Hyaluronic acid and derivatives for modulation of cellular activity

FIELD OF THE INVENTION The present invention relates to modifications of cellular activity, in particular human cellular activity, using specific molecular amounts and fractions of hyaluronic acid forms, including, for example, sodium hyaluronate and various molecules that mimic the hyaluronic acid forms.

Cell adhesion molecules, originally thought to serve only to keep cells in tissues together, are now increasingly believed to play a more complex role in the functioning of the human body [see, e.g., Biotech

260 (1993)].

New therapeutic methods based on cell adhesion molecules have been developed. One class of endothelial adhesion molecules is ICAM-1 endothelial adhesion molecule. It has been found that this ICAM-1 molecule delivers human rhinoviruses, which cause about 5% of colds, to cells in the body. Rhinoviruses enter the body cells by attaching to the ICAM-1 adhesion molecule.

Previously, it has been proposed to block cell infection using soluble ICAM-1. The soluble ICAM, on the other hand, had poor adhesion, so this idea was not found for market use.

In one of the attempts to prevent common colds, genetic engineering techniques have been used to attach antibody fragments to the rhinovirus binding portions of ICAM-1.

Another adhesion molecule, called CD44, is normally found on lymphocytes. However, the CD44 adhesion molecule also occurs on pancreatic tumor cells that are metastatic. CD44 molecules can mistakenly mask tumor cells and thus allow the free circulation of tumor cells in the bloodstream.

In our studies, we have discovered that ICAM-1 acts as a cell surface receptor for hyaluronic acid (hyaluronan). ICAM-1 is expressed (produced and delivered to the cell surface) in liver endothelial cells and corneal epithelial cells. In inflammation, cancer and infection, ICAM-1 is overexpressed.

It also functions as a cell surface receptor for hyaluronan called HARLEC (Hyaluronic Acid (Hyaluronan) Receptors Liver Endothelial Cells; liver endothelial cell hyaluronic acid (hyaluronan) receptors], which is also expressed (produced and delivered to the cell surface) in liver endothelial cells and corneal epithelial cells. (In our opinion, the two are one and the same adhesion molecule, or at least so closely related to one another that the two are almost the same molecule.)

CD44 and RHAMM (HA-Mediated Motility Receptor) are also cell surface receptors for hyaluronic acid (hyaluronan). THE

RHAMM is a regulatory molecule. CD44, on the other hand, is an adhesion molecule.

The objects of the present invention can be summarized as follows: novel methods for treating diseases and conditions; new uses of the hyaluronate forms (hyaluronan, HA); new dosage amounts of pharmaceutical compositions; and novel pharmaceutical compositions suitable for use in said treatment methods and said uses of HA forms.

In addition, the present invention also provides novel methods for treating diseases and conditions using molecules that mimic hyaluronic acid and its pharmaceutically acceptable salts (e.g., sodium hyaluronate) in the sense that they bind to the same receptors (e.g. bind to high affinity cell surface receptors for hyaluronic acid), to which the forms of hyaluronic acid bind; new uses of these hyaluronic acid mimetics; new dosage amounts of pharmaceutical compositions containing such molecules; and novel pharmaceutical compositions suitable for use in said treatment methods and said uses of mimicking molecules.

Based on the following detailed teaching and the abstract of the invention, one of ordinary skill in the art will appreciate the additional features of this invention.

To modify the cellular activity of tissues and cells expressing a high affinity cell surface receptor for hyaluronic acid, such as an adhesion molecule (such as ICAM-1, HARLEC and CD44) and a regulatory molecule (such as RHAMM). The method of the invention provides the human patient with a non-toxic, effective amount of a medicament tolerated by the human body. .

A form of hyaluronic acid, e.g., hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g., less than 750,000 daltons, such as 50,000, is administered in an art vehicle or vehicle, e.g., sterile water. sodium hyaluronate having an average molecular weight of less than about 100,000 daltons and having an average molecular weight of about 150,000 to about 225,000 daltons from Hyal Pharmaceutical Corporation, and 08/143983. sodium hyaluronates as well as various molecular weight fractions of a sodium hyaluronate form, such as those disclosed in U.S. Patent No. 1,205,031. (Fidia), for example

000 - fractions of molecular weight between 100,000 daltons, 250,000-350,000 daltons and 500,000-730,000 daltons, or other molecular weight fractions, such as less than 50,000 daltons, or homologues, analogs, derivatives, complexes, esters of hyaluronic acid , fragments and / or subunits thereof and / or combinations thereof, preferably a hyaluronic acid having a molecular weight of less than 750,000 daltons, a pharmaceutically acceptable salt thereof, such as sodium hyaluronate and combinations thereof, and molecules that mimic the aforementioned forms of hyaluronic acid in the sense that they are capable of binding to the same receptors as the hyaluronic acid form to modify the cellular activity of tissues and / or cells that express a molecule , which acts as a high affinity cell surface receptor for hyaluronic acid, such as * ··································································································

β is, for example, an adhesion or regulatory molecule.

Where an inflammatory response may occur in an area of ​​injury or injury, inflammatory cells (e.g., neutrophils, macrophages, or other white blood cells) may migrate to said area. Damage caused by an inflammatory response may be greater than actual injury or trauma injury. This is the case, for example, with fibrosis. Upon administration of the hyaluronic acid form, the hyaluronic acid form binds to the HA receptor (the high affinity cell surface receptor) of the molecule, for example, an 'adhesion molecule' or a regulator molecule, thereby modifying the body's response and subsequent damage. Similarly, when molecules that mimic the hyaluronic acid form are administered in the sense that they are capable of binding to the same receptors to which the hyaluronic acid form is attached, the mimetic molecules bind to the HA receptor. Examples of molecules that mimic the hyaluronic acid form include, but are not limited to, antibodies that bind to the active site of the receptor, peptides that attach to the receptor site, and synthetic compounds that attach to the receptor site.

The agents for treating the disease or condition may also be used in combination with the hyaluronic acid form and the molecules that mimic the hyaluronic acid form; in this case, the active ingredient enhances the modulation of the HA form and the molecule that mimics HA activity. In addition, the HA form directs the active ingredient to the adhesion molecule (e.g., ICAM-1, HARLEC, and CD44) or to the RHAMM regulator molecule.

