. See, for instance, <patcit id="pcit1243" dnum="US5981194A"><text>U.S. Patent No. 5,981,194</text></patcit>. Many reagents and cross linkers can be used to prepare bioconjugates of an active agent and a biopolymer. See, for instance, <nplcit id="ncit0060" npl-type="b"><text>Hermanson, GT et al. Bioconjugate Techniques, Academic Press, (1996</text></nplcit>).

Production of Chimeric Proteins

0163The chimeric protein can be produced using host cells expressing a single nucleic acid encoding the entire chimeric protein or more than one nucleic acid sequence, each encoding a domain of the chimeric protein and, optionally, an amino acid or amino acids which will serve to link the domains. The chimeric proteins can also be produced by chemical synthesis.

A. Host Cells

0164Host cells used to produce chimeric proteins are bacterial, yeast, insect, non-mammalian vertebrate, or mammalian cells; the mammalian cells include, but are not limited to, hamster, monkey, chimpanzee, dog, cat, bovine, porcine, mouse, rat, rabbit, sheep and human cells. The host cells can be immortalized cells (a cell line) or non-immortalized (primary or secondary) cells and can be any of a wide variety of cell types, such as, but not limited to, fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), ovary cells (e.g., Chinese hamster ovary or CHO cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), muscle cells, hepatocytes and precursors of these somatic cell types.

0165Cells which contain and express DNA or RNA encoding the chimeric protein are referred to herein as genetically modified cells. Mammalian cells which contain and express DNA or RNA encoding the chimeric protein are referred to as genetically modified mammalian cells. Introduction of the DNA or RNA into cells is by a known transfection method, such as electroporation, microinjection, microprojectile bombardment, calcium phosphate precipitation, modified calcium phosphate precipitation, cationic lipid treatment, photoporation, fusion methodologies, receptor mediated transfer, or polybrene precipitation. Alternatively, the DNA or RNA can be introduced by infection with a viral vector. Methods of producing cells, including mammalian cells, which express DNA or RNA encoding a chimeric protein are described in co-pending <patcit id="pcit1244" dnum="US33479794A" dnum-type="L"><text>patent applications U.S. Ser. No. 08/334,797</text></patcit>, entitled "In Vivo Protein Production and Delivery System for Gene Therapy", by Richard F Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov. 4,1994); <patcit id="pcit1245" dnum="US08334455B"><text>U.S. Ser. No. 08/334,455</text></patcit>, entitled "In Vivo Production and Delivery of Erythropoietin or Insulinotropin for Gene Therapy", by Richard F Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov. 4,1994) and <patcit id="pcit1246" dnum="US08231439B"><text>U.S. Ser. No. 08/231,439</text></patcit>, entitled "Targeted Introduction of DNA Into Primary or Secondary Cells and Their Use for Gene Therapy", by Douglas A. Treco, Michael W. Heartlein and Richard F Selden (filed Apr. 20,1994).

B. Nucleic Acid Constructs

0166A nucleic acid construct used to express the chimeric protein can be one which is expressed extrachromosomally (episomally) in the transfected mammalian cell or one which integrates, either randomly or at a pro-selected targeted site through homologous recombination, into the recipient cell's genome. A construct which is expressed extrachromosomally comprises, in addition to chimeric protein-encoding sequences, sequences sufficient for expression of the protein in the cells and, optionally, for replication of the construct. It typically includes a promoter, chimeric protein-encoding DNA and a polyadenylation site. The DNA encoding the chimeric protein is positioned in the construct in such a manner that its expression is under the control of the promoter. Optionally, the construct may contain additional components such as one or more of the following: a splice site, an enhancer sequence, a selectable marker gene under the control of an appropriate promoter, and an amplifiable marker gene under the control of an appropriate promoter.

0167In those embodiments in which the DNA construct integrates into the cell's genome, it need include only the chimeric protein-encoding nucleic acid sequences. Optionally, it can include a promoter and an enhancer sequence, a polyadenylation site or sites, a splice site or sites, nucleic acid sequences which encode a selectable marker or markers, nucleic acid sequences which encode an amplifiable marker and/or DNA homologous to genomic DNA in the recipient cell to target integration of the DNA to a selected site in the genome (targeting DNA or DNA sequences).

C. Cell Culture Methods

0168Mammalian cells containing the chimeric protein-encoding DNA or RNA are cultured under conditions appropriate for growth of the cells and expression of the DNA or RNA. Those cells which express the chimeric protein can be identified, using known methods and methods described herein, and the chimeric protein isolated and purified, using known methods and methods also described herein; either with or without amplification of chimeric protein production. Identification can be carried out, for example, through screening genetically modified mammalian cells displaying a phenotype indicative of the presence of DNA or RNA encoding the chimeric protein, such as PCR screening, screening by Southern blot analysis, or screening for the expression of the chimeric protein. Selection of cells having incorporated chimeric protein-encoding DNA may be accomplished by including a selectable marker in the DNA construct and culturing transfected or infected cells containing a selectable marker gene under conditions appropriate for survival of only those cells which express the selectable marker gene. Further amplification of the introduced DNA construct can be effected by culturing genetically modified mammalian cells under conditions appropriate for amplification (e.g., culturing genetically modified mammalian cells containing an amplifiable marker gene in the presence of a concentration of a drug at which only cells containing multiple copies of the amplifiable marker gene can survive).

0169Genetically modified mammalian cells expressing the chimeric protein can be identified, as described herein, by detection of the expression product. For example, mammalian cells expressing chimeric protein in which the carrier is p97 can be identified by a sandwich enzyme immunoassay. The antibodies can be directed toward the LRP portion or the active agent portion.

EXAMPLES

0170The examples provide exemplary protocols for assessing transcytosis <i>in vitro</i> and for characterizing the interaction of p97 and MTf-R receptor modulators or ligands with the p97 receptor or the blood-brain barrier:

Example I : <i>In vitro</i> model of the blood-brain barrier.

0171<img file="EP2147679B1_D0004.tif" />

0172A. Astrocytes. Primary cultures of mixed astrocytes were prepared from newborn rat cerebral cortex (<nplcit id="ncit0061" npl-type="s"><text>Dehouck, et al., Journal of Controlled Release (1992</text></nplcit>)). Briefly, after removing the meninges, the brain tissue was forced gently through a 82 µm nylon sieve. Astrocytes were plated on six-well microplates at a concentration of 1.2 x 10⁵ cells/ml in 2 ml of optimal culture medium (DMEM) supplemented with 10% fetal heat inactivated calf serum. The medium was changed twice a week.

0173B. <b>BBCEC.</b> Bovine brain capillary endothelial cells (BBCECs) were obtained from Cellial Technologies. The cells were cultured in the presence of DMEM medium supplemented with 10% (v/v) horse and 10% heat-inactivated calf serum, 2 mM glutamine, 50 µg/ml gentamycin, and 1 ng/ml basic fibroblast growth factor, added every other day.

0174C. <b>BBB.</b> The <i>in vitro</i> model of BBB was established by using a co-culture of BBCECs and astrocytes, basically as described in <nplcit id="ncit0062" npl-type="s"><text>Dehouck, et al., Eur. J. Pharm. Sci., 3:357-365 (1995</text></nplcit>); and <nplcit id="ncit0063" npl-type="s"><text>Cecchelli, et al., Adv. Drug Deliv. Rev., 36:165-178 (1999</text></nplcit>)). In this model, the luminal side corresponds to the apical or serum facing side of the blood-brain barrier. The abluminal side corresponds to the basolateral side of the BBB, <i>i</i>.<i>e</i>., the side facing the neurons. It is noted that this terminology is used throughout the specification. Prior to cell culture, plate inserts (Millicell-PC 3.0 µM; 30-mm diameter) were coated on the upper side with rat tail collagen. They were then set in the six-multiwell microplates containing the astrocytes prepared as described above, and BBCECs were plated on the upper side of the filters in 2 ml of co-culture medium. This BBCEC medium was changed three times a week. Under these conditions, differentiated BBCECs formed a confluent monolayer 7 days later. Experiments were performed between 5 and 7 days after confluence was reached. The number of cells at confluence was 400 000 cells/4.2 cm² or 90 µg of protein/4.2 cm², as evaluated by a micro-BCA assay from Pierce (Rockford, IL).