Suitable active ingredients include, but are not limited to, the following: free radical scavengers (e.g. ascorbic acid (vitamin C)), anticancer agents, chemotherapeutic agents, antiviral agents such as a nonionic surfactant such as nonoxynol-9 (nonylphenoxy polyethoxyethanol) in the Delien ™ contraceptive cream, surfactants such as cetylpyridinium chloride, cationic surfactants such as benzalkonium chloride, non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, naproxen, diclofenac and (+/-) - ketorolac tromethamine salt sold under the trademark Toadol ™, steroidal anti-inflammatory drugs, antifungal agents, detoxifying agents such as enema, rectal anesthetics, antibacterial agents, bronchodilator antibiotics, agents for the treatment of vascular ischaemia (such as diabetes and Berger's disease), antibody monoclonal agents, topical agents, minoxidil, diuretics such as furosemide under the trademark Lasix ™, immunosuppressants such as cyclosporins, lymphokines such as interleukin-2, etc., interferon alpha and interferon, etc.

Another object of the present invention is to provide a molecular fraction of a form of hyaluronic acid, such as hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g. ·· - *

·..· ··:· ...· .

- an average molecular weight fraction of 150,000 to 225,000 Daltons from 8 Cal Corporation; (Fidia), for example, fractions having a molecular weight of 50,000 to 100,000 Daltons, 250,000 to 350,000 Daltons, and 500,000 to 730,000 Daltons, or homologues, analogs, derivatives, complexes, esters of hyaluronic acid, fragments and / or subunits thereof and / or combinations thereof and molecules which mimic the aforementioned forms of hyaluronic acid in the sense that they are capable of binding to the same receptors, as bound to the hyaluronic acid form refers to the use of modifying the cellular activity of tissues and / or cells expressing high affinity cell surface receptors in the human body. The hyaluronic acid form may be used together with a pharmaceutically acceptable carrier or carrier (e.g., sterile water). Tissue or cellular modulation is increased in patients and / or patients. The HA and / or HA mimicking molecule binds to the HA receptors of the molecule, such as an adhesion or regulatory molecule, such as the high affinity cell surface receptor for the hyaluronic acid form. The HA form and the HA mimicking molecule may be used with a suitable active ingredient (as described above). The HA form, the HA mimicking molecule, and the active ingredient are used in effective non-toxic amounts.

The HA form and the HA mimicking molecule can also be used to prevent a disease or condition (e.g., a cold) in which the HA form and / or the HA mimicking molecule is 9 bound to ICAM-1 adhesion molecules (not rhinovirus bound or linked to ICAM-1). -1 adhesion molecules), we prevent the entry of human rhinovirus into body cells.

Accordingly, the present invention provides a novel method of preventing a disease and / or condition in a human by modifying the cellular activity of a high affinity cell surface receptor for hyaluronic acid, such as by expressing an adherent or regulatory molecule and / or cellular activity in a human. in a non-toxic, effective amount, in a pharmaceutical carrier or excipient tolerated by the human body, for example, a sterile water form of a hyaluronic acid, such as hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g., less than 750,000 daltons, such as less than 50,000 daltons, having an average molecular weight of about 100,000 daltons and 150,000 or 225,000 daltons; Sodium hyaluronate having an average molecular weight of 150,000 to 225,000 daltons from Corporation; Sodium hyaluronate and various molecular weight fractions of a sodium hyaluronate form, e.g.

205 Fractions, or other fractions, or homologues, analogs, derivatives, complexes, esters of hyaluronic acid, as disclosed in Canadian Patent No. 031 (Fidia), for example between 50,000-100,000 daltons, 250,000-350,000 daltons, and 500,000-730,000 daltons. , fragments and / or subunits and / or · · · ·

Combinations thereof, and molecules that mimic the aforementioned forms of hyaluronic acid in the sense that they are capable of binding to the same receptors as the form of hyaluronic acid. The HA form or the mimicking molecule is preferably administered in a pharmaceutically acceptable carrier (e.g., sterile water). In this way, for example, common colds can be prevented. The agents for the prevention of the disease and / or condition may also be administered with the hyaluronic acid form and / or the hyaluronic acid mimetic. For example, acetylsalicylic acid may be administered together with the hyaluronic acid form and / or the hyaluronic acid mimetic.

For example, administration of the hyaluronic acid form and the hyaluronic acid mimicking molecule in the human body inhibits cellular adhesion and / or modifies cellular activity. The HA and the mimicking molecule bind to the HA receptors at the high affinity cell surface receptor for hyaluronic acid, such as an adhesion molecule (such as ICAM-1, HARLEC and CD44) and / or a regulatory molecule (such as RHAMM). . Upon administration of the hyaluronic acid form and the mimetic molecule, the hyaluronic acid form and the mimetic molecule bind to the HA receptor of the molecule, thereby preventing the disease or condition (e.g., preventing rhinovirus from binding to the ICAM-1 adhesion molecule and thus preventing the rhinovirus bound to ICAM-1, enter cells of the body).

Where therapeutic agents are used to prevent a disease and / or condition, the active compounds are in the form of hyaluronic acid.

- can be used with 11 to prevent a disease or condition. For example, people today take effective amounts of acetylsalicylic acid (aspirin) to prevent stroke. The active ingredient may be associated with an effective non-toxic amount of a suitable form of hyaluronic acid (e.g., a sodium hyaluronate having a molecular weight of less than 750,000 daltons) and / or an effective non-toxic amount of a mimicking molecule. In our opinion, the combination of the two active ingredients reduces the risk of stroke to a greater extent than the individual receiving aspirin alone. In addition, the hyaluronic acid form directs the active agent to areas requiring treatment because the hyaluronic acid form binds to a high affinity cell surface receptor for the hyaluronic acid form, such as an adhesion molecule (e.g., ICAM-1). , HARLEC and CD44) and a regulator molecule (such as RHAMM) and thus modifies their activity.

Administration can be, inter alia, intravenous, intraarterial, intraperitoneal, intrapleural and percutaneous. In addition, the active ingredient may be administered intradermally to the skin, for example, to target the epidermis, or may be applied topically to an effective amount, e.g., to the mucous membranes, whereby the active ingredient is administered intranasally or rectally; in the latter case, for example, by enema or topical application in specific areas of the rectum. In addition, the active ingredient may be administered by direct injection.

- 12 forms.

The form of hyaluronic acid to be administered may be administered in various effective doses, for example, in the range of 10 mg to 1000 mg, for a person weighing 70 kg. Optimal dosages for a person weighing 70 kg are between 50 mg and 500 mg. Since hyaluronic acid has no toxic effect in humans, large amounts of hyaluronic acid (e.g. up to 3000 mg for a 70 kg person) can be used without adverse effects.

When applied topically, for example, when the active ingredient is applied to the skin or mucous membrane, the hyaluronic acid form should be applied in an amount of at least 5 mg per square centimeter of skin or tissue (including mucous membrane).

In the case of analgesia, the hyaluronic acid form should preferably be applied topically in an amount of at least 10 mg / cm.

The HA mimicking molecules may be administered in effective dosage amounts known to those skilled in the art.