0175D. Sucrose permeability. The permeability coefficient for sucrose was measured to verify the endothelial permeability and the integrity and tightness of the BBCEC monolayers, as previously described, using uncoated filters or coated with endothelial cells. Briefly, the results were plotted as the clearance of [¹⁴C]-sucrose (µl) as a function of time (min). The permeability coefficient (Pe) was calculated as: 1/Pe =(1/PSt-1/PSf)/filter area (4.2 cm²), where PSt is the permeability x surface area of a filter of the co-culture; PSf is the permeability of a filter coated with collagen and with astrocytes plated on the bottom side of the filter.

0176Brain EC monolayers grown on inserts were transferred to 6-well plates containing 2 ml of Ringer/Hepes per well (basolateral compartment). Ringer/Hepes solution was composed of 150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl₂, 0.2 mM MgCl₂, 6 mM NaHCO₃, 5 mM Hepes, 2.8 mM Hepes, pH 7.4. In each apical chamber, the culture medium was replaced by Ringer/Hepes containing the labeled [¹⁴C]-sucrose. At different times, inserts were placed into another well. At the end of the experiments, amounts of the radiotracers in the basolateral compartment were measured in a liquid scintillation counter. The permeability coefficient (Pe) for sucrose was calculated as previously described (<nplcit id="ncit0064" npl-type="s"><text>Dehouck, et al., J. Neurochem., 58:1790-1797 (1992</text></nplcit>)) using filters coated or noncoated with EC. At the end of the experiments, amounts of the radiotracers in the basolateral compartment were measured in a liquid scintillation counter. The results were plotted as the clearance of [¹⁴C]-sucrose (µl) as a function of time (min). PSt= permeability x surface area of a filter of the coculture; PSf= permeability of a filter coated with collagen and astrocytes plated on the bottom side of the filter. The permeability coefficient (Pe) was calculated as: <ol id="ol0002" compact="compact" ol-style=""><li>1) <maths id="math0003" num=""><math display="block"><mi>Clearance</mi><mfenced><mi mathvariant="normal">μl</mi></mfenced><mo>=</mo><mfrac><mrow><msub><mfenced open="[" close="]"><mi mathvariant="normal">C</mi></mfenced><mi mathvariant="normal">A</mi></msub><mo>×</mo><msub><mi mathvariant="normal">V</mi><mi mathvariant="normal">A</mi></msub></mrow><msub><mfenced open="[" close="]"><mi mathvariant="normal">C</mi></mfenced><mi mathvariant="normal">L</mi></msub></mfrac></math><img file="EP2147679B1_D0005.tif" /></maths><ul id="ul0004" list-style="none" compact="compact"><li>[C]_A = Abluminal tracer concentration</li><li>V_A = Volume of abluminal chamber</li><li>[C]_L= Luminal tracer concentration</li></ul></li><li>2) <maths id="math0004" num=""><math display="block"><mn>1</mn><mo>/</mo><mi>Pe</mi><mo>=</mo><mfenced separators=""><mn>1</mn><mo>/</mo><mi>PSt</mi><mo>-</mo><mn>1</mn><mo>/</mo><mi>PSf</mi></mfenced><mo>/</mo><mi>filter area</mi><mspace width="1em" /><mfenced separators=""><mn>4.2</mn><mo>⁢</mo><msup><mrow><mspace width="1em" /><mi>cm</mi></mrow><mn>2</mn></msup></mfenced></math><img file="EP2147679B1_D0006.tif" /></maths></li></ol>

0177<figref idref="f0001">Figure 1</figref> and <figref idref="f0002">Figure 2</figref> set out control experiments.

0178Briefly, in <figref idref="f0001">Figure 1</figref>, coated or non-coated filters with BBCE cells were transferred to 6-well plates containing 2 ml of Ringer/Hepes per well (basolateral compartment) for 2 hrs at 37°C. In each apical chamber, the culture medium was replaced by 1 ml Ringer-Hepes containing labeled [¹⁴C]-sucrose. At different times, inserts were placed into another well. At the end of the experiments, amounts of the radiotracers in the basolateral compartment were measured in a liquid scintillation counter. The difference in the slopes for PSf and PSt demonstrates that the monolayer of cells provides significant resistance to sucrose permeability, thus confirming the presence of tight junctions between cells regardless of preincubation.

0179In <figref idref="f0002">Figure 2</figref>, <figref idref="f0001">1</figref> micromolar p97 protein (provided by Synapse Technologies Inc, Vancouver, Canada) was added to the luminal side to determine its effect on sucrose permeability. Sucrose permeability in the absence of p97 was 1.21 X 10-3 cm/min, whereas in the presence of p97 sucrose permeability was 1.35 X 10-3 cm/min. The results show that there was no significant change in sucrose permeability. A control experiment in the absence of cells demonstrates the relative effectiveness of the BBB model compared to the filter alone.

0180In <figref idref="f0001">Figures 1</figref> and <figref idref="f0002">2</figref>, Psf and Pst refer to flow rates through the membrane with and without cells. They are used to calculate the permeability of the membrane with cells. Definitions for Psf and Pst are in found in <nplcit id="ncit0065" npl-type="s"><text>J. Neurochem., 58:1790-97 (1992</text></nplcit>). The definition of PSt and PSf are simply the slope of the clearance curves for the co-culture and for the control filter respectively. The PS value for the endothelial monolayer alone is defined as PSe where: <maths id="math0005" num=""><math display="block"><mn>1</mn><mo>/</mo><mi>PSe</mi><mo>=</mo><mn>1</mn><mo>/</mo><mi>PSt</mi><mo>-</mo><mn>1</mn><mo>/</mo><mi>PSf</mi></math><img file="EP2147679B1_D0007.tif" /></maths> Permeability of the endothelial monolayer alone is defined as Pe where:<maths id="math0006" num=""><math display="block"><mi>Pe</mi><mo>=</mo><mi>PSe</mi><mo>/</mo><mi mathvariant="normal">A</mi><mo>,</mo></math><img file="EP2147679B1_D0008.tif" /></maths><ul id="ul0005" list-style="none" compact="compact"><li>wherein A is the area of the membrane.</li></ul>

Example II: Binding of p97 with BBCECs and Rat Brain Endothelial Cells

0181B. Binding studies of p97. Binding of p97 was performed with BBCECs and Rat Brain Endothelial Cells that were pre-incubated 2 hrs in Ringer/Hepes to avoid any interference from the astrocytes.

0182<figref idref="f0003">Figure 3</figref> demonstrates the competitive binding of p97 with cold p97, transferrin and lactoferrin. For the binding experiments, cells were incubated for 2 h at 4°C in Ringer/Hepes in the presence of [¹²⁵I]-p97 (25 nM) and increasing concentrations of cold-p97 or high (7.5 micromolar) concentrations of transferrin or lactoferrin. At the end of the incubation, the filters were gently washed at 4°C three times with 4 ml of cold-PBS. Then the associated radioactivity of endothelial cells was determined by removing the membrane of the culture insert and counting it in a gamma counter.

0183The results of <figref idref="f0003">Figure 3</figref> demonstrate that [^12SI]-p97 binding to the BBCECs was competitively inhibited by cold-p97 and by lactoferrin, but significantly, transferrin did not block or reduce the binding of [^12SI]-p97 to its receptor on the BBCECs. This data establishes for the first time that [¹²⁵I]-p97 is not binding to the transferrin receptor (Tf-R) as previously hypothesized.

C. Binding of p97 in rat brain endothelial cells.

0184As with <figref idref="f0003">Figure 3</figref>, <figref idref="f0004">Figure 4</figref> demonstrates a comparative study, but this time using Rat Brain Endothelial-4 cells (RBE4 supplied commercially by ATCC). RBE4 cells were grown in monolayers in 24 wells plastic tissue culture flasks at 37°C under 5% CO₂ in minimum essential medium Alpha and Ham's F10 (1:1) supplemented with 10% heat inactivated fetal bovine serum. For p97 binding experiments, RBE4 cells were pre-incubated at 37°C for 2 hrs in Ringer/Hepes. ¹²⁵I-p97 in 200 µl of Ringer/Hepes was added to RBE4 cells for 2 hrs at 4°C in the presence or absence of high concentration of cold-p97, human holo-transferrin or human lactoferrin. After the incubation, the cells were washed 4 times with PBS and the ¹²⁵I-p97 associated with the cells was measured.

0185<figref idref="f0004">Figure 4</figref> demonstrates that cold p97 (10 micromolar) and lactoferrin (10 micromolar) both competitively inhibit [¹²⁵I]-p97 binding to RBE4 cells, whereas transferrin (10 micromolar) does not.

Example III: Interaction of p97 with human brain capillaries.

0186A. <b>Isolation of human brain capillaries.</b> Capillaries of the blood-brain barrier were isolated from human brain cortex by a procedure previously described by <nplcit id="ncit0066" npl-type="s"><text>Dallaire, et al., J. Biol. Chem, 267:22323-22327 (1992</text></nplcit>) with slight modifications. Human brains were obtained post-mortem. The brain was cleared of meninges, superficial large blood vessels and choroid plexus. All the following procedures were performed at 4°C. The cerebral cortex was homogenized in 5 volumes of Ringer/Hepes solution with a Polytron (Brinkman Instruments, Rexdale, Ontario, Canada). The homogenate was mixed with an equal volume of Dextran T-70 (27g in 100 ml of Ringer/Hepes). The suspension was centrifuged at 25,000 g for 10 min. The pellet was resuspended in 30 ml of Ringer/Hepes and passed through a 250 µm nylon mesh screen. The nylon mesh was rinsed and the filtrate was concentrated by centrifugation at 25,000 g for 10 min. The pellet was resuspended in 30 ml of cold-Ringer/Hepes and passed through a 2.5 cm x 4.0 cm glass beads column [40/60-mesh (0.25 mm) glass beads]. The columns were washed twice with 25 ml of Ringer/Hepes. The glass beads were transferred to a beaker and swirled vigorously (15 min at 4°C) in Ringer/Hepes to separate the microvessels from the beads. The beads were allowed to settle and the supernatant was decanted and kept at 4°C. The beads were swirled for another 15 min in Ringer/Hepes. The supernatants were pooled and the microvessels were collected by centrifugation at 25,000 g for 10 min. Brain capillaries were kept at -80°C until used.

0187<b>B. p97 accumulation in human brain capillaries.</b> A rapid filtration technique was used to measure the accumulation of [¹²⁵I]-p97 in human brain capillaries. Accumulation of [¹²⁵I]-p97 was measured at 37°C for 1 h in isolated human brain capillaries (100 µg/assay). The incubation medium contained [¹²⁵I]-p97 and a final concentration of 100 nM p97 in Ringer/Hepes solution. The accumulation of [¹²⁵I]-p97 was performed in presence or in absence of 5 µM of cold-p97, holo-transferrin or lactoferrin. After incubation, the accumulation was stopped by addition of 1 ml-cold stop solution (150 mM KCl, 0.1% BSA and 5 mM Hepes, pH 7.5). The suspension was filtered under vacuum through a 0.45µM pore size Millipore filter. The filter was rinsed with 8 ml of stop solution, and the radioactivity was counted. Nonspecific binding of the radioactivity to the capillaries was determined by the addition of the ice-cold stop solution to the capillaries before adding the incubation medium. This value was subtracted from accumulation values obtained following an 1 h incubation. The results were expressed as ng of [¹²⁵I]-p97 accumulated per µg of brain capillaries.

0188<figref idref="f0005">Figure 5</figref> demonstrates the results of the experiments. Consistent with the findings in <figref idref="f0003">Figures 3</figref> and <figref idref="f0004">4</figref>, it is found that cold p97 (5 micromolar) or lactoferrin (5 micromolar) significantly inhibits competitive [¹²⁵I]-p97 binding to human brain capillary cells, whereas transferrin (5 micromolar) shows no significant competition. This finding confirms that p97 binding to its receptor is not blocked by transferrin.

0189<figref idref="f0006">Figure 6</figref> is a repeat experiment, performed identically to the experiment of <figref idref="f0005">Figure 5</figref>, except this time with the additional competition assay for β-amyloid peptide. Evidently, the β -amyloid peptide 1-40 competes with p97 for receptor binding, along with lactoferrin, but not with transferrin. This finding indicates that the receptor responsible for the p97 binding and transport across the BBB is RAGE and/or LRP1.

0190<figref idref="f0007">Figure 7</figref> shows the results of experiments where the ligands p97, Lf and Tf were heated or not heated prior to the binding study. In all cases, binding experiments were conducted in the transwell apparatus as described previously, with the exception that binding was conducted at either 4°C or at 37°C. For the 37°C trial, a separate experiment was conducted where the ligand was boiled for 30 mins then rapidly cooled prior to administration on the transwell plates. Results demonstrate that the heat denatured p97 protein had a significantly lower accumulation in the cell monolayer compared to normal p97; although both forms at 37°C bound to a higher degree than at 4°C. Similarly, heat denatured Lf had significantly lower accumulation in the BBB model cells than its natural counterpart. Transferrin itself had very little accumulation to speak of.

0191In <figref idref="f0007">Figure 7</figref>, it is important to clarify the overall striking difference in accumulation of Lf and p97, as Lf is almost twice as high at the end of the experiment. It must be remembered that p97 is being substantially transcytosed through the BBB model cells and secreted into the abluminal region, whereas Lf is not. This effect leads to the incorrect conclusion that Lf may be taken into the BBB at a higher rate than p97.

Example IV: Transcytosis of p97

0192<img file="EP2147679B1_D0009.tif" />

0193Transcytosis experiments were performed as follows. One insert covered with BBCECs was set into a transwell apparatus containing a six-well microplate with 2 ml of Ringer/Hepes and pre-incubated for 2 h at 37°C. Plates were slowly adjusted to the indicated temperatures (4°C or 37°C). [¹²⁵I]-p97 (250 nM) was added to the upper side of the filter covered with cells. At various times, the insert was transferred to another well to avoid a possible reendocytosis of p97 by the abluminal side of the BBCECs. At the end of experiment, [¹²⁵I]-p97 was assessed in 500 µl of the lower chamber of well by TCA precipitation. p97 was also measured in 50 µl of the lower chamber of the well by Western blots using mAb L235.

0194<figref idref="f0008">Figure 8</figref> demonstrates that p97 transcytosis was significantly higher at 37°C than at 4°C. This result demonstrates that p97 is actively transported in an energy dependent process across this blood-brain barrier model in a temperature dependent fashion, presumably by receptor mediated uptake.

0195<figref idref="f0009">Figure 9</figref> confirms that transcytosis of p97 is also a saturable phenomenon, thus further implicating a specific MTf-receptor protein in this model of the blood-brain barrier. These experiments were conducted as previously described. Measurements of the amount of transcytosis were made at the time points indicated.