For modes of administration other than topical application, an effective dosage amount of a form of hyaluronic acid in a subject weighing 70 kg may be at least about 10 mg and may be greater than 1000 mg. When a person weighing 70 kg is administered an NSAID, such as indomethacin (dissolved in N-methylglucamine), together with more than 200 mg of hyaluronic acid such as sodium hyaluronate, during which the NSAID (e.g. amount of person 1 ·· · ···· ··

1-2 mg / kg body weight does not cause any major toxic side effects such as gastrointestinal disorder, neurological abnormality, depression, etc., even when indomethacin is used in larger amounts when needed. If the amount of hyaluronic acid is reduced below this value, the usual side effects of using NSIAD compounds may reappear. For topical administration, the dosage amounts should contain at least about 5 'mg of hyaluronic acid form per 1 square centimeter of skin, mucous membrane, or tissue to be treated.

The active compounds used in combination with the hyaluronic acid form and / or the HA mimicking molecule are formulated into a formulation containing the active ingredient in an effective non-toxic amount for the particular treatment.

One form of hyaluronic acid and / or pharmaceutically acceptable salts thereof (e.g., sodium salt) and homologues, analogs, derivatives, complexes, esters, fragments and subunits thereof, preferably hyaluronic acid and salts thereof, for use in the present invention. , the form sold by Hyal Pharmaceutical Corporation. This form contains sodium hyaluronate in a 15 ml vial at a concentration of 20 mg / ml (300 mg / vial - 2F3). The form corresponds to a 2% solution of sodium hyaluronate having a molecular weight of about 225,000 daltons. The vial is distilled three times in the desired amount for US injection preparations.

- Contains 14 United States Pharmacopoeia (USP) compliant sterile water. Vials for hyaluronic acid and / or salts thereof are vials made of borosilicate glass type I which are closed with a butyl stopper which does not react with the contents of the vial.

The fraction / amount of hyaluronic acid and / or salts (e.g., sodium salt) can be described by the following characteristics:

purified, substantially pyrogen-free, hyaluronic acid fraction obtained from natural sources and having at least one of the following characteristics:

<td>(I)</td><td>a molecular weight of between 150,000 and 225,000;</td>

<td>(Ii)</td><td>about 1.25% by weight based on total weight, less than sulfated mucopolysaccharides;</td>

<td>(Iii)</td><td>about 0.6% by weight of the total weight, less than protein;</td>

<td>(Arc)</td><td>at about 150 ppm based on total weight contains less iron;</td>

<td>(V)</td><td>at about 15 ppm based on total weight contains less lead;</td>

<td>(Vi)</td><td>Less than 0.0025% by weight of glucosamine;</td>

<td>(Vii)</td><td>Less than 0.025% glucuronic acid;</td>

<td>(Viii)</td><td>Less than 0.025% by weight of ΔΓ-acetylglucosamine;</td>

<td>(Ix)</td><td>Less than 0.0025% by weight of amino acids;</td>

<td>(X)</td><td>UV extinction less than about 0.275 at 257 nm has a coefficient;</td>

<img file="HUT76846A_D0001.tif" />

(xi) has a UV extinction coefficient of less than about 0.25 at 280 nm; and (xii) a pH of between 7.3 and 7.9.

Preferably, the hyaluronic acid is mixed with water and the hyaluronic acid fraction has a molecular weight of between 150,000 and 225,000. More preferably, the hyaluronic acid fraction has at least one feature selected from the group consisting of:

(i) less than about 1% by weight of sulfated mucopolysaccharides based on total weight;

(ii) less than about 0.4% protein by weight;

(iii) less than about 100 ppm iron by weight;

(iv) less than about 10 ppm lead by weight;

(v) less than 0.00166% by weight of glucosamine;

(vi) less than 0.0166% by weight of glucuronic acid;

(vii) less than 0.0166% by weight of W-acetylglucosamine;

(viii) less than 0.00166% by weight of amino acids;

(ix) having a UV extinction coefficient of less than about 0.23 at 257 nm;

(x) has a UV extinction coefficient of less than about 0.19 at 280 nm; and (xi) a pH of between 7.5 and 7.7.

We can also recommend LifeCore '^IM Sodium hyaluronate produced and marketed by Biomedical, Inc. 16, which has the following characteristics,

Typical appearance odor viscosity average molecular weight

UV / Vis spectrum (190-820 nm) Optical Density (OD) (260 nm) Hyaluronidase Sensitivity

IR spectrum pH (10 mg / g solution) water content protein content (1 mg sodium hyaluronate) acetate content (1 mg has:

DETAILED DESCRIPTION White-cream granules no detectable odor &lt;750,000 Daltons corresponding to known spectrum &lt;0.25 OD units positive response according to known spectrum

6.2-7.8 Up to 8% &lt;0.3 gg &lt;0.3 gg in sodium hyaluronate) ······· · ···

- 17 maximum heavy metal content (ppm)

As Cd Cr Co Cu Fe Pb Hg Ni

2,0 5,0 5,0 10, 0 10,0 25,0 10,0 10, 0 5,0

<td>microbial load</td><td>no observable amount</td>

<td>endotoxin content (1 mg</td><td>&lt;0.07 EU</td>

<td>Sodium hyaluronate)</td><td></td>

<td>biosecurity</td><td>enough for the rabbit eye</td>

<td>testing</td><td>toxicity test</td>

<td>Skymart Enterprises, Inc.</td><td>One called Hyaluronan HA-M5070</td>

<td>another sodium hyaluronate</td><td>- sells a format that follows -</td>

<td>has initial features:</td><td></td>

Specification test

Results

<td>serial</td><td>HG1004</td>

<td>pH</td><td> 6,12</td>

<td>chondroitin sulphate</td><td>is not detected</td>

<td>protein</td><td> 0,05 %</td>

<td>heavy metals</td><td>up to 20 ppm</td>

<td>arsenic</td><td>up to 2 ppm</td>

<td>drying loss</td><td> 2,07 %</td>

<td>combustion residue</td><td> 16, 69 % '·</td>

intrinsic viscosity

12.75 dl / s (XW: 679,000)

<img file="HUT76846A_D0002.tif" />

nitrogen analysis microbiological

E. coli mold and yeast

3,14 %

104,1 %

80 / g negative up to 50 / g

Other forms of hyaluronic acid and / or salts (e.g., sodium salts) and hyaluronic acid homologues, analogs, derivatives, complexes, esters, fragments and subunits thereof, preferably hyaluronic acid and salts thereof, available from other vendors, such as those known in the art. The following references refer to hyaluronic acid as well as sources, methods of production and recovery of hyaluronic acid.