0196The effect of potentially competitive ligands on transcytosis was assessed in a series of experiments. In <figref idref="f0010">Figure 10a</figref>, transcytosis of ¹²⁵I-p97 was compared in the presence of cold p97 (5 micromolar), Lf(5 micromolar), and Tf (5 micromolar). At these concentrations, only the cold p97 successfully reduced transcytosis of the labelled p97. In <figref idref="f0010">Figure 10b</figref>, β -amyloid protein (5 micromolar) also failed to slow or reduce transcytosis of labelled p97. Higher amounts of ligands do interfere with p97 transcytosis (data not shown). In <figref idref="f0011">Figure 10c</figref>, RAP, a known polypeptide inhibitor of the LDL-Receptor family was applied to the cells (25 micrograms/ml). RAP significantly inhibited the transcytosis of p97, thus directly implicating the LDL-receptor family, especially LRP1 as the MTf-R.

Example V. Accumulation and transcytosis of p97 in brain.

0197<b>A. Brain uptake and in situ brain perfusion.</b> To measure the brain uptake of [¹²⁵I]-p97, mice were each given approximately 4 pmol of [¹²⁵I]-p97, [¹²⁵I]-BSA or human [¹²⁵I]-holo-transferrin in 200 µl of injection solution through the jugular vein. After 1 hour, animals were sacrificed and perfused with buffer via cardiac aorta. The serum and brain samples were collected and the levels of radioactivity were measured. In situ brain perfusion was performed as previously described (<nplcit id="ncit0067" npl-type="s"><text>Dagenais, C., Rousselle, C., Pollack, G.M. &amp; Scherrmann, J.M. J. Cereb. Blood Flow Metab. 20: 381-386 (2000</text></nplcit>)). Briefly, the right hemisphere of the brain was perfused with 10 nM of [¹²⁵I]-p97 or [¹²⁵I]-holotansferrin in Krebs-bicarbonate buffer (pH 7.4 with 95 % O 2 and 5 % CO 2 at a flow rate of 2.5 ml/min for 10 min) via a catheter inserted in the right common carotid artery following ligation of the external branch. Mice were decapitated to terminate perfusion and the right hemisphere was isolated on ice before subjected to capillary depletion (<nplcit id="ncit0068" npl-type="s"><text>Triguero, D., Buciak, J. &amp; Pardridge, W.M. J Neurochem., 54: 1882-1888 (1990</text></nplcit>)). Aliquots of homogenates, supernatants, pellets and perfusates were taken to measure their contents in [¹²⁵I]-proteins by TCA precipitation and to evaluate their apparent V_D.

0198<b>B. Iodination of proteins.</b> p97, bovine holo-transferrin and bovine lactoferrin were iodinated with standard procedures using iodo-beads from Sigma. Bovine holo-transferrin and bovine lactoferrin were diluted in 0.1M phosphate buffer, pH 6.5 (PB). p97 obtained from Synapse Technologies in neutralized citrate at pH 7.0 was dialyzed against this PB. Two iodo-beads were used for each protein. These beads were washed twice with 3 ml of PB on a Whatman filter and resuspended in 60 µl of PB. ¹²⁵I (1 mCi) from Amersham-Pharmacia biotech was added to the bead suspension for 5 min at room temperature. The iodination for each protein was initiated by the addition of 100 µg (80-100 µl). After an incubation of 10 min at room temperature. The supernatants were applied on a desalting column prepacked with 5 ml of cross-linked dextran from Pierce and ¹²⁵I-proteins were eluted with 10 ml of PBS. Fractions of 0.5 ml were collected, and the radioactivity in 5 µl of each fraction was measured. Fractions corresponding to ¹²⁵I-proteins were pooled and dialyzed against Ringer/Hepes, pH 7.4. The efficiency of radiolabeling was between 0.6-1 x 10⁸ cpm/100 µg of protein.

0199<b>Transcytosis and binding experiments.</b> One insert covered with BBCECs was set into a six-well microplate with 2 ml of Ringer-Hepes and was pre-incubated for 2 h at 37 °C. [¹²⁵I]-p97 was then added to the upper side of the insert. At various times, the insert was sequentially transferred into a fresh well to avoid possible reendocytosis of p97 by the abluminal side of the BBCECs. At the end of the experiment, [¹²⁵I]-p97was quantitated in 500 µl of the lower chamber of each well by TCA precipitation. We also measured p97 in 50 µl of the lower chamber of each well by SDS-PAGE according to the method of Laemmli (<nplcit id="ncit0069" npl-type="s"><text>Laemmli, U.K. Nature; 227: 680-685 (1970</text></nplcit>)). Proteins were separated on 7.5% acrylamide gels, stained with Coomassie Blue, dried and analysed by densitometry. For the binding experiments, cells were treated with or without saponin (0.5% wt/vol) to permeabilize cellular membranes as perviously described (<nplcit id="ncit0070" npl-type="s"><text>Descamps, L., Dehouck, M.P., Torpier, G. &amp; Cecchelli, R. Am. J. Physiol. 270: H1149-H1158 (1996</text></nplcit>)). After 2 hrs at 4oC, ECs were gently washed, and the [¹²⁵I]-p97 attached to the ECs was quantified in a liquid scintillation counter.

0200<b>p97 accumulation in human brain capillaries.</b> Human brain capillaries were isolated by a procedure previously described (see <nplcit id="ncit0071" npl-type="s"><text>Dallaire, L., Tremblay, L. &amp; Beliveau, R Biochem. J. 276: 745-752 (1991</text></nplcit>), <nplcit id="ncit0072" npl-type="s"><text>Demeule, M. et al. Int. J. Cancer. 93: 62-66 (2001</text></nplcit>)). [¹²⁵ I]-p97 was incubated with capillaries (100 µg) in Ringer/Hepes solution in the presence or absence of unlabelled p97, holo-transferrin or lactoferrin. The uptake was stopped by the addition of ice-cold stop solution (150 mM KCl, 0.1% bovine serum albumin (BSA) and 5 mM Hepes, pH 7.5) and the suspension was filtered under vacuum through a 0.45 µM pore size Millipore filter. The filter was rinsed with 8 ml of stop solution, and the radioactivity was assayed. Nonspecific binding of radioactivity to the capillaries was determined by addition of the ice-cold stop solution to the capillaries before adding the incubation medium. This value was subtracted from the values obtained following a 1 h incubation.

Example VI. Exemplary Methods.

0201<b>Cell culture.</b> Cells were grown in monolayer at 37°C under 5% CO2 in DMEM supplemented with 10% fetal heat inactivated calf serum (normal astrocytes); DMEM high glucose, 1mM sodium pyruvate supplemented with 10% calf serum (CTX); DMEM high glucose supplemented with 10% calf serum (RG2); RPMI-1640 supplemented with 10% calf serum and 2 mM glutamine (CNS-1); Ham's F12 supplemented with 10% calf serum (C6); MEM, 1mM sodium pyruvate supplemented with 10% calf serum (U-87, U-138).

0202<b>Uptake of [¹²⁵I]-p97.</b> Cells were grown in monolayer in six-multiwell microplates at 37°C under 5% CO2. Uptake of [¹²⁵I]-p97 was measured at 37°C for 2 h in astrocytes and astrocytomas. The incubation medium contained [¹²⁵I]-p97 and a final concentration of 50 nM P97 in Ringer/Hepes solution. The uptake of [¹²⁵I]-p97 was performed in presence or in absence of 5 µM cold-P97. After incubation, the cell monolayer was wash three times with cold Ringer/Hepes solution. Triton X-100 0.1% was added and the [¹²⁵I]-p97 uptake was assessed in the Triton X-100 soluble fraction by TCA precipitation.