U.S. Patent 4,141,973 discloses hyaluronic acid fractions (including the corresponding sodium salts) having the following characteristics:

(a) having an average molecular weight greater than about 750,000, preferably greater than about 1,200,000, i.e., having an intrinsic viscosity of about. 1400 cm³g / g and preferably greater than about 2000 cm / g;

(b) having a protein content of less than 0.5% by weight;

(c) an ultraviolet absorbance of a 1% sodium hyaluronate solution of less than 3.0 at 257 nanometers and less than 2.0 at 280 nanometers;

- 19 (d) 1% physiological buffered sodium hyaluronate3 2

kinetic viscosity of the solution is greater than about 1 x 10 m / s2 (1000 centistokes), preferably greater than 1 x 10 m / s (10,000 centistokes);

(e) a molar optical rotation of a 0.1-0.2% physiological buffered sodium hyaluronate solution at less than -11 x 10 degrees-cm / mole disaccharide at less than 220 nanometers;

(f) if approximately half of the vitreous monkey vitreous humor is replaced by ml of 1% physiological buffered saline, there is no significant cellular infiltration in the vitreous humor and anterior chamber, no opacification or opacification in the aqueous humor. light reflection, and no pathological changes in the cornea, lens, iris, retina, and choroid;

(g) sterile and pyrogen-free; and (h) is not antigenic.

Canadian Patent No. 1,205,031 (U.S. Patent 4,141,937, which discloses prior art) discloses 50,000 and

100 Describes fractions of hyaluronic acid with an average molecular weight of between 000, 250,000 and 350,000, and between 500,000 and 730,000, as well as methods for preparing the fractions.

If high molecular weight hyaluronic acid (or en ··· · ··

- 20 -, -, -, - or other forms of the same), dilution should be used to permit administration and dilution to avoid intramuscular coagulation. .

The present invention relates to a dosage amount of a pharmaceutical composition which expresses tissue expressing a high affinity cell surface receptor for hyaluronic acid, such as an adhesion molecule (such as ICAM-1, HARLEC and CD44) and a regulatory molecule (such as RHAMM). and to modify the cellular activity of cells in the human body, which is a non-toxic, effective amount, a pharmaceutically acceptable salt of hyaluronic acid, e.g., hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, such as sodium hyaluronate having an average molecular weight of less than 750,000 daltons, e.g. sodium hyaluronate having an average molecular weight of 150,000 to 225,000 Daltons; sodium hyaluronates and various molecular weight fractions of a sodium hyaluronate form, such as those described in Canadian Patent No. 1,205,031 (Fidia), e.g. 50,000-100,000 daltons,

250 Fractions of molecular weight between 000,000-350,000 daltons and 500,000-730,000 daltons, or other molecular weights, e.g. less than 50,000 daltons or between 100,000-150,000 daltons, or -haluronic acid homologues, analogs, derivatives, complexes, esters, fragments and / or subunits and / or combinations thereof, and HA mimicking molecules that mimic the aforementioned forms of hyaluronic acid in the sense that they are capable of binding to the same receptors, such as the hyaluronic acid form, so that the dosage amount modifies the cellular activity of tissues and / or cells that express a molecule that functions as a high affinity cell surface receptor for hyaluronic acid, such as an adhesion or regulatory molecule.

As a result of treatment with the dosage amount, tissue and cellular modulation in the individual suffering from the disease or condition is enhanced. For example, modulation and / or regulation of cellular adhesion and / or cellular activity is inhibited as a result of administration of the hyaluronic acid form (HA) or the HA mimicking molecule. Both the hyaluronic acid and the HA mimicking molecule bind to the HA receptors of the molecule (e.g., adhesion molecule, regulator molecule, etc.). In this way, a disease or condition can be prevented. Unexpectedly, high affinity between HA and the HA mimicking molecule and cell surface HA receptors allows modulation of the disease or condition, such as modulation of the inflammatory process, fibrosis, and oncogenic control (preventing cancer and metastases).

The dosage amount of the pharmaceutical composition may also comprise, in an effective, non-toxic amount, a pharmaceutical agent or therapeutic agent containing a hyaluronic acid form or a hyaluronic acid form for the prevention of a disease or condition.

- 22 Used to treat a disease and / or condition associated with a mimicking molecule. The active ingredient can thus enhance the modulation of the activity of the HA form and of the molecule mimicking the HA form. In addition, the HA form directs the drug to the cell surface receptor of the molecule, such as ICAM-1, HARLEC and CD44, and the RHAMM regulatory molecule.

According to the present invention, the dosage amount of a pharmaceutical composition may comprise the following components:

i) a non-toxic, therapeutically effective amount of a pharmaceutical and / or therapeutic agent for treating a disease or condition;

ii) a non-toxic effective amount of a hyaluronic acid form, such as hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, such as a sodium hyaluronate having an average molecular weight of less than 750,000 daltons, e.g. dalton sodium hyaluronate average molecular weight fraction; and 08/143983; Sodium hyaluronates and various molecular weight fractions of a sodium hyaluronate form, e.g.

205 Canadian Patent No. 031 (Fidia), e.g., 50,000 to 100,000 Daltons,

250 000 to 350,000 Daltons to 500,000 to 730,000 Daltons, or other, such as less than 50,000 Daltons

4444 ·*

- 23 • ν · • 4 · ·

4 ···» • 4 · · ·

4« « ·

Fractions having a molecular weight of 4 or 100,000 to 150,000 Daltons, or homologues, analogs, derivatives, complexes, esters, fragments and / or subunits of hyaluronic acid and / or combinations thereof, and HA mimicking molecules mentioned above imitates the hyaluronic acid forms in the sense that they are capable of binding to the same receptors as the hyaluronic acid form, the component modifying the cellular activity of tissues and / or cells in the human body that express a molecule that functions as a high affinity cell surface receptor for hyaluronic acid, such as an adhesion or regulatory molecule; and iii) a pharmaceutically acceptable carrier (e.g., sterile water);

wherein component (ii) is in such a form that when administered in a dosage amount of the pharmaceutical composition, component (ii) is capable of modifying the cellular activity of tissues and cells which, for example, have an adhesion molecule (e.g., ICAM-1, HARLEC- et CD44et) and a regulatory molecule (RHAMM), wherein the modification of cellular activity is based on the fact that component (ii) binds to a receptor for the hyaluronic acid form and / or the HA mimicking molecules, and component (ii) is immediately capable of transporting component (i) to the body or skin , ha. component ii) is a hyaluronic acid for * ··· ··

- 24 ma.