0203<b>RNA extraction.</b> Cells in six-wells plate or in 75 cm² plastic tissue culture flasks were grown at 37°C under 5% CO2 with optimal culture medium to 80-90% confluence. Total cellular RNA was preserved in Trizol (Gibco BRL, Burlington, ON). The solution are frozen at -80 oC until extraction. The solution was defroze and mixed with 5:1 chloroform for 3 minutes at room temperature. The suspension was centrifugated at 12000 g at 4oC. The clear supernatant was collected then mixed with 1:1 of isopropanol for 15 minutes at room temperature. The mix was centrifugated at 12000 x g for 10 min at 4oC. The pellets was washed with 70% ethanol and dried before resuspended in RNase Free H2O.

0204<b>RT-PCR.</b> RT-PCR was performed for members of the LDL-R family: LRP, LRP1B, megalin, LDL, VLDL, LRP8, LR11, RAP, LR3, cubulin and P97. DNA (cDNA) synthesis was performed with 1 (g of total RNA using a cDNA one step synthesis kit (Invitrogen,USA) following the manufacturer's protocol. (1x of reaction mix, RNA 1(g, 0.2(M of both primers, 1(1 of RT/Platinum Tag mix). The cDNA generated was amplified using primers produced with MacVector 7.0 ( Oxford molecular Ltd, Oxford, UK). All the subsequent assays were then performed under conditions that produced amplifications of cDNA within a linear range. RT inverse-transcription was performed at 50oC for 30 min. PCR amplification for 35 cycles for all was performed as follows: denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. Finalisation stage was performed at 72oC for 30 min. Tubes containing all the ingredients except templates were included in all runs and served as negative controls. The amplified PCR products were electrophoresed on a 2% agarose gel in TAE (40 mM Tris, 360 mM acetic acid, 1 mM EDTA, 12.5 fM Ethium bromide) and were visualized under ultraviolet light followed by densitometric analysis.

0205<b>Western blot.</b> Cell lysates (25 µg of protein) were subjected to SDS-PAGE and electroblotted onto PVDF membranes. Membranes were blocked over night at 4°C in 5% non fat dry milk in TBS (NaCl 125 mM, Tris 20 mM, pH 7,5) with 0.1 % Tween. Successive incubations with proper primary antibody and horseradish peroxidase-conjugated secondary antibody were carried out for 60 min at room temperature. All incubations with antibodies were done after 3x15 minutes washes with TBS-Tween 0.1% The immunoreactive proteins were detected using the ECL system (Amersham-Pharmacia, Baie d'Urfé, Que).

0206<b>Ligand-binding.</b> Briefly, cells were incubated with [¹²⁵I]-p97in Ringer/Hepes solution, in presence or in absence of 5 µM cold-P97 for 1 h at 4°C. After incubation, the cell monolayer was washed three times with cold Ringer/Hepes solution. After the ligand binding at 4°C, cell monolayer was washed three times with cold Ringer/Hepes solution and was incubated with DSS (Disuccinimidyl suberate) or Lomant's reagent DSP (Dithiobis(succinimidyl propionate)) from Pierce. After the cross-link, cells were lysed and proteins were separated by SDS-PAGE electrophoresis. Gels were fixed, dryed and exposed to Kodak films at -80°C for about 3 weeks before developping. The cross-link was performed exactly as the manufacturer's protocol.

0207<b>BIAcore analysis.</b> MAb L235 was covalently coupled to a CM5 sensor chip via primary amine groups using the N-hydroxysuccinimide (NHS) /N-ethyl-N'-(dimethylaminopropyl)carbodiimide (EDC) coupling agent as previously described (<nplcit id="ncit0073" npl-type="s"><text>Johnsson, B., Lofas, S., &amp; Lindquist, G. Anal. Biochem. 198: 268-277 (1991</text></nplcit>)).

0208Briefly, the carboxymethylated dextran was first activated with 50 µl of NHS/EDC (50 mM/200 mM) at a flow rate of 5 µl/min. The mAb L235 (5 µg) in 10 mM acetate buffer, pH 4.0 was then injected and the unreacted NHS-esters were deactivated with 35 µl of 1 M ethanolamine hydrochloride, pH 8.5. Approximately 8000 to 10000 relative units of mAb 235 were immobilized on the sensor chip surface. The Ringer/Hepes buffer was used as the eluent buffer to monitor the signal plasmon resonance (SPR). p97 diluted in the same eluent buffer was boiled for various lengths of time, cooled to room temperature and injected onto the sensor chip surface. The SPR obtained was compared to that of unboiled p97.

0209The brain uptake of human [¹²⁵I-p97 in mice, one hour after i.v. injection was evaluated and and compared to that obtained for [¹²⁵I-BSA or human [¹²⁵I]-transferrin (<figref idref="f0012">Fig. 11a</figref>). The brain/serum ratio for p97, BSA and holo-transferrin is respectively 0.025, 0.002 and 0.008 indicating a higher brain accumulation for p97. To determine whether this observation is related to a greater brain penetration, we measured the apparent volume of distribution (V _D) of p97 and transferrin by <i>in situ</i> brain perfursion in mice (<figref idref="f0012">Fig. 11b</figref>). After a 10 min perfusion, the apparent V _D for both proteins was calculated for the whole brain homogenates as well as for brain capillaries and brain parenchyma. Under these conditions the apparent V_D of transferrin in the brain parenchyma is 2.4 ml/100g which is slightly higher than the brain V_D for the vascular marker [¹⁴C]-inulin at 1.7 ml/100g (data not shown). Importantly, the apparent V_D of p97 in the brain parenchyma is 17.2 ml/100g, 8.8-fold higher than for transferrin, indicating a greater passage through brain capillaries. To further investigate the transport of p97 across the BBB, the passage of [^I25I -p97 across an <i>in vitro</i> model of the BBB was measured at 37 °C and at 4 °C. (<figref idref="f0012">Fig. 11c</figref>). A dramatic reduction in the transport from the apical to the basolateral surface of BBCEC monolayers of [¹²⁵I -p97 is observed at 4 °C, indicating that the transcytosis of p97 requires an active mechanism.

0210Transcytosis of [¹²⁵I -p97 at37 °C was measured both in the apical-to-basolateral direction and in the basolateral-to-apical direction across BBCEC monolayers to ascertain any vectorial transport of p97 (<figref idref="f0012">Fig. 11d</figref>). This figure demonstrates that p97 transport in the BBCEC model is highly vectorial. In this experiment, [¹²⁵I -p97 (25 nM) was added to the luminal or abluminal side of the BBCECs. After 2 hours of incubation at 37°C, the amount on the opposing side of the membrane was measured. Results demonstrate that p97 transport is substantially directed from the luminal to the abluminal side, corresponding <i>in vivo</i> to the delivery of p97 from the blood/serum face of brain capillaries (<i>i</i>.<i>e</i>., inside the capillaries) to the neural cells of the brain. After 2 hrs, [¹²⁵I -p97 transport is about 3-fold higher when measured in the apical-to-basolateral direction, indicating a substantial preferential transport of p97 towards the brain.

Localization and saturability of the p97 binding activity.

0211To assess the presence and extent of intracellular p97 binding sites, BBCECs were treated with saponin (<figref idref="f0013">Fig. 12a</figref>). The saponin permeabilization of ECs increased the amount of [¹²⁵I]-p97associated with BBCECs 4-fold. Moreover, the binding of [¹²⁵I]-p97 after saponin treatment decreased in the presence of unlabelled p97 (<figref idref="f0013">Fig. 12b</figref>). A 200-fold molar excess of unlabelled p97 inhibited radiolabel binding by approximately 50%, showing that much of the interaction of p97 with ECs is saturable.

0212Values for specific p97 binding were calculated by subtracting the non-specific binding of p97 measured in the presence of a high concentration of unlabelled p97 and are expressed in a Scatchard plot (<figref idref="f0013">Fig. 12c</figref>). Analysis of this plot is consistent with a single-binding site for p97 with a Kd of about 1 µM and 4 x 106 sites/cell.

0213Comparison of p97, transferrin and lactoferrin binding on BBCEC monolayers. <tables id="tabl0005" num="0005"><table frame="none"><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="38mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="49mm" /><thead><row><entry valign="top" /><entry align="center" valign="top"><b>Kd</b></entry><entry align="center" valign="top"><b>Number of binding sites/cells</b></entry></row><row><entry valign="top" /><entry align="center" valign="top"><b>(nM)</b></entry><entry valign="top" /></row></thead><tbody><row><entry /><entry /><entry /></row><row><entry><b>P97 (+saponin)</b></entry><entry align="right"><b>1400</b></entry><entry align="right"><b>6 000 000</b></entry></row><row><entry><b>Transferrin (+saponin)</b></entry><entry align="right"><b>11</b></entry><entry align="right"><b>35 000</b></entry></row><row><entry><b>Lactoferrin (-saponin)</b></entry><entry align="right" /><entry align="right" /></row><row><entry><b>Site 1</b></entry><entry align="right"><b>35</b></entry><entry align="right"><b>35 000</b></entry></row><row><entry><b>Site 2</b></entry><entry align="right"><b>1900</b></entry><entry align="right"><b>900 000</b></entry></row></tbody></tgroup></table></tables>

0214In the above table, p97 results from <figref idref="f0013">figure 12</figref> (Kd and number of binding sites/cells) are compared to published values for transferrin and lactoferrin (see <nplcit id="ncit0074" npl-type="s"><text>Descamps et al., Am. J. Physiol. 270:H1149-H1158, 1996</text></nplcit>; <nplcit id="ncit0075" npl-type="s"><text>Fillebeen et al., J. Biol. Chem. 274 :7011-7017, 1999</text></nplcit>).

Efficiency of p97 transcytosis

0215The efficiency of p97 transcytosis was assessed by comparing the passage of both p97 and bovine holo-transferrin under identical conditions (<figref idref="f0014">Fig. 13a</figref>). Transport of p97 from the apical to the basolateral surface of ECs is much higher than for transferrin at 37°C (<figref idref="f0014">Fig. 13a</figref>). Heat-denaturation reduced the passage of both p97 and holo-transferrin through the BBCEC monolayers, indicating that their transcytosis is conformation-dependent. As p97 is resistant to heat denaturation in Ringer/Hepes solution (<figref idref="f0014">Fig. 13b</figref>), it was necessary to determine the denaturing conditions. The conformation of the protein was assessed using the biological interaction analysis in real-time between p97 and the monoclonal antibody (mAb) L235, which recognizes a conformational epitope on p97 since no enzymatic activity has yet been defined for this protein. For this analytical approach, mAb L235 was immobilized on the surface of a sensor chip and exposed to native p97 as well as to p97 which had been boiled for 5, 10, 20 or 30 min. The surface plasmon resonance signal of native proteins, the accumulation of p97 in BBCE cells is 5.7 µg/cm² whereas no significant accumulation is observed for bovine transferrin. These results show that the p97 transport system has much greater capacity than has the transferrin transport system.

p97 stability and BBCEC monolayer integrity following transendothelial transport

0216To examine p97 integrity after transcytosis at 37 °C and 4 °C, 50 µl of the lower compartments of the wells were recovered after 30, 60, 80 and 120 min. Proteins were then separated by SDS-PAGE and visualized by gel staining (<figref idref="f0015">Fig. 14a</figref>). Time-dependent transcytosis of recombinant p97 is observed, with no apparent degradation. Transcytosis of this protein is much higher when the experiment is performed at 37 °C than at 4 °C. The low molecular weight proteins observed at 30 min are only serum proteins remaining in the assay. Furthermore, the gels were scanned and the amount of p97 that passed through the BBCEC monolayers was evaluated using known quantities of p97 (<figref idref="f0015">Fig. 14b</figref>). The total amount of intact p97 after transendothelial transcytosis is 35 µg/cm², very similar to the amount shown in <figref idref="f0014">Fig. 13a</figref> after TCA precipitation, indicating that the iodination of p97 does not interfere with its transcytosis. Since p97 is transported much faster than is transferrin, the permeability to [¹⁴C]-sucrose was measured in the presence of a high concentration of p97 (<figref idref="f0015">Fig. 14c</figref>). No significant increase in the clearance of sucrose is detectable in the presence of p97. In addition, the permeability coefficient (Pe) for sucrose in the presence of p97 is 1.04 ± 0.15 x10_-3 cm/mitr, not significantly different from the value of 1.07 ± 0.19 x 10_-3cm/min measured in the absence of p97 (<figref idref="f0015">Fig. 14d</figref>). These data indicate that the rapid passage of p97 is unrelated to changes in the integrity of BBCEC monolayers.

Effect of p97 and transferrin on [¹²⁵I]-p97 transcytosis

0217To establish whether this p97 transport is saturable, and whether it involved the transferrin receptor, apical-to-basal transport of [¹²⁵I]-p97 across BBCEC monolayers was measured in the presence of a 200-fold molar excess of p97, bovine holo-transferrin or human holo-transferrin (<figref idref="f0016">Fig. 15</figref>). An excess of unlabelled p97 reduced the transport of [¹²⁵I]-p97 by 69% (<figref idref="f0016">Fig. 15a</figref>) whereas the presence of either bovine or human holo-transferrin had no impact (<figref idref="f0016">Fig. 15b</figref>). This indicates that p97 transcytosis is a saturable process that does not employ the transferrin receptor. This assumption is supported by the fact that mAb OX-26, which binds to the transferrin receptor, does not significantly reduce p97 transcytosis as compared to transcytosis measured in the presence of nonspecific IgGs (<figref idref="f0016">Fig. 15c</figref>).

Identification of LRP as a potential receptor for p97

0218We also assessed the uptake of [¹²⁵I]-p97 into isolated human brain capillaries incubated for 1 h at 37 °C (<figref idref="f0017">Fig. 16a</figref>). A 50-fold molar excess of unlabelled p97 inhibited the uptake of [¹²⁵I]-p97 by 60%. Human lactoferrin caused a similar inhibition of [¹²⁵I]-p97 uptake whereas human holo-transferrin had no effect. These results indicate that LRP, which binds and transports lactoferrin across BBCEC monolayers is also involved in the uptake of [¹²⁵I]-p97 into brain capillaries and in the transcytosis of p97.

0219To further investigate the role of LRP in the transport of p97, transcytosis experiments were performed in the presence of the receptor-associated protein (RAP), a protein chaperone that regulates LRP (<figref idref="f0017">Fig. 16b</figref>). Recombinant RAP (25 µg/ml) reduced the initial rate of [¹²⁵I]-p97 transport across BBCEC monolayers by more than 50%. In addition, the transcytosis of bovine [¹²⁵I]-lactoferrin is inhibited by more than 75% by a 200-fold molar excess of unlabelled p97 (<figref idref="f0017">Fig. 16c</figref>).

0220<figref idref="f0018">Figure 17</figref> further illustrates the effect of LRP ligands RAP, Aprotinin and BSA on p97 transcystosis and the effect of p97 on lactoferrin transcytosis. The inhibition by RAP but not BSA distinguishes the p97 from megalin, the chylomicron receptor found in the liver, and the receptor for advanced glycation end products which can be found on lung endothelial cells, neurons, atrocytes, and glomeruli.