Suitable active ingredients include, but are not limited to, the following: free radical scavengers [e.g. ascorbic acid (vitamin C)], anticancer agents, chemotherapeutic agents, antiviral agents such as a nonionic surfactant such as nonoxyno-1-9 (nonylphenoxy polyethoxyethanol) in the Delien ™ contraceptive cream. , anionic surfactants such as cetylpyridinium chloride, cationic surfactants such as benzalkonium chloride, non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, naproxen, diclofenac and (+/-) - ketorolac tromethamine salt sold under the trademark Toadol ™, steroidal anti-inflammatory drugs, antifungal agents, detoxifying agents such as enema, rectal anesthetics, antibacterial agents, bronchodilator antibiotics, agents for the treatment of vascular ischaemia (such as diabetes and Berger's disease), antibody monoclonal agents, topical agents, minoxidil, diuretics such as furosemide under the trademark Lasix ™, immunosuppressants such as cyclosporins, lymphokines such as interleukin-2, etc., interferon alpha and beta, and the like.

Dosage amounts of the pharmaceutical compositions may be stored in multi-dose containers, from which the individual dosage amount may be withdrawn.

The invention is illustrated by the accompanying drawings.

Figure 1 a) CCI 218 tumor immunohistochemical dye • ·

25 shows the use of polyclonal antibodies to the HARLEC HA receptor. Figure 1 (b) depicts a control obtained using preimmune antibodies. (See Materials and Methods for details).

Figure 2 shows the clearance of radioactivity from the blood following intravenous injection of 125 µg of IT-HA into rats. Inner graph: Radioactivity in various organs of rat 10 minutes after intravenous injection of 5 µg IT-HA into the rat. KC is a Kuppfer cell; PC is a parenchymal cell; and LEEC is a liver endothelial cell.

Figure 3 shows a scintigraphic view of a naked rat 3 weeks after a CCL in one of the rats

218 human colon cancer was inoculated and 24 hours after injection of 1.5 mg HA containing 2.5 MBq IT-HA into the rat. In Figure 3 (b), the liver is covered with a lead shield. The arrow points to the tumor.

Figure 4 a) depicts immunohistochemical staining of a CCI 218 tumor after intravenous IT-HA injection using polyclonal antibodies to the HARLEC HA receptor.

Figure 4 (b) shows immunohistochemical staining 4 hours after treatment with hyaluronidase.

Figure 4 (c) and (d) are the same as Figure (a) and (b), but preimmune (control) antibodies were used in these cases.

Figure 4 (e) and (f) are the tumors according to (a) to (d)

- 26 shows the staining of HA using biotinylated HA binding protein.

Figure 5 (a) shows a scintigraphic image of a naked rat carrying a CCI 218 tumor that was 3.5 gg (0.75 MBq) intradumoral one day prior to capture. ¹²⁵Received IT-HA.

Figure 5 (b) shows a scintigraphic image of a naked rat carrying a CCI 218 tumor at 3.5 gg (0.75 g) intraperitoneally 15 days prior to capture.

MBq) ¹²⁵Received IT-HA.

125

Figure 6 shows the inhibition of IT-HA and LÉC cell association in culture and at 37 ° C. Results are mean ± standard deviation values; n = 3 (for control medium) and n = 6 (for 1A29 medium). (See Materials and Methods for details).

Figure 7 shows the depletion of radioactivity in tumor tissue and control muscle tissue 18-20 hours after IT injection of 2 mg IT-HA. Results are mean ± standard deviation values; n = 3. (See Materials and Methods for details).

Figure 8 shows frozen sections of tumor tissue stained for HA and HARLEC / ICAM-1, 18-20 hours after IT-HA by intravenous injection. (See Materials and Methods for details).

8th Figure a): HA staining;

8th b): staining for HARLEC / ICAM-1;

• · • · · « · · ·· ·· ··· · • «···· · · ·· · ··· ···

Figure 8 c) Staining to HARLEC / ICAM-1 after treatment with hyaluronidase.

Further details of the present invention will be provided by the following examples^-we introduce it.

First EXAMPLE

Hyaluronan (hyaluronic acid; HA or HYA) polysaccharide is rapidly cleared from the circulation, primarily by endothelial cells of the liver, via receptor-mediated endocytosis. The HA receptor on these cells has already been characterized; the receptor was purified from rat liver endothelial cells (LEC) and raised polyclonal antibodies. Other cell surface receptors for HA have also been described previously, such as the lymphocyte directing CD44 receptor and the HA-mediated motility receptor (RHAMM) of fibroblasts. HA tumors have been reported to be enriched in many tumors, and HA binding sites on cells derived from such tumors have also been described. Previous observations have shown that transformed tissues such as murine mastocytomas and human breast carcinomas give positive staining for the 'HA receptors originally found on liver endothelial cells.

The present work was started to determine whether HA binding sites are available in vivo in tumor tissue, and to determine the relationship between these potential sites and the HA binding proteins described above.

- 28 MATERIALS AND METHODS

polysaccharides

HA for labeling, uptake and cycle assays was extracted from avian tissue (Hyal Pharmaceutical Corporation, Toronto, Ontario, Canada). The molecular weight distribution of HA was determined by chromatography on a calibrated Sephacryl HR column of porosity 400, 1000 and 2000 (Pharmacia, Uppsala, Sweden), 0.25 M sodium chloride, 0.05% chlorobutanol.

The HA content of each fraction was monitored by measuring the absorbance at 214 nanometers. Radioactivity was measured by gamma counting on a Packard automotive gamma counter.

(The average molecular weight is about 450,000 daltons (derived from 500,000 to 800,000 medium molecular weight powders)).

Indication of HA

Following the activation of the polysaccharide in bromocyano by HA, the method of Glabe et al., Preparation and Properties of Fluorescent Polysaccharides, Anal. Biochem. 130: 287-294 (1983)] with DL-tyrosine (Sigma Chemical

Company, St. Louis, Missouri, USA). 15 mg of HA were then activated at pH 11 with 8 mg of bromocyanine for 1 minute. The activated polysaccharide was equilibrated with Sephadex G25 (PD 10, 0.2 M borate buffer, pH 8.0).

Pharmacia, Uppsala, Sweden) was isolated from the reaction mixture by a short column. Activated HA was incubated overnight with 1 mg tyrosine (T). Tyrosine was bound to HA (T-HA) and then T-HA was separated from unbound tyrosine;

• · ·· ·· ··· · • · · · · « * ·· · · · · · · ·

- 29 this separation was performed on a PD10 column previously prepared with 8 g / l sodium chloride, 0.2 g / l potassium chloride, 0.2 g / l potassium dihydrogen phosphate and 1.15 g / l disodium equilibrated with phosphate buffered saline (pH 7.5) containing phosphate buffered saline (PBS) containing hydrogen phosphate.

The T-HA. part of it was iodinated with I. 100 µg T-HA and 0.5 mCi I were placed in a small test tube and then 10 µg 1,3,4,6-tetrachloro-3a, 6a-diphenylglycouryl (Sigma Chemical Company, St. Louis, Missouri, USA). It is not built¹²⁵I was removed on a PD10 column equilibrated with PBS. The iodinated THA (¹²⁵IT-HA) was stored at 5 ° C. The specific radioactivity is usually 1500-5000 dpm / ng.