Time course of p97 uptake in BBCE.

0221The time course for the internalization of p97 is illustrated in <figref idref="f0019">Figure 18</figref> which depicts the movement of p97 at 30 minutes and 60 minutes and its accumulation in early endosome. <figref idref="f0020">Figure 19</figref> illustrates the conditions for studying the rate of p97 internalization and transcytosis in the BBB model. <figref idref="f0021">Figure 20</figref> shows the results of such a study. Transcytosis is fast as in 10 minutes, 80% of membrane bound p97 has transcytosed.

Pathway for p97 endocytosis.

0222Upon binding to a LRP to a growing brain capillary endothelial cell, LDL is classically internalized by a clathrin dependent pathway wherein the LDL is directed toward lysosomes and degraded so as to provide cholesterol to the growing cell. This pathway is sensitive to filipin. In differentiated BCECs, the LDL is transcytosed. Evidence indicates the same receptor is involved in both pathways. (see <nplcit id="ncit0076" npl-type="s"><text>Dehouck et al., J. of Cell Biology 138(4) 877-889 (1997</text></nplcit>).

0223The possibility that p97 endocytosis involved a clathrin-dependent mechanism or a clathrin independent mechanism (i.e., caveolae) was examined in BBCE. p97 was found to co-localize with clathrin much more similarly than with caveolin (data not shown). Transcellular localization of p97 and clathrin was examined in BBCE as the p97 moved from the luminal to abluminal side of the BBB model (<figref idref="f0022">Figure 21</figref>). The data are indicative of a co-migration of p97 and clathrin. As a control for the effect of the Alexa label, the behavior of p97 and p97-alexa were compared. (data not shown). Although p97-alexa also labeled other vesicular structures, p97 and p97-alexa had a similar localization indicating that the label did not change the behavior of p97.

0224Following endocytosis, the transport fate of p97 depends upon the cell type. As shown in <figref idref="f0023">Figure 22</figref>, p97 may be transported across the cell as occurs with brain endothelial cells lining the brain capillaries or else it may be transported to a lysosome. In the first instance, p97 is useful as a means of delivering therapeutic agents such as p97 therapeutic agent conjugates across the blood brain barrier. In the second instance, p97 is useful as a means of delivering therapeutic agents to the intracellular compartment, particularly the lysosome. In this instance, conjugates with enzymes (e.g., an enzyme deficient in a patient with a lysosomal storage disease) are particularly of interest.

LRP Ligands and receptor family expression.

0225<figref idref="f0024">Figure 23</figref> illustrates an LRP receptor α and β subunits with respect to the cell membrane and some LRP ligands. <figref idref="f0026">Figure 25</figref> presents the relative amounts of LRP/LRP1B proteins in various cell types, including astrocytomas, normal astrocytes, and brain capillaries. As a contrast, the distribution of megalin among astrocytes and astrocytomas is presented in <figref idref="f0027">Figure 26</figref>. As shown in <figref idref="f0028">figures 27</figref> and <figref idref="f0029">28</figref>, the expression of LRP1B in astrocytes and astrocytomas as determined by (RT-PCR) is increased in the astrocytomas over the astrocytes. This result is result is consistant with the greater uptake of the labeled p97 by the astrocytomas. By way of contrast, and in confirmation that megalin is not involved in the p97 uptake, the pattern ofmegalin expression among these cells is not at all similar to the updtake pattern for p97 for these cells (see <figref idref="f0027">Figure 26</figref>). <figref idref="f0029">Figure 28</figref> more clearly illustrates the correlation between p97 uptake and LRP1B expression.

0226<figref idref="f0033">Figure 32</figref> shows the expression of members of the LDL receptor family by RT-PCR. In glioblastoma cell line U87 LRP, LRP1b, LDL, and LRP8 are highly expressed. Cubilin and RAP are expressed to a much lesser extent. In human capillaries, LRP1B, megalin, LDL, LRP8, and LRP are highly expressed as compared to cubilin.

Association of p97 with LRP/LRP1B

0227<figref idref="f0030">Figure 29</figref> shows that LRP/LRP1B migrates as a high molecular weight dissociable complex in the presence of p97, indicating the association of the two molecules. Exposing the complex to reducing conditions such as β-mercaptoethanol induces the release of p97 from the high molecular weight complex (see <figref idref="f0031">Figure 30</figref>). p97 similarly forms high molecular weight complexes upon contact with glioblastomas and human brain capillaries (see <figref idref="f0032">Figure 31</figref>).

Effect of p97 on cell expression of members of LDL-receptor family in human glioblastoma.

0228To examine the ability of p97 to be taken up by astrocytoma cells and astrocytes, cells of each type were incubated with ¹²⁵I p97. As shown in <figref idref="f0025">Figure, 24</figref>, the specific uptake of p97 in such cells was greatly increased in the astrocytoma cells over the astrocytes. As shown in <figref idref="f0025">figure 24</figref>, astrocytoma cells take up p97 to a much greater extent than astrocytes. This finding indicates that p97 conjugates to anticancer agents would be particularly useful in the treatment of gliomas. <figref idref="f0035">Figure 34</figref> shows the effect of p97 and RAP treatments on LRP and LRP1B expression in U87 cells. While p97 does induce the expression of LRP1B, it has no discernable effect on the morphology of the U-87 cells (see <figref idref="f0034">figure 33</figref>). <figref idref="f0036">Figure 35</figref>, shows the dose response effect of p97 for LRP1Bb, LRP, LDL-R, Megalin, and Cubilin as measured by RT-PR in U-87 cells. p97 induces the expression of the LRP1B receptor and the Cubilin receptor, but not the LRP or Megalin receptor. RAP appears to antagonize the effect of p97 on those receptors. p97 also appears to induce its own expression as shown by RT-PCR methods (see <figref idref="f0037">Figure 36</figref>). <figref idref="f0038">Figures 37</figref> and <figref idref="f0039">38</figref> summarize the quanitated effects of p97 and RAP on the expression of LRP1B, LRP, LDL-R, Cubilin, p97, and Megalin in the glioblastoma U87 cell line as determined by RT-PCR.

Effect of p97 on Fibroblast Cells.

0229The fibroblast cell line MG1391 was used to assess the potential of p97 and other LRP ligand conjugates as carriers for transcytosis or endocytosis in fibroblasts. <figref idref="f0040">Figure 39</figref> shows the expression of members of the LDL receptor family in MG1391 cells. LRP, LDL, and to a lesser extent, LRP1B and Cubilin are expressed as measured by RT-PCR. Further more, p97 particularly induces the expression of the LRP1B as shown in <figref idref="f0041">Figures 40</figref> and <figref idref="f0042">41</figref>.

Expression of LDL receptor Family members in human endothelial cells and BBCE cells.

0230As shown in <figref idref="f0043">Figure 42</figref>, LRP, LRP1B, and LDL-R as well as LRP8 are highly expressed in human endothelial cells. The expression of LDL receptor family members in the absence of astrocytes in largely that of LRP5. However, the presence of astrocytes induces the expression of LRP1B and LRP8 in the BBCE cells (see <figref idref="f0044">Figure 43</figref>).