125

IT-HA retained its higher molecular weight profile during gel-permeation chromatography. We found that the 125

IT-HA was cleared from the circulation by the same kinetics and organ distribution as described previously for high molecular weight HA biosynthetically labeled. Isolated rat liver endothelial cells took up 125 of the polysaccharide labeled in vivo and in vitro, indicating that binding did not affect binding to specific cell surface receptors on these cells (1,2).

immune Painting

All staining was performed using the ABC-elite method (Vectastain® Elite ABC). Frozen sections of 6 gm were prepared and the sections were placed on glass slides and filled with gelatine • · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

30-chromalun mixture {gelatin-chromalun mixture was prepared so that 0.5 g of chromalun [KCr (S0₄) ₂^X12H₂A mixture of O 2 and 750 ml of distilled water or 5 g of gelatin and 250 ml of distilled water were heated to 56 ° C each and the two solutions were then mixed together. To remove non-specific binding to serum proteins, antiserum and preimmune serum were adsorbed on rat serum protein-coupled (6 mg protein / ml gel) Sepharose® 4B gel (Pharmacia LKB Technology, Uppsala, Sweden). The coupling was performed according to the manufacturer's specifications (Affinity Chromatography; principles &amp; methods; Pharmacia LKB Technology, Uppsala, Sweden). Equal volumes of serum and gel were mixed and the resulting mixture was incubated for 6 hours at 4 ° C. Frozen sections were fixed in cold methanol for 10 minutes, dried for 10 minutes and then washed with phosphate buffered saline (PBS). The peroxidase was blocked with 0.3% methanolic hydrogen peroxide solution and the sections were washed repeatedly in PBS. An avidin / biotin blocking kit (Vector laboratories) was used to block endogenous biotin and biotin binding activity. The sections (S) were then in 4% goat serum in PBS (Vectastain

Elite ABC) were incubated for 30 minutes. HARLEC antiserum was diluted 1: 300 with PBS containing 4% goat serum and incubated for one hour. The sections were washed with PBS and then diluted with PBS in a 1: 200 dilution with a second goat anti-rabbit antibody (Vectastain Elite ABC) for 30 minutes. · · ·· · · · ·

- 31 incubated. After incubation with the second antibody, sections were washed and incubated with ABC-Elite Complex (Vectastain Elite ABC). For color development, a solution of 10 mg of 3-amino-9-ethylcarbazole in 6 ml of dimethyl sulfoxide (DMSO) was mixed with 45 ml of 15 mM acetate buffer (pH 5) and 4 μΐ Perhydroll (Merck), and the mixture was stirred for 7.5 minutes. incubations were performed. After washing the sections

Mayer was stained with hematoxylin. The glass slides were placed in Kelser glycerol gelatin (Merck).

Treatment of sections with hyaluronidase

After methanol fixation and washing in PBS, sections were incubated at 37 ° C for 2 hours with 5 U / ml Streptomyces hyaluronidase (Amano Pharmaceutical Co., Ltd, Japan), 1 mg / ml pepstatin (Sigma Chemical). Company, St. Louis, Missouri, United States), 0.1 M W ethyl ethyl maleimide, 0.1 M EDTA (Merck), and 5 KIE / ml

Trasylol (Bayer). Subsequently, we followed the usual staining procedures.

Examination of in vivo recording

14 to 30 days prior to the experiments, under ether anesthesia, four female naked (Nu / Nu) rats weighing 200 to 250 g were exposed to the CCL 218 human cell line (American β) in one hind paw.

Approximately 3x10 cells of Tissue Type Collection) were inoculated subcutaneously (in 0.5 mL RPMI medium). The animals were anesthetized with 45 mg / kg body weight pentobarbital, then applied to the tail vein in 2 or directly into approximately 1-2 cm tumors at 3.4-1500 pg (5-15χ10 pg).⁶ cpm) ¹²⁵IT-HA was injected. The specific • · ·

- Unlabeled HA was also occasionally administered to reduce 32 activity and to achieve a higher chemical content of HA (0.05-1.0 mL of 0.15 M sodium chloride and 10 mM sodium dihydrogen phosphate, pH 7.4).

After the test, the animals were sacrificed and the tumor and organs were analyzed for radioactivity. Data were processed on a Macintosh SE / 30 or a Macintosh Ilsi computer (Apple Computer Inc., Cupertion, California, USA) and charted using Cricket Graph (version 1.3, Cricket Software, Malvern, Pennsylvania, USA) and a Canvas (3.0.2. version, Deneba Systems Inc., Miami, Florida, USA).

Scintigraphic examinations

The rats were anesthetized and injected as described above. In dynamic assays, rats were injected on a standard medium resolution collimator of the gamma camera. Images were captured with a Gamma 11 system (Philips) with a 34 keV and 80% window setting, in word mode, in a 64x64 pixel (pixel) matrix. The dynamic sequence was preset at 1 fpm. Fifteen minutes later, still images were taken at various times after injection. The images were then transferred to the Hermes system (Nuclear Diagnostics, Hagersten, Sweden) and the regions of interest (ROI: 6) were drawn. In some cases, the area may be larger than the tumor or liver due to scattering

- 33 blanket ROIs had to be drawn.

RESULTS

Immunohistochemical studies have shown that polyclonal antibodies to HARLEC recognize structures in the tumors of the CCL 218 human colon cancer cell line in nude rats. Staining was mainly localized in vascular endothelium and tumor cells, where it was enhanced towards the edge of the tumor [1. Preimmune antibodies showed virtually no staining [Fig. 1 a]. Figure b), which indicated that HARLEC staining was specific to this system.

125

When trace amounts of IT-HA were injected intravenously into the animals, radioactivity was rapidly transferred from the blood to the liver and, after a few minutes, only a small amount of radioactivity remained in the blood (Figure 2).

Because of the high binding capacity of the liver, few tracers were left in the blood after passing through the liver, and only very small amounts could reach other tissues. As a result, the animals were injected with amounts in excess of the hepatic binding capacity. At a dose of 1.5 mg HA, the substance was still present in the general circulation after 24 hours and, according to a scintigraphic examination, radioactivity accumulated in the tumors (Figure 3). Most of the radioactivity was detected above the liver, but significant background activity was also observed above the tumor [3. Figure a)]. The activity above the tumor was many times higher than would have been expected by the tumor size alone. Covering the liver with lead radiation shields, scintigraphic images were obtained that showed 50% of the liver today. · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··

34 x levels were well matched to tumor size [Fig. 3 Figure b).

Immunohistochemical analysis of treated animals showed that treatment of sections with hyaluronidase caused a specific increase in HARLEC immunoreactivity [4. Figures a) and b) without increasing the preimmune response [Fig. 4 (a) and (b)]. Figures c) and d)]. Extensive receptor staining coincides with staining for HA [4. (e) and (f)], HA staining was most pronounced in the marginal zone as well as in and around the vessels, although other receptor stained structures were found to be positive for HA.

In order to reduce hepatic uptake and to attempt a therapeutic response, two animals received a single mtratumoral IT-HA injection. One rat received 3.4 pg of high specific activity (0.25 MBq / gg) labeled ΗΆ, more than 90% of which remained in the tumor. Small amounts reached the general circulation, which was then taken up by the liver [Fig. 5 Figure a)]. Fifteen days after injection, the ratio of tumor to liver radioactivity was similar to that observed immediately after injection [5. Figure b). The animal's response was to reduce the size of the tumor from about 2 cm to about 1 cm within 7 days. The tumor gradually diminished and softened until day 14, when the tumor was no longer palpable. The 15th. day, the animal was sacrificed, and subsequent histochemical analysis showed that the tumor was approximately

He was always present at a size of 0.5 cm. The other rat, although 250 µg, had only 0.025 MBq of HA

- 35 received intratumoral, no response, and approximately 5 cm tumor was found in the animal's body at the time of killing (two rats killed at the same time).

DISCUSSION

The fact that tumors untreated with hyaluronidase had a clear immunohistochemical staining of HAR'LEC (Figure 1) indicated that the binding sites were to some extent free, that is, that there was potential for uptake of circulating HA. This observation is consistent with that observed for liver receptors.

Second EXAMPLE

HA (hyaluronic acid and its pharmaceutically acceptable salts) is eliminated from the bloodstream by receptor mediated endocytosis primarily by hepatic endothelial cells (LECs). The HA receptor on the LEC (HAR) was characterized by HA affinity chromatography of the surface labeled rat LEC and purified from LEC membranes to homogeneity. This receptor (HARLEC for short) is in the range of 85 kD to 100 kD with an average molecular weight of about 90 kD and about

It has a pl value of 6.7. A monospecific polyclonal antibody (in short, anti-HARLEC) has been developed that is capable of inhibiting the binding of HA to LEC and LEC membranes. Using this antibody, 85-90 kD protein specimens have also been detected with the antibody in kidney, spleen, thymus and lymph nodes. The same tissues - with the liver - are anti-· · ·

- 36 -HARLECs give positive staining, immunohistochemical staining is mainly restricted to vascular regions such as the sinusoids of the liver, spleen and lymph nodes, as well as to the capillaries of the small intestine, although it appears in some specific structures such as thymicus reticular cells . CEC staining is inhibited if the corneas are treated with HA prior to staining, while staining intensity can be restored by treatment with hyaluronidase.

HARLEC was purified from rat rat liver by a series of affinity chromatography steps. In the final step, virtually all HA bound to HA-Sepharose, and specific elution using HA oligosaccharides was performed. Trypsin digestion of purified HARLEC resulted in several peptides which were separated by reverse phase chromatography. For four peptides, the sequence was determined and found to be identical

Intracellular adhesion molecule-1 (ICAM-1). ICAM-1 is a glycosylated, single-chain 80-114 kD protein having a 55 kD polypeptide nucleus. It is normally expressed only in small amounts, but ICAM-1 has already been detected in the normal liver endothelium of the sinusoids and in the endothelium of lymph nodes, spleen and certain kidneys, and corneal endothelial cells. The immunohistochemical localization of ICAM-1 is in good agreement with known results previously obtained with HARLEC staining. ICAM-1 is also localized in tissues in which the former • · · • · · · ····

- 37 banana HA binding and HA uptake were detected.

HARLEC / ICAM-1 is expressed on endothelium in tumor mastocytomas of mice and is capable of binding to intravenously radiolabelled HA. Significant increases (approximately 5 times the control) in tumor tissue were observed.

During immunohistochemical examination, we found that HA is localized in areas that also give positive staining for ICAM-1, i.e., mainly in blood vessels. After treatment of the sections with hyaluronidase, ICAM-1 immunoreactivity increased dramatically.

Further evidence of the HA receptor of HARLEC / ICAM-1 is that HA also targets human tumors in nude rats and that control is mainly through binding to HARLEC / ICAM-1 on the tumor endothelium.

MATERIALS AND METHODS

polysaccharides

HA for labeling, uptake and cycle assays was extracted from avian tissue (Hyal Pharmaceutical Corporation, Toronto, Ontario, Canada). The average molecular weight was approximately 450,000 daltons.

Indication of HA

HA was labeled with DL-tyrosine (Sigma Chemical Company, St. Louis, Missouri, USA).

• ·

<img file="HUT76846A_D0003.tif" />

- 38 Monoclonal antibodies

Rat monoclonal antibody to ICAM-1 (clone 1A29) was obtained as a gift from Professor M. Miyasaka (Osaka University, Japan).

Cultivation of endithelial cells

Rat liver endothelial cells (LEC) were isolated after perfusion of rat liver collagenase and cultured in serum-free RPMI medium.

Antibody inhibition experiments

100 Between 000 and 200,000 liver endothelial cells cultured for 4-5 hours were incubated with 0.5 μg / ml IT-HA, whereas in competing experiments, unlabeled HA or 1: 2 diluted anti-ICAM-1 supernatant medium was used. incubation. Since t-anti-ICAM-1 contained 115 ng / ml HA, a control medium containing 115 ng / ml HA was also prepared and used at a 1: 2 dilution as a control to inhibit HA-related binding activity in the antibody supernatant. . Competitors were added prior to the addition of radiolabeled HA. Total incubation time was 35-40 minutes. After incubation was completed, the media was removed, the cells were washed, and the radioactivity was analyzed as described previously (10).

immune Painting

All staining was done using the ABC-elite method (Vectastain Elite ABC). Frozen sections of 6 μιη were prepared, and the sections were placed on glass slides and coated with a gelatin-chromalun mixture. Serum Protein Link • «· · · ·

- HARLEC antiserum was adsorbed on Sepharose 4B gel (Pharmacia LKB Technology, Uppsala, Sweden) coupled with rat serum proteins (6 mg protein / ml gel) to remove 39 dots of non-specific binding.

Frozen sections were fixed in cold methanol for 10 minutes, dried for 10 minutes, and then washed with phosphate-buffered saline (PBS). The peroxidase was blocked with 0.3% methanolic hydrogen peroxide solution and the sections were washed again in PBS. An avidin / biotin blocking kit (Vector laboratories) was used to block endogenous biotin and biotin binding activity. The sections were then incubated in PBS (Vectastain® Elite ABC) containing 4% goat serum for 30 minutes. HARLEC antiserum was diluted 1: 300 with PBS containing 4% goat serum and incubated for one hour. The sections were washed with PBS and incubated with PBS diluted 1: 200 in goat anti-rabbit antibody (Vectastain Elite ABC) for 30 minutes.

HA staining was performed on frozen sections of cartilage frozen biotinylated hyaluronan binding proteins (b-HABP) treated with methanol and avidin / biotin blocking kit (Vector Laboratories) to block endogenous biotin binding activity.

After incubation with the second antibody or b-HABP, sections were washed and incubated with ABC-Elite Complex (Vectastain Elite ABC). For the development of color, a solution containing 3,3-diaraobenzidine or 3-amino-9-ethylcarbazole • · · ···

40 sets of oxidase substrates (Vector Laboratories) were used and sections were incubated for 5-10 minutes in the mixture. After washing, sections were re-stained with Mayer hematoxylin for 1.5 minutes. The glass slides were placed in Kelser glycerol gelatin (Merck).

Treatment of sections with hyaluronidase

After methanol fixation and washing in PBS, sections were incubated at 37 ° C for 2 hours with 5 U / ml Streptomyces hyaluronidase (Amano Pharmaceutical Co., Ltd, Japan), 1.8 µg / ml pepstatin ( Sigma Chemical Company, St. Louis, Missouri, United States), 1.8 mM EDTA (Merck), 1.8 µg / ml soybean trypsin inhibitor (Sigma), 2.0 mM iodoacetic acid (Sigma), 0, 18 mM 6-amino-n-hexanoic acid (Sigma) and 9.0 mM benzamide (Sigma). Subsequently, we followed the usual staining procedures.

Examination of in vivo recording

14 to 30 days prior to the experiments, under ether anesthesia, CCL 218 human cell line z in one hind leg of approximately 200 g of female bare (Rowette Nu / Nu) rats was:

(American Tissue Type Collection) approximately 3x10 cells were inoculated subcutaneously [in μΐ s-MEM medium (Gibco)]. The animals were anesthetized and then injected intravenously with 2 mg (2-3x10⁶ cpm) ¹²⁵The animals received IT-HA. From time to time, unlabeled HA (0.05-1.0 mL of 0.15 M sodium chloride and 10 mM sodium dihydrogen phosphate (pH) was also administered to reduce specific activity and to achieve a higher chemical content of HA). 7.4).

·· * ··»» «· • · · · · · • · ···» · ·

The animals were sacrificed after 18-20 hours and examined for radioactivity as described above (9) for tumors and organs.

RESULTS

To further characterize HA HARLEC / ICAM-1 binding, experiments were performed with isolated rat liver endothelial cells (LEC), radiolabeled HA, and a well-characterized rat

With a monoclonal antibody to ICAM-1 [Ialenti, A. and Di

Rosa, M., Hyaluronic acid modulates acute and chronic infiam1? R matlon, Agents Actions, £ 3, 44-47]. The cell association between IT-HA and LEC at 37 ° C was inhibited by approximately 60% (Figure 6). However, approximately 20% of the inhibition was derived from HA in the antibody tissue culture medium, as indicated by the inhibition seen in a control experiment using the same amount of HA but without the antibody (Figure 6).

In order to examine whether HARLEC / ICAM-1 expressed on endothelial cells of tissues other than rat has the same HA binding capacity as was observed for LEC, one of the hind legs has human tissue distribution in human colon colon carcinoma. It was found that 18-20 hours after the intravenous injection of 2 mg IT-HA, most of the radioactivity was as expected in the liver, but only a very small amount of radioactivity in the blood (not shown). The radioactivity in the tumor was more than three times that of the control muscle tissue (wet weight

- 42 • · · »· · · · *« · · · · · · · · · · ·················. This increase was statistically significant (p = 0.001, n = 3) (Figure 7).

When tumor tissue was analyzed by histochemical methods, it was found that HA in the tumor was mainly found in the tumor connective tissue skeleton (stroma) and blood vessels [8. Figure a)], whereas ICAM-1 is predominantly localized in blood vessels in the same areas [Figure 8 Figure b). ICAM-1 staining, although clearly visible, was surprisingly poor [Fig. 8 Figure b). However, staining was dramatically enhanced after treatment with hyaluronidase [8. Figure c)].

DISCUSSION

The observation that rat ICAM-1 monoclonal antibodies are capable of inhibiting the cellular association of labeled HA and cultured rat liver endothelial cells provides further evidence that ICAM-1 is an essential cell surface receptor for HA. Although inhibition is only about 50%, however, since cellular association was studied at 37 ° C, inhibition was not complete due to the continuous binding of free receptors from within the cells. High molecular weight HA exhibits an extremely high affinity for receptors on the liver endothelial cell, and the labeled HA is expected to be a major competitor for binding to free receptor sites. It should also be noted that other HA binding sites not affected by 1A29 may be present on ICAM-1.

Monoclonal antibodies used in the assay have previously been shown to inhibit leukocytes in the liver.

- 43 ································································································································································································································································································· · Because antibodies cause inhibition of binding between HA and hepatic endothelial cells, HA binds to a site close to the leukocyte binding site on ICAM-1. Binding of HA to endothelial cells inhibits the adhesion of leukocytes to the same cells. Thus, systemic administration of HA may have a beneficial effect on inflammatory conditions. Similar effects to the anti-inflammatory effects achieved by systemic treatments with ICAM-1 antibodies have been described previously in animal models of acute and chronic inflammation.

Increased uptake of radiolabeled intravenously administered HA into tumor tissues (Figure 7) indicates that the tumor has structures for binding HA. HA staining with highly specific biotinylated HA binding protein indicated that HA was localized in and around blood vessels. As expected, HARLEC / ICAM-1 staining was also found in blood vessels, although it was significantly weaker [8. Figure b). Application of HA to corneal endothelial cells (CEC) resulted in reduced immunohistochemical staining of CEC receptors, which, in turn, has been shown to be reversible by treatment with hyaluronidase. In view of this, we have attempted to perform such hyaluronidase treatment on tumor sections. As a result, a

HARLEC / ICAM-1 staining, indicating that HA bound to HARLEC / ICAM-1 caused inhibition of specific antibody binding.

Intravenously administered HA binds predominantly to endothelial cells via ICAM-1. This suggests that HA

<img file="HUT76846A_D0004.tif" />

as a carrier for delivering cytotoxic drugs to tumors expressing this type of HA receptor.

In the preferred embodiments of the invention described above, many changes can be made within the scope of the invention. It is to be understood that the materials used in the above-described embodiments are illustrative only and are not intended to limit the scope of the invention in any way.