0231The above examples show that the brain p97 uptake, in vivo, is much higher than that of other proteins such as BSA and transferrin. The in vitro model of the BBB used here to characterize the transcytosis of p97 has been used previously for such proteins as transferrin, lactoferrin, low density lipoproteins and insulin (see <nplcit id="ncit0077" npl-type="s"><text>Dehouck, B. et al. J. Cell. Biol. 138: 877-889 (1997</text></nplcit>), <nplcit id="ncit0078" npl-type="s"><text>Fillebeen, C. et al. J. Biol. Chem. 274: 7011-7017 (1999</text></nplcit>); <nplcit id="ncit0079" npl-type="s"><text>Descamps, L., Dehouck, M.P., Torpier, G. &amp; Cecchelli, R. Am. J. Physiol. 270: H1149-H1158 (1996</text></nplcit>)); <nplcit id="ncit0080" npl-type="s"><text>Frank, H.J., Pardridge, W.M., Morris, W.L., Rosenfeld, R.G. &amp; Choi, T.B. Diabetes 35: 654-661 (1986</text></nplcit>)). As was seen with these proteins, transendothelial transport of p97 requires energy and is concentration-dependent, indicating a receptor-mediated endocytosis mechanism for p97. In addition, preferential transport of p97 from the apical to the basolateral surface of BBCECs is observed with no detectable degradation of p97. The conformation of p97 also seems to be very important for its transcytosis because heat-denaturation considerably reduced the transendothelial transport of this protein. Thus, the in vitro results strongly confirm and support the in vivo observations on high p97 uptake in the brain.

0232The results show the presence of a low affinity receptor for p97 with a high capacity. Since all the experiments comparing bovine transferrin with human p97 are performed in a heterologous system, we can expect that the binding constant for the p97 receptor would be even greater in a human homologous system. It has been postulated that p97 is an alternate ligand for the transferrin receptor because p97 shares many properties with human transferrin and because the transferrin receptor has been detected in the same tissues as p97. However, our results strongly support that a different mechanism than that involving the transferrin receptor. First, the transcytosis, binding and accumulation of p97 are much higher than those for transferrin indicating that the p97 receptor has a much higher capacity and lower affinity than those previously reported for the transferrin receptor. Second, the transcytosis of p97 is unaffected by either bovine or human transferrin, indicating that p97 does not compete with transferrin for its receptor. Third, the mAb OX-26 directed against the transferrin receptor, which was previously shown to inhibit the uptake of transferrin, has no effect on p97 transport. In addition to the transcytosis experiments using BBCEC monolayers, the competition of [125I]-p97 uptake by unlabelled p97 in isolated human brain capillaries confirmed the presence of a receptor for p97. Moreover, lactoferrin competed [125I]-p97 uptake efficiently, better than transferrin or any other tested proteins, indicating that lactoferrin and p97 share a receptor. The receptor for lactoferrin transcytosis in brain ECs is LRP 6, a member of the large LDL-receptor family. (see <nplcit id="ncit0081" npl-type="s"><text>Bu, G. &amp; Rennke, S. J. Biol. Chem. 271: 22218-2224 (1996</text></nplcit>)). To further investigate whether LRP could be involved in p97 transcytosis, experiments were performed with BBCEC monolayers in the presence of RAP, a protein which inhibits the binding of ligand to members of the LDL-receptor family (see <nplcit id="ncit0082" npl-type="s"><text>Bu, G. &amp; Rennke, S. J. Biol. Chem. 271: 22218-2224 (1996</text></nplcit>); <nplcit id="ncit0083" npl-type="s"><text>Willnow, T.E, Goldstein, J.L., Orth, K., Brown, M.S. &amp; Herz, J. J. Biol. Chem. 267: 26172-26180 (1992</text></nplcit>); <nplcit id="ncit0084" npl-type="s"><text>Bu, G. &amp; Schwartz, A.L. Trends Cell Biol. 8: 272-276 (1998</text></nplcit>); and <nplcit id="ncit0085" npl-type="s"><text>Herz, J. &amp; Strickland, D.K. J. Clin.Invest. 108: 779-784 (2001</text></nplcit>).

0233Known members of this family also include LDL-R, LRP1B, megalin, VLDL-R, apoE-receptor 2 and the mosaic LDLR-related protein (LR11) (see <nplcit id="ncit0086" npl-type="s"><text>Hussain, M.M. Front. Biosci. 6: D417-D428 (2001</text></nplcit>); and <nplcit id="ncit0087" npl-type="s"><text>Liu, C.X., Li, Y., Obermoeller-McCormick, L.M., Schwartz, A.L. &amp; Bu, G. J. Biol. Chem. 276: 28889-28896 (2001</text></nplcit>)). Among these receptors, megalin, is also known to bind lactoferrin (see <nplcit id="ncit0088" npl-type="s"><text>Hussain, M.M. Front. Biosci. 6: D417-D428 (2001</text></nplcit>); and <nplcit id="ncit0089" npl-type="s"><text>Willnow TE. Biol. Chem. 379: 1025-1031 (1998</text></nplcit>)). However, megalin is mainly expressed in the kidney whereas the major site of LRP expression is in brain. Thus, the diminution of p97 transcytosis by RAP and the inhibition of lactoferrin transcytosis by p97 also indicates that LRP transports p97 across BBCECs.

0234The concept of using receptor-mediated endocytosis to deliver peptides into the brain was initially described with the findings on the transendothelial transport of insulin across the blood brain barrier (BBB). Subsequent studies demonstrated that a neuropeptide could be delivered into the CNS using receptor-mediated endocytosis by targeting the transferrin receptor with the mAb OX-26 (see <nplcit id="ncit0090" npl-type="s"><text>Bickel, U., Yoshikawa, T. &amp; Pardridge, W.M. Adv. Drug Deliv. Rev. 46: 247-279 (2001</text></nplcit>) and <nplcit id="ncit0091" npl-type="s"><text>Pardridge, W.M., Buciak, J.L. &amp; Friden, P.M. J. Pharmacol. Exp. Ther. 259: 66-70 (1991</text></nplcit>)). The development of chimeric proteins containing this mAb, specific linkers and a neurotropic peptide has permitted delivery into the brain of significant levels of this peptide (see <nplcit id="ncit0092" npl-type="s"><text>Bickel, U., Yoshikawa, T. &amp; Pardridge, W.M. Adv. Drug Deliv. Rev. 46: 247-279 (2001</text></nplcit>);<nplcit id="ncit0093" npl-type="s"><text> Pardridge, W.M., Wu, D., &amp; Sakane, T. Pharm. Res. 15: 576-582 (1998</text></nplcit>); and <nplcit id="ncit0094" npl-type="s"><text>Zhang, Y. &amp; Pardridge, W.M. Brain Res. 889: 49-56 (2001</text></nplcit>)). In addition, the transendothelial transport of mAb OX-26 was also reported in these studies to be similar to the transport of human transferrin across the BBB. Our results show that p97 passes across the blood brain barrier (BBB) at least as well as does OX-26. Another advantage of using p97 is its very low concentration in the serum (100 000-fold lower than transferrin) (see <nplcit id="ncit0095" npl-type="s"><text>Jefferies, W.A. et al. Brain Res. 712: 122-126 (1996</text></nplcit>), and <nplcit id="ncit0096" npl-type="s"><text>Kim, D.K. et al. S Neurapsychopharmacology 25: 84-90 (2001</text></nplcit>)), which indicates that it would deliver p97-conjugate(s) directly into the CNS.

0235The results show that intact p97 can cross brain ECs without affecting the integrity of the BBB and with a much higher rate than is seen with transferrin. The inhibition of p97 transcytosis by RAP in BBCEC monolayers and the competition of p97 uptake in brain capillaries by human lactoferrin show that LRP, a member of the LDL-R family, is involved in the transendothelial transport of p97. The results indicate that p97 and, more generally, ligands of the LRP and LRPIB receptors are preferred carriers for conjugation with active agents and preferred modulators for the transport of such conjugates via the LRP or LRP1B receptor. Each publication, patent application, patent, and other reference cited in any part of the specification is incorporated herein by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Based on the invention and examples disclosed herein, those skilled in the art will be able to develop other embodiments of the invention. The examples are not intended to limit the scope of the claims set out below in any way. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended