Targeting vector-phospholipid conjugates

191850/2

FIELD OF THE INVENTION

[0002] The present invention relates to targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates, which are useful in. therapeutic anddiagnostic compositions and methods of preparation of tlie same. The invention includestargeted ultrasound contrast agents, and particularly targeted microbubbles which includesuch targeting vector-phospholipid conjugates.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis, tlie formation of new blood vessels, occurs not only during embiyonic development and normal tissue growth and repair, but is also involved in thefemale reproductive cycle, establishment and maintenance of pregnancy, and repair ofwounds and fractures. In addition to angiogenesis that occurs in the normal individual,angiogenic events are involved in a number of pathological processes, notably tumor growthand metastasis, and other conditions in which blood vessel proliferation is increased, such asdiabetic retinopathy, psoriasis and arthropathies. In addition, angiogenesis is important in thetransition of a tumor from hypeiplastic to neoplastic growth. Consequently, inhibition ofangiogenesis has become an active cancer therapy research field.

[00041 Tumor-induced angiogenesis is thought to depend on the production of pro- angiogenic growth factors by the tumor cells, which overcome other forces that tend to keepexisting vessels quiescent and stable. The best characterized of these pro-angiogenic agentsor growth factors is vascular endothelial growth factor (VEGF) (Cohen et al., FASEB J., 13: 1 191850/2 9-22 (1999)). VEGF is produced naturally by a variety of cell types in response to hypoxiaand some other stimuli. Many tumors also produce large amounts of VEGF, and/or inducenearby stromal cells to make VEGF (Fukumura et al., Cell, 94: 715-725 (1998)). VEGF, alsoreferred to as VEGF-A, is synthesized as five different splice isoforms of 121, 145, 165, 189,and 206 amino acids. VEGF121 and VEGF165 are the main forms produced, particularly intumors (see Cohen et al. 1999, supra). VEGF121 lacks a basic domain encoded by exons 6and 7 of the VEGF gene and does not bind to heparin or extracellular matrix, unlike VEGFiss.

[0005] VEGF family members act primarily by binding to receptor tyrosine kinases.

In general, receptor tyrosine kinases are glycoproteins having an extracellular domain capableof binding one or more specific growth factors, a transmembrane domain (usually an alphahelix), a juxtamembrane domain (where the receptor may be regulated, e.g., by phosphorylation), a tyrosine kinase domain (the catalytic component of the receptor), and acarboxy-terminal tail, which in many receptors is involved in recognition and binding ofthesubstrates for the tyrosine kinase. There are three endothelial cell-specific receptor tyrosinekinases known to bind VEGF: VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), and VEGFR-3(Flt4). Flt-1 and KDR (also known as VEGFR-2 or Flk-1, which are used interchangeablyherein) have been identified as the primary high affinity VEGF receptors. While Flt-1 hashigher affinity for VEGF, KDR displays more abundant endothelial cell expression (Bikfalviet al., J. Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to dominate theangiogenic response and is therefore of greater therapeutic and diagnostic interest (see Cohenet al. 1999, supra). Expression of KDR is highly upregulated in angiogenic vessels,especially in tumors that induce a strong angiogenic response (Veikkola et al., Cancer Res.,60:203-212 (2000)). The critical role of KDR in angiogenesis is highlighted by the complete 2 WO 2007/067979 PCT/US2006/061793 lack of vascular development in homozygous KDR knockout mouse embryos (Folkman et al.,Cancer Medicine, 5th Edition (B.C. Decker Inc.; Ontario, Canada, 2000) pp. 132-152).

[0006] KDR (kinase domain region) is made up of 1336 amino acids in its mature form. The glycosylated form of KDR migrates on an SDS-PAGE gel with an apparentmolecular weight of about 205 kDa. KDR contains seven immunoglobulin-like domains inits extracellular domain, of which the first three are the most important in VEGF binding(Cohen et al. 1999, supra). VEGF itself is a homodimer capable of binding to two KDRmolecules simultaneously. The result is that two KDR molecules become dimerized uponbinding and autophosphorylate, becoming much more active. The increased kinase activityin turn initiates a signaling pathway that mediates the KDR-specific biological effects of VEGF.

[0007] Thus, not only is the VEGF binding activity of KDR in vivo critical to angiogenesis, but the ability to detect KDR upregulation on endothelial cells or to detectVEGF/KDR binding complexes would be extremely beneficial in detecting or monitoringangiogenesis.

[0008] It is well known that gas filled ultrasound contrast agents are exceptionally efficient ultrasound reflectors for echography. Such ultrasound contrast agents include, forexample, gas-filled microvesicles such as gas-filled microbubbles and gas filledmicroballoons. Gas filled microbubbles are particularly preferred ultrasound contrast agents.(In this disclosure the term of "microbubble" specifically designates a gaseous bubblesurrounded or stabilized by phospholipids). For instance injecting into the bloodstream ofliving bodies suspensions of air- or gas-filled microbubbles in a carrier liquid will stronglyreinforce ultrasonic echography imaging, thus aiding in the visualization of internalanatomical structures. Imaging of vessels and internal organs can strongly help in medicaldiagnosis, for instance for the detection of neoplastic, cardiovascular and other diseases. 3 191850/1 [0009] For both diagnostic and therapeutic purposes it would be particularly beneficial to incorporate into gas filled ultrasound contrast agents, targeting vectorcompositions which exhibit high binding affinity for a desired target (such as, forexample, KDR or the VEGF/KDR complex). For example, targeting vector -phospholipid conjugates and particularly targeting peptide-phospholipid conjugates maybe used to prepare targeted, gas filled ultrasound contrast agents. In addition, it would beparticularly beneficial to have methods for large scale production of highly purified formsof such targeting vector - phospholipid conjugates. Such compositions and methodswould allow for production of compositions for use in diagnostic or therapeuticapplications such as, for example, precise targeting of reporter moieties, tumoricidalagents or angiogenesis inhibitors to the target site.

[0009a] US 20050100963 provides polypeptides, peptide dimer, and multimericcomplexes comprising at least one binding moiety for KDR or VEGF/KDR complex,which have a variety of uses wherever treating, detecting, isolating or localizingangiogenesis is advantageous. Particularly disclosed are synthetic, isolated polypeptidescapable of binding KDR or VEGF/KDR complex with high affinity (e.g., having aΚο<1μΜ), and dimer and multimeric constructs comprising these polypeptides.

[0009b] US 20050250700 provides, inter alia, peptides, peptide dimer, andmultimeric complexes comprising at least one binding moiety for KDR or VEGF/KDRcomplex, which have a variety of uses wherever treating, detecting, isolating or localizingangiogenesis is advantageous. Particularly disclosed are synthetic, isolated polypeptidescapable of binding KDR or VEGF/KDR complex with high affinity (e.g., having aΚβ<1μΜ), and dimer and multimeric constructs comprising these polypeptides.

SUMMARY OF THE INVENTION

[0009c] The present invention provides a peptide-phospholipid conjugate selectedfrom the group consisting of: 3a 191850/1

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3b 191850/1

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3c 191850/1

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[0009d] The present invention also provides an ultrasound contrast agentcomposition comprising a conjugate as described above, wherein the contrast agentcomprises a gas-filled microvesicle.

[0009e] The present invention also provides a method for imaging KDR-containing tissue in a mammal, for detecting or imaging angiogenic processes in amammal and for detecting or imaging tumor tissue containing KDR in a mammalcomprising administering an effective amount of a composition described in [0009d] tothe mammal and imaging the mammal.

[0009f] The present invention also provides a ultrasound contrast agent composition comprising one or more monomers selected from the group consisting ofAc-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Ar- g-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2 (SEQ ID NO 5); Ac-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-Hi- s-Ala-Phe-Leu-Ser 3d 191850/2 -Gly-Gly-Gly-Gly-Gly-Lys-NH2 (SEQ ID NO 6); Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Th- r-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic(6-13) disulfide (SEQ ID NO 7), and Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gl- y-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12)disulfide (SEQ ID NO 8).

[0009g] The present invention also provides a method for imaging KDR-containing tissue in a mammal, for detecting or imaging angiogenic processes in amammal and for detecting or imaging tumor tissue containing KDR in a mammalcomprising administering an effective amount of a composition described in [0009f] tothe mammal and imaging the mammal. ADDITIONAL ASPECTS OF THE APPLICATION[0010] The present invention provides targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates which are useful in thepreparation of gas filled ultrasound contrast agents. In a preferred embodiment thetargeting peptide-phospholipid conjugates include targeting peptides which exhibit highKDR binding affinity and thus are useful components of contrast agents for imaging ofangiogenesis processes.

[0011] The present invention also provides monomelic and dimeric peptidephospholipid conjugates (also referred to herein as lipopeptides) which are useful inpreparing gas filled ultrasound contrast agents, and particularly in preparing ultrasoundcontrast agents which target KDR and may be used for imaging of angiogenesisprocesses.

[0012] The present invention also provides methods and processes for the largescale production of highly pure monomelic and dimeric peptide phospholipid conjugates,particularly monomelic and dimeric peptide phospholipids conjugates having high KDRbinding affinity. 4 WO 2007/067979 PCT/US2006/061793 [0013] The present invention also provides methods and processes for the large scale production of highly pure dimeric peptide phospholipid conjugates having minimal levels oftriflouroacetic acid (TFA).

[0014] The present invention also provides methods for synthesizing monomeric peptides in high purity and the construction of peptide phospholipid conjuages from multiplepeptide sub-units.

[0015] The present invention also provides monomeric peptides which bind KDR or the VEGF/KDR complex with high affinity, as well as methods of synthesizing and usingsuch monomeric peptides.

[0016] The present invention also provides targeted ultrasound contrast agents prepared from such targeting vector-phospholipid conjugates. Such targeted ultrasoundcontrast agents are useful for imaging target-bearing tissue. In a preferred embodiment, thetargeted ultrasound contrast agents are targeted microbubbles and the targeting vector-phospholipid conjugates include targeting peptides which exhibit high KDR binding affinityand thus are useful components of contrast agents for imaging KDR-bearing tissue andparticularly for imaging of tumors and angiogenesis processes. Methods of preparing andusing such targeted ultrasound contrast agents are also provided.

BRTEF DESCRIPTION OF THE DRAWINGS

[0017] FIGURE 1 illustrates a method for the production of a monomeric peptide phospholipid conjugate (1) from a linear peptide monomer (2).

[0018J FIGURE 2 illustrates a monomeric peptide phospholipid conjugate (1) including a peptide with high binding affinity for KDR.

[0019] FIGURE 3 illustrates a method for the production of a precursor dimer peptide (16) from peptide monomers. 5 WO 2007/067979 PCT/US2006/061793 [0020] FIGURE 4 illustrates a method for the conjugation of the precursor dimer peptide shown in Figure 1 to DSPE-PEG2000-NH2 to form a dimeric peptide phospholipidconjugate (11) containing peptides which bind with high affinity to KDR.

[0021] FIGURE 5 illustrates a dimeric peptide-phospholipid conjugate (11) containing peptides which bind with high affinity to KDR.

[0022] FIGURE 6 illustrates a method for the production of dimer peptide- phospholipid conjugates (such as (21)) having minimal levels of TFA.

[0023] FIGURE 7 illustrates another method for the production of dimer peptide- phospholipid conjugates (such as (21)) having minimal levels of TFA.

[0024] FIGURE 8 illustrates another method for the production of dimer peptide- phospholipid conjugates having minimal levels of TFA.

[0025] FIGURE 9 illustrates another representative monomeric peptide (32) having a high binding affinity for KDR.

[0026] FIGURE 10 illustrates anothermonomeric peptide-phospholipid conjugate (31) which includes the monomeric peptide shown in FIGURE 9.

[0027] FIGURES 11A-C show images obtained by using the dimer peptide- phospholipid conjugate (11) (shown in FIGURE 52) in a contrast agent at: 1) baseline (FIG. 11 A); 2) after 25 minutes (FIG. 11B); and 3) after subtraction of the baseline and freecirculating bubbles (FIG. 11C).

[0028] FIGURES 12A-C show images obtained by using the monomeric phospholipid peptide conjugate (1) (shown in FIGURE 2) in a contrast agent at baseline(FIG. 12A); after 25 minutes (FIG. 12B); and after subtraction of the baseline and freecirculating bubbles (FIG. 12C). 6 WO 2007/067979 PCT/US2006/061793

DETAILED DESCRIPTION

[0029] Applicants have unexpectedly discovered peptide phospholipid conjugates, which are useful in producing targeted ultrasound contrast agents and which have exceptionalKDR binding efficiency. Two of these compounds are monomeric peptide phospholipidconjugates which include a linear peptide monomer which hinds with high affinity to KDRwhile the other is a dimeric peptide phospholipid conjugate which includes two distinctmonomer subunits, each binding to KDR. In addition, highly efficient methods for largescale production of purified forms of these conjugates and precursor materials have beendiscovered. Such methods include the production of dimeric peptide phospholipid conjugateshaving minimimal levels of TFA.

[0030] The phospholipid may be selected from the group consisting of: phosphatidylethanolamines and modified phosphatidylethanolamines Particularly preferredphospholipids include phosphatidylethanolamines modified by linking a hydrophilic polymerthereto. Examples of modified phosphatidylethanolamines are phosphatidylethanolamines(PE) modified with polyethylenglycol (PEG), in brief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEGmolecule of variable molecular weight (e.g. from 300 to 5000 daltons), such as DPPE-PEG,DSPE-PEG, DMPE-PEG or DAPE-PEG. DSPE-PEG2000, DSPE-PEG3400, DPPE-PEG2000 and DPPE-PEG3400 are preferred, with DSPE-PEG2000 particularly preferred.Note that a salt form of the phospholipid may be used, such as, for example, the trimethylammonium salt, the tetramethylammonium salt, the triethylammonium salt, sodium salt, etc.[0031J These compounds may be incorporated into gas Allied ultrasound contrast agents, such as, for example, gas filled microbubbles to form contrast agents that provideexcellent imaging of target-bearing tissue. In a preferred embodiment, targeting vector-phospholipid conjugates which include targeting peptides which bind with high affinity to 7 WO 2007/067979 PCT/US2006/061793 KDR are incorporated into targeted microbubbles. As shown herein, such targetedmicrobubbles selectively localize at KDR-bearing tissue, permitting imaging of such tissue,and, in particular imaging of tumors and angiogenic processes, including those processesassociated with neoplastic development.

Monomer Conjugates

Generally [0032] Table 1 provides a description for the identification labels shown in Figures 1, 2, 9 and 10.

Table 1 1 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ IDNO. 1) 2 Ac-RAQDWYYDETLSMADQLRHAFLSGGGGGK-NH2 (SEQ TDNO. 2) 3 mono-NHS ester of glutaryl-peptide monomer (2) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(NHS-Glut)-NHZ (SEQ ID NO. 3) 4 DSPE-PEG2000-NH2 phospholipid l,2-distearoyl-in-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-anrine 31 Ac-AQDWYYDE1LSMADQJLRHAFLSGGGGGK(DSP£-PEG2OOO-NH-Glut)-NH2 (SEQ ID NO. 4) 32 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 5) [0033] As shown if Figures 1 and 2 the monomeric peptide phospholipid conjugate (1) N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-Ll-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl- {N6-[ 1,2-distearoyl-in-glycero-3- phosphoethanolaminocarbonyIoxy-(PEG2000)-aminoglutaryI]}-L-lysinamide, is aphospholipid conjugate. This conjugate is also referred to as Ac- 8 WO 2007/067979 PCT/US2006/061793 RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ IDNO. 1) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH2. Itcomprises a 29 amino acid linear peptide monomer (2) N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-sermyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-Ll-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide, also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2(SEQ ID NO. 2) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-He-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2. This novel peptidemonomer binds with high affinity to KDR. It should be understood that analogs andderivatives of the monomeric peptide phospholipid conjugate (1) and the linear peptidemonomer (2) are intended to be included within the scope of the present invention.

[0034] Figure 10 provides the structure of another monmeric peptide phospholipid t conjugate (31), N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-'L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seTyl-glycyl-glycyl-glycyI-glycyl-glycyl-{N6-[1,2-distearoyl-sn-glycero-3- phosphoethanolaminocarbonyloxy-(PEG2000)-aTninoglutaryl]}-L-lysine-amide, aphospholipid conjugate. This conjugate is also referred to as Ac- AQDWYYD£1LSMADQLRHAFLSGGGGGK(DSPE-PEG2OOO-NH-Glut)-NH2 (SEQ IDNO. 4) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH2. Asshown in Figure 9, the conjugate comprises a 28 amino acid linear peptide monomer (32), N-acctyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L- 9 191850/3 glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide, which is also referred to as Ac- AQDWYYDEILSMADQLRHAFLSGGGGGK-NH2(SEQIDNO. 5) and Ac-Aia-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-GIy-Gly-Gly-Gly-Gly-Lys-NbU. As shown in published U.S. Application No. 2005-0100963A1, filed September 11,2003, this peptide monomer binds with high affinity toKDR. It should be understood that analogs and derivatives of the monomeric peptidephospholipid conjugate and the linear peptide monomer are intended to be included withinthe scope of the present invention.

[0035] As shown in the Examples, ultrasound contrast agents such as gas filled microbubbles formulated with the monomeric peptide phospholipid conjugates (1) and (31)displayed high KDR binding which was confirmed using echographic examination of VX2 tumors in rabbits.

[0036] Ideally, to facilitate production of the monomeric peptide phospholipid conjugate (1) or (31), the linear peptide monomer (2) or (32) should be prepared in bulk.Then conjugation of the purified linear peptide monomer (2) or (32) to the phospholipid,such as, for example, a pegylated phospholipid in salt form, e.g., DSPE-PEG2OOO-NH2phospholipid ammonium salt (4) via the linker disuccinimidyl glutarate (DSG), maybe usedto provide monomeric peptide phospholipid conjugates (1) or (31).

Methods of Preparation of Monomer Peptide-Phospholipid Conjugates10037] In preparing monomeric peptide phospholipid conjugates (1) and (31), methods according to the present invention provide at least the following advantages:increased yield of peptide synthesis; reduced extent of racemization; avoidance of previouslyobserved piperidine amide formation during synthesis, efficient purification of peptide 10 WO 2007/(167979 PCT/US2006/061793 monomers (2) and (32), development of a procedure for conjugation of peptide monomers (2) and (32) on larger scale; and development of purification protocols that would allow the ready separation of the monomeric peptide phospholipid conjugates (1) and (31) from the starting DSPE-PEG2000-NH2 phospholipid ammonium salt (4), [0038] Monomeric peptide phospholipid conjugates may be prepared as described below. It should be appreciated that the numerical values referred to in this representativedescription of the synthesis of monomeric peptide phospholipid conjugates are representative.[0039] Linear peptide monomers may be prepared by SPPS. The sequence of the linear peptide monomers may be constructed as a C-terminal carboxamide on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Peptide synthesis may be accomplished using Fmocchemistry on a SONATA®/Pilot Peptide Synthesizer. Problems previously observed with thisprocess have been racemization, incomplete couplings and piperidine amide formation, eachof which contribute to suboptimal yield and purity. A dramatic decrease in the formation of'the piperidine amide may he attained by the use of 25% piperidine in DMF containing HOBt(0.1M) as the reagent for Fmoc removal. Racemization may be considerably reduced byusing DIC/HOBt as the activator for most couplings; a 3 h coupling time using a four-foldexcess of pre-activated Fmoc-amino acid with an intervening wash with anhydrous DMF(6x). N“-Fmoc amino acids may be dissolved just before their coupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to the reaction vessel. This may ·be accomplished on the Sonata instrument by loading the solid Fmoc-amino acids into theamino acid vessels of the instrument and then programming the instrument to add DMF,HOBt/DMF and DIC/DMF sequentially with bubbling of the solution.

[0040] To optimize the yield, the problem of aggregation of the resin during the synthesis of longer peptides, which can be devastating even when optimal coupling reagentsare employed, may be. addressed. To reduce aggregation during peptide assembly the 11 WO 2007/067979 PCT/US2OO6/O61793 strategy of using pseudoproline dipeptides to incorporate Χ-Thr or X-Ser as dipeptides instead of sequential couplings of X and Thr or X and Ser, may be employed. For linear peptide monomers sequential couplings of Leu1'-Ser12 and Leu22-Ser23 may be replaced by the single coupling of the pseudoproline dipeptide, Fmoc-Leu-Ser(\/Me,Mt:pro)-OH.

Additional optimization may be accomplished by reducing the number of couplings by usingFmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling of Fmoc-Gly-OH.Activation of-Gly-Gly-OH segments may lead to cyclization of the activated acid functionwith the distal amide function, to produce an inactive diketopiperazine; this may reducecoupling yields in a time dependant manner. This problem may he avoided by addition ofFmoc-Glyn-OH (n = 2, 3) to the reaction vessel and sequential addition of HOBt and DIC; theactivated Fmoc-Glyn-OH may be intercepted by the resin-bound amino group beforeappreciable cyclization to the diketopiperazine takes place. With these improvements, thesynthesis of linear peptide monomers may be completed on the Sonata Peptide Synthesizeron a 10 mmol synthesis scale.

[0041] After chain elongation, the Fmoc may he removed from the N-terminus. The peptide and the free amino group may be acetylated.. Then the peptide sequence may becleaved from the resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. After the cleavage reactionthe crude peptide may be isolated as a solid by evaporation of the volatiles, trituration of theresidue with diethyl ether and washing of the solid thus obtained using the same solvent. Inanother variation the peptide may be precipitated from the reaction mixture by addition ofdiethyl ether to the reaction mixture, collecting the solid thus formed and washing with thesame solvent.

[0042] Linear peptide monomers may be purified as described below. Again, the numerical references arc representative. Crude linear peptide monomers (0.5 g) may be 12 WO 2007/067979 PCT/US2006/061793 dissolved in CH3CN (40 mL/g) and this solution may be diluted to a final volume of 100 mLwith water. The solution may then be filtered. The filtered solution may be loaded onto thepreparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300A, 50 x 250 mm)equilibrated with 10% CH3CN in water (0.1% TFA). After loading, the composition of theeluent may then be ramped to 20% CHaCN-water (0.1 %TFA) over 1 min, and a lineargradient may be initiated at.a rate of 0.6%/min of CH3CN (0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may be checked for purity on an analyticalreversed phase Cl8 column (Waters XTerra MS-C18, 10μ, 120A, 4.6 x 50 mm) and fractionscontaining the product in >95% purity may be combined and freeze-dried. For eachpurification of 0.5 g of crude peptide 0.12 g (24%) of linear peptide monomer may beconsistently isolated, and will provide the peptide in the same yield and. purity.

[0043] Synthesis of monomeric peptide phospholipid conjugates may be performed as described below. The numerical references are again representative. The last step in thesynthesis may be the conjugation of the phospholipid, such as, for example, a pegylatedphospholipid such as DSPE-PEG2OOO-NH2 phospholipid ammonium salt to a linear peptidemonomer. The PEG2000 moiety of DSPE-PEG2OOO-NH2 phospholipid ammonium salt (4)is nominally comprised of 45 ethylene glycol units. It should be understood, however, thatthis material is a distribution of PEG containing species whose centroid is the nominalcompound containing 45 ethylenoxy units. The conjugation of a linear peptide monomerwith DSPE-PEG2OOO-NH2 phospholipid ammonium salt may be accomplished bypreparation of the glutaric acid monoamidc mono NHS ester of a linear peptide monomerand reaction of this with the free amino group of the phospholipid ammonium salt. Thus alinear peptide monomer may be reacted with DSG (4 cq.) in DMF in the presence of DIEA (5eq.) for 30 min. The reaction mixture may be diluted with ethyl acetate, which may result inprecipitation of the peptide glutaric acid monoamide mono-NHS ester. The supernatant 13 WO 2007/067979 PCT/GS2006/061793 containing un-reacted DSG may be decanted and the intermediate peptide mono-NHS estermay be washed several times with ethyl acetate to remove traces of DSG. Mass spectral dataconfirms the formation of the peptide mono-NHS ester as a clean product. The solid mono-NHS ester may be dissolved in DMF and reacted with DSPE-PEG2OOO-NH2 phospholipidammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for.24 h. The linear peptidemonomer glutaric acid monoamide mono-NHS ester may be used in excess to maximize theconsumption of the phospholipid ammonium salt because free phospholipid ammonium saltmay complicate the isolation of monomeric peptide phospholipid conjugates in highly pure form.

[0044] The reaction mixture may be diluted with a 1:1 mixture of water (0.1%TFA) and CH3CN-CH3OH (1:1, v/v) (0.1%TFA) (~100 mL), applied to a reversed phase C2column (Kromasil® Prep C2, 10 μ, 300A, 50 x 250 mm, flow rate 100 mL/min) and thecolumn may be eluted with a 3:1 mixture of water (0.1%TFA) and CH3CN-CH3OH (1:1, v/v)(0.1%TFA) to remove hydrophilic impurities. Then the product may be eluted using agradient of CH3CN-CH3OH (1:1) (0.1 % TFA) into water (0.1% TFA) (see ExperimentalSection for details). The collected fractions may be analyzed by reversed phase HPLC usingan ELS detector which allows the detection of the desired product and the often difficxilt-to-separate DSPE-PEG2000-NH2 phospholipid which has very little UV absorbance. Thisindicates the clear separation of the monomeric peptide phospholipid conjugates and DSPE-PEG2OOO-NH2 phospholipid. The pure product-containing fractions may be collected,concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried toprovide monomeric peptide phospholipid conjugates as a colorless solid. In order to preparethe required quantity of the monomeric peptide phospholipid conjugates, several runs may beconducted employing 0.5 g to 1.0 g of linear peptide monomer. In all cases the target 14 WO 2007/067979 PCT/US2006/061793 monomeric peptide phospholipid conjugates may be were isolated in high yield and purity (e.g., 57-60% yield and >99% purity).

Dimer Conjugate

Generally [0045] Table 2 provides a description for the identification labels shown in Figures 3,4 and 5.

Table 2 11 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH2 cyclic (2-12)disulfide]-NH2 cyclic (6-13) disulfide 12 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (6-13) disulfide 13 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12) disulfide 14 mono-NHS ester of glutaryl-peptide 12 Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH2 cyclic (6-13)disulfide 15 ivDdc-bc'aring dimer Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(ivDde)l-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide 16 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide 17 Mono-NHS ester of glutaryl-peptide 16 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(NHS-Glut)]-NH2 cyclic (2-12) disulfide]-NH2 cyclic (6-13)disulfide 18 DSPE-PEG2000-NH2 phospholipid [0046] As shown in those figures the dimeric peptide phospholipid conjugate (11)

Acctyl-L-alanyl-glycyl-L-prolyl-L-thrconyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl- L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-l-lcucyl-L- phcnylalanyl-glycyl-L-thrconyl-glycyl-glycyl-glycyl-L-lysyl[Acctyl-L-valyl-L-cystinyl-L- tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L- 15 WO 2007/067979 PCT/US2006/06.1793 cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glirtaryl-L-lysyl) amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide , consists of two monomeric peptide chains which bind KDR: a 21 amino acidcyclic disulfide peptide monomer (13) Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl)amide cyclic (2-12) disulfide, and a 22 amino acidcyclic disulfide peptide monomer (12) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysinamide cyclic 6-13 disulfide tethered by a glutaryl linker. It should be understood thatanalogs and derivatives of the dimeric peptide phospholipid conjugate (11) and the cyclicdisulfide peptide monomers (12) and (13) are intend ed to be included, within the scope of thepresent invention.

[0047] Ultrasound contrast agents (e.g. gas filled microbubbles) formulated with the dimeric peptide phospholipid conjugate (11) displayed high KDR binding which wasconfirmed using echographic examination of VX2 tumors in rabbits.

Methods of Preparation of Dimer-Phospholipid Conjugates[0048] To accomplish synthesis ofthe dimeric peptide phospholipid conjugate (11), the monomers used for this purpose optimally should be prepared in bulk. Then themonomers may be tethered to each other using di-succinimidyl glutarate as a linker to formthe precursor dimer peptide (16), Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-E-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl- 16 WO 2007/067979 PCT/US2006/061793 L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seiyl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide.. Thenconjugation of the purified precursor dimer peptide (16) to a DSPE-PEG2OOO-NH2phospholipid ammonium salt (18) again, via disuccinimidyl glutarate may be used in order toprovide the target dimeric peptide phospholipid conjugate (11).

[0049] In preparing dimeric peptide phospholipid conjugate (11), methods according to the present invention provide at least the following advantages: increased yield ofautomated chain elongation of the peptide sequences; reduced extent of racemizationencountered during synthesis; avoidance of previously observed piperidine amide formationduring synthesis of peptide monomer (13); cyclization of linear di-cysteine containingpeptide precursors of (12) and (13) using procedures amenable to multigram scale yetallowing efficient and practical sample handling; efficient purification of monomer peptides(12) and (13); maximized yield and purity of precursor dimer peptide (16); development of aprocedure for conjugation of the precursor dimer peptide (16) on larger scale; anddevelopment of purification protocols that would allow the ready separation of the targetdimeric peptide phospholipid conjugate (11) from phospholipid ammonium salt (18).

[0050] The dimeric peptide phospholipid conjugate (11) may be prepared by automated synthesis of the peptide monomers (12), Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Tip-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13)disulfide, and (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu.-Val-Cys-Ph.e-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide, their efficient couplingusing disuccinimidyl glutarate (DSG) to give an ivDde-protected dimer, its deprotection andsubsequent coupling to DSPE-PEG2000-NH2, also via a glutaryl linkage. Using procedures 17 WO 2007/067979 PCT/US2006/061793 according to the present invention, monomer peptides may be synthesized on a 10 mmol scale without complication and after HPLC purification may be obtained in about 20% yield and >95% purity. Such methods allow dimer formation reactions and the subsequent conjugation to the phospholipid component providing formation of dimeric peptide phospholipid conjugate (11) to be carried out on a gram scale. The precursor dimer peptide (16) may be obtained from the monomer peptides routinely in about 32% yield and >95%

I purity. The dimeric peptide phospholipid conjugate (11) may be produced from the precursordimer peptide (16) in 57-60% yield and >99% purity.

[0051] Dimeric peptide phospholipid conjugates may be prepared as described below.

It should be appreciated that the numerical values referred to in this representative descriptionof the synthesis of dimeric peptide phospholipid conjugates are representative.

[0052] Described below is a representative method for the solid phase synthesis and disulfide cyclization of a peptide monomer (12) Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13)disulfide, and a peptide monomer (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide.

[0053] The peptides may be constructed as their C-terminal carboxamides on Pal-

Peg-PS-resin (substitution level: 0.2 mmol/g). Chain elongation maybe accomplished usingFmoc chemistry employing optimized deprotection and coupling protocols on a SONATA®/Pilot Peptide Synthesizer on a 10 mmol synthesis scale. The optimized synthesis of thepeptides by automated SPSS may be developed by study of peptide impurities and the effectof changes of particular elements of the protocols on the overall yield and purity of thepeptides obtained.

[0054] Analysis of the impurities obtained from nonoptimized syntheses of the monomer peptides indicates that the major problems arc racemization, incomplete couplings 18 WO 2007/067979 PCT/US2006/061793 and piperidine amide formation (presumably via an intermediate aspartimide or glutarimideintermediate), each of which contributes to suboptimal yield and purity. A dramatic decreasein formation of the piperidine amide may be attained by the use of 25% piperidine in DMFcontaining HOBt (0.1M) as the reagent for fmoc removal. Racemization may beconsiderably reduced by using DIC/HOBt as the activator for most couplings; and a 3 hcoupling time using a four-fold excess of pre-activated Fmoc-amino acid with an interveningwash with anhydrous DMF (6x). N-aFmoc amino acids may be dissolved just before theircoupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to thereaction vessel. This may be accomplished on the Sonata instrument by loading the solidFmoc-amino acids into the amino acid vessels of the instrument and then programming theinstrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution after each addition.

[0055] To optimize the yield, the problem of aggregation of the resin during the synthesis of longer peptides, which can be devastating even when optimal coupling reagentsare employed, may be addressed. To reduce aggregation during peptide assembly thestrategy of using pseudoproline dipeptides to incorporate X-Thr or X-Ser (X refers to the n-1amino acid of the sequence) as dipeptides instead of sequential couplings of X and Thr or Xand Ser, may be employed. Thus, for the monomer (12), Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-G1y-Gly-Lys(ivDde)-NH2 cyclic (6-13) disulfide, sequential coupling of suitably protected Thr and Gly (shown in bold above)may be replaced by the single coupling of the pseudoproline dipeptide, Fmoc-Gly-Thfrii/^’^proj-OH. Similarly, in the synthesis of the monomer (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Ttp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide, the pseudoproline dipeptide, Fmoc-Asp(OtBu)-Ser(yMs,Mepro)-OH may be employed to replace the sequential coupling of suitably protected Ser and Asp 19 WO 2007/067979 PCT/US2006/061793 (shown in bold font above). Further optimization may be accomplished by reducing thenumber of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu ofserial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OH segments can lead tocyclization of the activated acid function with the distal amide function to produce an inactivediketopiperazine; this may reduce coupling yields in a time dependant manner. This problemmay be avoided by addition of Fmoc-Glyn-OH (n = 2, 3) to the reaction vessel and sequentialaddition of HOBt and DIC; the activated Fmoc-GlyB-OH may be intercepted by the resin-bound amino group before appreciable cyclization to the diketopiperazine takes place. Afterchain elongation is completed the N-terminal Fmoc protecting group may be removed fromeach of the peptides and the free amino group may be acetylated.

[0056] The pseudo-orthogonally protected derivative, Fmoc-Lys(ivDde)-OH may be used to enable the selective unmasking of the e-amine of the C-terminal lysine of themonomer and dimer peptides and their subsequent functionalization, which also may beoptimized. The ivDde group on the e-amine of the C-terminal lysine of each of the peptidemonomers may be removed using 10% hydrazine in DMF. Then Fmoc-Adoa, for monomer(13) or Lys(ivDde) for monomer (12) may he appended, to the exposed lysine e-amino groupusing 4 equivalents of the Fmoc amino acid and 4 equivalents each of DTC and HOBt inDMF for 10 h. After completion of the synthesis, the peptide sequence may be cleaved fromthe resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2,v/v/w/v) for 4 h. After the cleavage reaction was complete the peptide may be precipitated,washed with diethyl ether and dried.

[0057] The following procedures for cyclization of the linear di-cysteine containing peptides may be used to provide optimal scale-up of monomer peptides. Generally the aerialoxidation of linear di-cysteine peptides may be carried out at ca 0.5-5 mg/mL (for thedisclosed peptide monomers -0.18-1.8 mM in peptide, -0.36-3.6 mM in cysteine thiol). In 20 WO 2007/067979 PCT/US2006/061793 order to work at significantly higher concentrations DMSO-assisted cyclization of di-cysteinepeptides allows the cyclization of ~Ί 0 g of the linear peptides in good yields in as little as ~50mL of solution. Therefore the crude linear di-cysteine peptides may be cyclized in 95%DMSO-H2O (5 mL/g) at pH 8.5 at ambient temperature. The progress of the cyclization maybe routinely followed by mass spectroscopy and HPLC. Although cyclization may beessentially complete in ~ 36 h, the reaction mixture may be generally stirred for up to 48 b.The cyclic disulfide peptides may be precipitated from the reaction mixture by dilution withCH3CN and the resulting off-white crude solid peptides may be collected by filtration. Thisis a convenient method for removing DMSO from the crude cyclic peptide.

[0058] Purification and isolation of monomer peptide (12), Ac- AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH2 may be accomplished as describedbelow. Note that as used herein the designation “C*” refers to a cysteine residue thatcontributes to a disulfide bond. Attempts to dissolve 0.5 g of the crude peptide in up to 300mL of 30% CH3CN in water (0.1 % TFA) have been unsuccessful. Therefore, as analternative, the crude peptide, (0.5 g) may be dissolved, in DMSO (5 mL/g) and this solutionmay be diluted to a final volume of 100 mL with 20% CH3CN-water. The solution may befiltered. The filtered solution may be loaded onto the preparative HPLC column (Waters,XTerra® Prep MS Cl 8, 10μ, 300A, 50 x 250 mm) equilibrated with 10% CH3CN (0.1 % TFA) in water (0.1 % TFA), and the column may be eluted with 10% CFT3CN (0.1 % TFA) inwater (0.1% TFA) to wash DMSO from the column. The composition of the eluent then maybe ramped to 35% CH3CN-water (0.1%TFA) over 1 min, and a linear gradient may beinitiated at.a rate of 0.5%/min of CH3CN (0.1% TFA) into water (0.1% TFA) and run for 50min. Eluted fractions may be checked for purity on an analytical reversed phase Cl 8 column(Waters XTerra MS-C18,10μ, 120A, 4.6 x 50 mm) and fractions containing the product in>95% purity may be combined and frcczc-dricd. For each purification of 0.5 g of crude 21 WO 2007/067979 PCT/US2006/061793 peptide 0.1 g (20%) for (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH2 may be isolated. Repeat purifications have been found to provide the peptide consistently in the same yield and purity.

[0059] The peptide monomer (13), Ac- VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 may be purified and isolated asdescribed for peptide monomer (12) except that the subjectpeptide may be dissolved in 20%CH3CN (0.1% TFA) in 0.1% aqueous TFA (0.5 g peptide/100 mL) instead of a DMSO-containing diluent. The resulting solution of crude peptide may be loaded onto thepreparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300A, 50 x 250 mm, flowrate 100 mL/min) equilibrated with 10% CH3CN in water (0.1% TFA). The column may heeluted with 10% CH3CN (0.1% TFA)/water (0.1% TFA) at 100 mL/min for 5 min. Then thecomposition of the eluent may be ramped to 30% CH3CN (0.1% TFA)/water (0.1%TFA)over 1 min and a linear gradient rate of 0.5%/min of CH3CN (0.1% TFA) into water (0.1%TFA) may he initiated, and maintained until the desired peptide is completely eluted from thecolumn. Product-containing fractions may he analyzed on a Waters XTerra analyticalreversed phase C-18 column (10μ, 120A) and fractions containing the product in >95%purity may be pooled and freeze-dried to afford the cyclic disulfide peptide monomer (13) (0.12 g, 24% yield) in >95% purity. The 10 g of crude peptide monomer may be purifiedserially in this manner.

[0060] Described below is a representative method for preparing the precursor dimer peptide (16), Ac-AGPTWC£DDWYYCWLFGTGGGK[Ac- VCW£DSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH2 cyclic (2-12) disulfide]-NH2cyclic (6-13) disulfide. The preparation of the precursor dimer peptide may he accomplishedby the tethering of the monomer peptides in a two step procedure. First, Ac- AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDdc)]-NH2 (12) may be reacted with 22 WO 2007/067979 PCT/US2006/061793 disuccinimidyl glutarate (DSG, 5 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min.The reaction mixture may be diluted with ethyl acetate, which results in precipitation of theglutaric acid monoamide mono-NHS ester of the peptide. The supernatant, containingunreacted DSG, may be decanted and the mono-NHS ester may he washed several times withethyl acetate to remove traces of DSG. Mass spectral data confirms the formation of themono-NHS ester as a clean product. This may be redissolved in DMF and reacted withmonomer peptide Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 (13) in thepresence of DIEA (5 eq). HPLC and MS results indicate the formation of the ivDde-bearingdimer, as a single major product. The ivDde protecting group on the e-amine of Lys of thedimer may be removed by stirring the reaction mixture with hydrazine (10%) in DMF for 20min. The solution then, may be acidified with TFA and diluted with 10% CH3CN (0.1%TFA)-water (0.1% TFA), applied to a preparative reversed phase Cl 8 HPLC column andpurified by a gradient elution of acetonitrile (0.1% TFA) into 0.1% aqueous TFA. In order toprovide the needed, quantity of the precursor dimer peptide, the reaction may be condu cted,employing from 0.5 g to as much as 1 g of each of the monomer peptides. In every case therequired precursor dimer peptide may be isolated in ~32% yield, and >95% purity confirmingthe reproducibility and scalability of the procedures.

[0061] The final step in the synthesis may he the conjugation of DSPE-PEG2000- NH2 phospholipid ammonium salt (18) to the precursor dimer peptide. As mentionedpreviously, the PEG2000 moiety of DSPE-PEG2000-NH2 is nominally comprised of 45ethylene glycol units. It should he understood, however, that this material is a distribution ofPEG containing species whose centroid is the nominal compound containing 45 ethylenoxyunits.

[0062] Conjugation of the DSPE-PEG2000-NH2 to the precursor dimer peptide may be accomplished by preparation of a glutaric acid monoamide mono NHS ester of the 23 WO 2007/067979 PCT/US2006/061793 precursor dimer and reaction of this with the free amino group of the phospholipidammonium salt. Thus the ivDde bearing precursor dimer peptide (16) may be reacted withDSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. As in the preparation of theprecursor dimer peptide the solution may be diluted with ethyl acetate to precipitate theglutaric acid monoamide mono-NHS ester of the dimer (17), as a solid. The supernatant maybe decanted to remove the un-reacted DSG. The solid glutaric acid monoamide mono-NHSester of the dimer peptide (17) may then be washed several times with ethyl acetate toremove traces of DSG. Mass spectral results confirm the formation of the glutaric acidmonoamide mono-NHS ester of the peptide dimer as a clean product.

[0063] The dimer glutaric acid monoamide mono-NHS ester (17) may be dissolved in DMF-CH2CI2 (8:2) and reacted with DSPE-PEG2OOO-NH2 phospholipid ammonium salt (0.9eq.) in the presence of DIEA (4 eq.) for 24 h. The NHS ester (17) may be used in excess tomaximize the consumption of the phospholipid ammonium salt because any freephospholipid may complicate the purification and isolation of the final product The reactionmixture may be diluted with water (0.1% TFA)-CH3CN-CH3OH (1:1) (0.1 %TFA) (-100mL), applied to a reversed phase C4 column (Kromasil® Prep C4, 10 μ, 300A, 50 x 250 mm,flow rate 100 mL/min) and the column may be eluted with water (0.1 %TFA)-CH3CN-CH3OH (1:1) (0.1%TFA) solvent mixture to remove hydrophilic impurities. Then theproduct may be eluted using a gradient of CH3CN-CH3OH (1:1) (0.1 % TFA) into water(0.1 % TFA). The collected fractions may be analyzed by reversed phase HPLC using anELS detector which allows the detection of the desired product and the often difficult toseparate DSPE-PEG2OOO-HH2 phospholipid ammonium salt which has no strong UVchromophore. This indicates the clear separation of dimeric peptide phospholipid conjugateand DSPE-PEG2OOO-NH2 phospholipid ammonium salt. The pure product-containingfractions may he collected, concentrated on a rotary evaporator (to reduce the content of 24 WO 2007/067979 PCT/US2006/061793 methanol) and freeze-dried to provide the dimer peptide phospholipid conjugate as a colorlesssolid.

[0064] In order to prepare the required quantity of the dimer peptide phospholipid conjugate, several runs may be conducted employing 0.5 g to 1.0 g of the precursor dimerpeptide. In all cases the target dimer peptide phospholipid conjugate may be isolated in 57-60% yield and in >99% purity. The bulk quantity of dimer peptide phospholipid conjugate,obtained from the serial runs described above may be obtained by dissolution of the productfrom the individual runs in t-butanol-acetonitrile-water (1:1:3) followed by lyophilization.

The procedure of Ellman for quantitative estimation, of free thiol may be applied to the bulksample of the dimeric peptide phospholipid conjugate; free thiol, if present will be below thelimit of detection. Amino acid composition analysis gives results within the acceptablelimits, supporting the assigned structure of the peptide derivative. MALDI-TOF massspectral analysis also supports the presumed structure of the dimeric peptide phospholipidconjugate.

Methods of Preparation of Dimer-Phospholipid. Conjugates Having Low orNegligible Levels of TFA

[0065] The present invention also provides methods for producing dimeric peptide- phospholipid conjugates having very low levels of TFA. While certain methods provide forthe synthesis and purification of such conjugates on a gram scale, formation of a lyso- versionof the conjugates has been observed upon storage of lyophilized material at 5°C or uponstorage of aqueous solutions of the conjugates. It is believed that the lyso- compound isformed by TFA-promoted acid hydrolysis of one of the phosphohpid fatty acid esters indimer peptide-phospholipid conjugates.

[0066] To obtain the phospholipid peptide as a stable material bearing a pharmaceutically acceptable counterion, highly efficient methods for obtaining dimer 25 WO 2007/067979 PCT/US2006/061793 peptide-phospholipid conjugates were discovered which convert the TFA salts of the dimer peptide-phospholipid conjugate, or any suitable precursor(s), to analogous pharmaceutical acetate salt(s). Representative embodiments of these methods are provided below.

[0067] Table 3 provides a description for the identification labels shown in Figures 6, 7 and 8.

Table 3 21 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac- VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH2 cyclic (2-12) disulfide}-NH2 cyclic (6-13) disulfide 22 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (2-12) disulfide•nTFA 23 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH2 cyclic (2-12)disulfide · xHOAc 24 mono-NHS ester of glutaryl-peptide 23 Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH2 cyclic (2-12) disulfide 25 Ac-VCW£DSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12) disulfide• yTFA 26 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH2 cyclic (2-12) disulfide• zHOAc 27 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac- VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH2 cyclic (2-12)disulfide]-NH2 cyclic (6-13) disulfide · X HOAc 28 ' Mono-NHS ester of glutaryl-peptide 27 Ac-AGPT WCEDD WYYC WLFGTGGGK {Ac- VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K(NHS-Glut)]-NH2cyclic (2-12) disulfide}-NH2 cyclic (6-13) disulfide 29 DSPE-PEG2000-NH2

Where m, n, x, y, z are variable depending on lyophilization conditions.

[0068] Referring now to Figures 6 and 7, in certain embodiments monomer peptide components of heterodimer peptide (27), namely TFA salts compounds (22) and (25), are 26 WO 2007/067979 PCT/US2006/061793 subjected to ion exchange chromatography on the macroporous sulfonic acid cation exchangeresin AG MP-50 using a step gradient of ammonium acetate to convert them to their acetatesalts. Then the two peptide monomer acetates (23) and (26) may be tethered through aglutaryl linker to form the dimer (27) as an acetate salt. Purification of the crude dimeracetate salt of (27), by C-18 preparative HPLC using a linear gradient method employingCH3CN/H2O each containing 10 mM NH4OAC provides the pure dimer acetate (27).Conjugation of this dimer to DSPE-PEG2OOO-NH2 (29) and final purification of the crudemixture by C-3 preparative HPLC using CH3CN/H2O/NH4OAC provides compound (21) asthe acetate salt.

[0069] More specifically, compounds (22), (25) and (27) all bear side-chain carboxylic acid and amino groups. AG MP-50, a macroporous cation-exchange resin, may beused to allow full penetration of the resin by the peptides and to exploit the immobilization ofthe peptides via their basic (amino and guanidine groups). TFA salts of the peptides may beadsorbed to an AG MP-50 column (sulfonic acid, form) and the column may be washed withwater and then eluted with a step gradient of NH/jOAc in 0 or 30% CH3CN/H2O, dependingon the solubility of the peptides. The peptides may be eluted, at about 600 mM NFLOAc andthe acetate form of the peptides then may be obtained in pure form. Both TC fluorine analysisand CE TFA counter-ion analysis consistently show very low TFA content of the peptides.[0070] Preferred methods also include redissolution/relyophilization of the final peptides several times to remove residual NH4OAC. Otherwise, residual traces of NH4OACpresent in the peptides may give rise to free ammonia in the presence of DJLEA. This mayresult in the formation of unwanted peptide-Glut-amide as a major product in subsequentpreparation of (27) from the monomers (23) and (26) or final phospholipid-peptide conjugate(21) from the acetate salt of (27). 27 WO 2007/067979 PCT/US2006/061793 [0071] Referring now to Figure 7, another embodiment provides the conversion of the TFA salt of dimer (27) to its analogous acetate salt by ion exchange chromatography on themacroporous sulfonic acid cation exchange resin AG MP-50. This dimer acetate then may beconjugated with DSPE-PEG2000-NH2 followed by purification of the crude material by C-3preparative column using CH3CN/H2O/NH4OAC to give the final compound (21) as anacetate salt.

[0072] While the methods described above and in Figures 6 and 7 provide excellent results, the second approach has the advantage of requiring fewer steps. Additional detailsare provided below in the Examples section.

[0073] Turning to Figure 8, another embodiment provides methods for providing dimeric conjugates having minimal amounts of TFA utilizing the size differential between thephospholipid-peptide conjugate (21) and TFA ions. In this embodiment 21· nTFA adductmay be eluted down a size exclusion column in the presence of ammonium bicarbonatebuffer. The crude 21· nTFA initially may be freed of the lyso- compound by preparativeHPLC on a Zorbax C-3 column using a linear gradient of acetonitrile into water. Both phasesmay be buffered with 10 mM ammonium acetate. This provides separation of the lyso-compound as indicated by analytical HPLC.

[0074] To further reduce the amount of TFA, the material may be applied to a

Sephadex G-25 column and eluted with aqueous ammonium bicarbonate solution. The eluatemay be monitored by HPLC. Product-containing fractions may be pooled and lyophilized toafford the desired material (21) essentially free of TFA and with high recovery rates.Additional detail is provided below in the Examples section.

[0075] Both the monomeric and dimeric peptide phospholipid conjugates described herein may be incorporated into ultrasound contrast agents such as, for example, gas filledmicrovcsiclcs. Such gas filled microvcsiclcs include, for example, gas filled microbubblcs, 28 191850/2 gas filled, microballoons, gas filled microcapsules, etc. In a preferred embodiment, thepeptide phospholipid conjugates may be incorporated into ultrasound contrast agentscomprising gas filled microbubbles. Methods of preparation of gas filled microbubbles fromphospholipids and phospholipid conjugates are known to those skilled in the art. Forexample, microbubbles according to the present invention can be prepared by methodsdescribed in any one of the following patents: EP 554213, WO 04/069284, U.S. Pat. No.5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No. 5,556,610, U.S.

Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833, U.S. Pat. No. 5,271,928, U.S. Pat.No. 5,380,519, U.S. Pat. No. 5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551,U.S. Pat. No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S. Pat. No.6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat. No.5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No. 5,908,610.

The methods disclosed in WO 04/069284 are particularly preferred.

[0076] Suitable phospholipids include esters of glycerol with one or two molecules of fatty acids (the same or different) and phosphoric acid, wherein the phosphoric acid residue isin turn bonded to a hydrophilic group, such as choline, serine, inositol, glycerol, ethanolamine, and the like groups. Fatty acids present in the phospholipids are in generallong chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from14 to 22, that may be saturated or may contain one or more unsaturations. Examples ofsuitable fatty acids are lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid,behenic acid, oleic acid, linoleic acid, and linolenic acid. Mono esters of phospholipids areknown in the art as the “lyso” forms of the phospholipid.

[0077] Further examples of phospholipids are phosphatidic acids, i.e., the diesters of glyccrol-phosphoric acid with fatty acids, sphingomyelins, i.e., those phosphatidylcholine 29 WO 2007/067979 PCT/US2006/061793 analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain, cardiolipins, i.e. the esters of 1, 3-diphosphatidylglycerol with a fatty acid, gangliosides, cerebrosides, etc.

[0078] As used herein, the term phospholipids includes either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures. ' [0079] Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins.Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivativesof the naturally occurring lecithins.

[0080] Examples of synthetic phospholipids are e.g., dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoyl-phosphatidylcholine(“DPPC”), diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine(“DSPC”), l-myristoyl-2-palmitoylphosphatid.ylcholine (“MPPC”), l-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1 -pahnitoyl-2-stearoylphosphatidylcholine(“PSPC”), 1 -stearoyl-2-palmitoyl-phosphatid.ylcholine (“SPPC”), dioleoylphosphatidylycholine (“DOPC”), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine(Ethyl-DSPC), dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts, dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts, dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts, distearolyphosphatidylglycerol(“DSPG”) and its alkali metal salts, dioleoylphosphatidylglycerol (“DOPG”) and its alkalimetal salts, dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts, dipalmitoylphosphatidic acid (“DPPA”) and its alkali metal salts, distearoyl phosphatidic acid (“DSPA”),diarachidoyl phosphatidic acid (“DAPA”) and its alkali metal salts, dimyristoyl phosphatidyl- 30 WO 2007/067979 PCT/US2006/061793 ethanolamin- e (“DMPE”), dipalmitoyl phosphatidylethanolamine (“DPPE”), distearoylphosphatidyl-ethanolamine (“DSPE”), dimyristoyl phosphatidylserine (“DMPS”),diarachidoyl phosphatidylserine (“DAPS”), dipalmitoyl phosphatidylserine (“DPPS”),distearoylphosphatidylserine (“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoylsphingomyelin (“DPSP”), and distearoyl sphingomyelin (“DSSP”).

[0081] Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer thereto. Examples of modified phospholipids arephosphatidylethanolamines (PE) modified wiih polyethylenglycol (PEG), in brief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linkedto a PEG molecule of variable molecular weight (e.g. from 300 to 5000 daltons), such asDPPE-PEG, DSPE-PEG, DMPE-PEG or DAPE-PEG (where DAPE is 1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine). The compositions also may contain other amphiphiliccompounds including, for instance, fatty acids, such as palmitic acid, stearic acid, arachidonicacid or oleic acid; sterols, such as cholesterol, or esters of sterols with fatty acids or withsugar acids; glycerol or glycerol esters including glycerol tripalmitate, glycerol distearate,glycerol tristearate, glycerol dimyristate, glycerol trimyristate, glycerol dilaurate, glyceroltrilaurate, glycerol dipalmitate; tertiary or quaternary alkyl-ammonium salts, such as 1,2-distearoyl-3-trimethylammoTiium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),and mixtures or combinations thereof.

[0082] Preferably, the formulation comprises at least one component bearing an overall net charge, such as, for instance, phosphatidic acid, PE-PEG, palmitic acid, stearicacid, Ethyl-DSPC or DSTAP, preferably in a molar amount of less than about 50%.Particularly preferred formulations may include mixtures of two or more of the followingcomponents: DSPC, DPPG, DPP A, DSPE-PEG1000, DSPE-PEG2000, palmitic acid andstearic acid. Some preferred phospholipids and formulations arc set forth in the examples 31 191850/3

Any of the gases disclosed herein or known to the skilled artisan may be employed; however,inert gases, such as SF6 or perfluorocarbons like CR, C3F8 and C4F10, are preferred,optionally in admixture with other gases such as air, nitrogen, oxygen or carbon dioxide[0083] The preferred microbubble suspensions of the present invention may be prepared from phospholipids using known processes such as a freeze-drying or spray-dryingsolutions of the crude phospholipids in a suitable solvent or using the processes set forth inEP 554213; WO 04/069284; U.S: Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No'. 5,846,518; U.S. Pat. No. 6,183,725; EP474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No. 5,531,980; U.S. Pat.No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat. No. 5,643,553; U.S. Pat. No. 5,911,972;U.S. Pat No. 6,110,443; U.S. Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S.Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat No. 6,187,288; and U.S. Pat. No.5,908,610. Preferably, as disclosed, in International patent application WO 04/069284, a microemulsion can beprepared which contains the phospholipids (e.g DSPC and/or DSP A) in admixture with alyoprotecting agent (such as, for instance, carbohydrates, sugar alcohols, polyglycols andmixtures thereof, as indicated in detail hereinafter) and optionally other amphiphilic materials(such as stearic acid), dispersed in an emulsion of water and of a water immiscible organicsolvent. Preferred organic solvents are those having solubility in water of 1.0 g/1 or lower, preferably lower than about 0.01 g/1, and include, for.instance, pentane, hexane, heptane, octane,nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopentane, cyclohexane, cyclooctane, 1-methyl-cyclohexane, benzene, toluene, ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl ether and di-isopropylketone, chloroform, carbon tetrachloride, 2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane), 2-chloro-2-(difluoromethoxy)-1,1,1-trifluorocthanc (isofluranc), tctrachloro-l,l-difluorocthanc, pcrfluoropcntanc, 32 WO 2007/067979 PCT/US2006/061793 perfluorohexane, perfluoroheptane, perfluorononane, perfluorobenzene, perfluorodecalin,methylperfluorobutylether, methylperfluoroisobutylether, ethylperfluorobutylether,ethylperfluoroisobutylether and mixtures thereof. The peptide-phospholipid conjugate of theinvention can he admixed together with the phospholipid forming the microvesicle’senvelope, in the microemulsion. Preferably, an aqueous suspension of the peptide-phospholipid conjugate and of a PE-PEG (e.g. DSPE-PEG2000) is first prepared, which isthen admixed together with an aqueous-organic emulsion comprising the phospholipid andthe lyoprotecting agent. Preferably said mixing is effected under heating, e.g. form about 40°C to 80°C.

[0084] Prior to formation of the suspension of microbubbles by dispersion in an aqueous carrier, the freeze dried or spray dried phospholipid powders are contacted with airor another gas. When contacted with the aqueous carrier the powdered phospholipids whosestructure has been disrupted will form lamellarized or laminarized segments that will stabilizethe microbubbles of the gas dispersed therein. This method permits production ofsuspensions of microbubbles that are stable even when stored for prolonged periods and areobtained by simple dissolution of the dried laminarized phospholipids (which have beenstored under a desired gas) without shaking or any violent agitation.

[0085] Alternatively, microbubbles can be prepared by suspending a gas into an aqueous solution at high agitation speed, as disclosed e.g. in WO 97/29783. A furtherprocess for preparing microbubbles is disclosed in WO 2004/069284 , herein incorporated byreference, which comprises preparing an emulsion of an organic solvent in an aqueousmedium in the presence of a phospholipid and subsequently lyophilizing said emulsion, afteroptional washing and/or filtration steps. Some preferred preparation methods are disclosed inthe examples. 33 WO 2007/067979 PCT/US2006/061793 [0086] The formulation for the preparation of the gas-filled microbubbles may advantageously further comprise a lyophilization additive, such as an agent withcryoprotective and/or lyoprotective effect and/or a bulking agent, for example an amino-acidsuch as glycine; a carbohydrate, e.g. a sugar such as sucrose, mannitol, maltose, trehalose,glucose, lactose or a cyclodextrin, or a polysaccharide such as dextran; or a polyglycol suchas polyethylene glycol (e.g. PEG-4000).

[0087] Any of these ultrasound compositions should also be, as far as possible, isotonic with blood. Hence, before injection, small amounts of isotonic agents may be addedto any of above ultrasound contrast agent suspensions. The isotonic agents are physiologicalsolutions commonly used in medicine and they comprise aqueous saline solution (0.9%NaCl), 2.6% glycerol solution, 5% dextrose solution, etc. Additionally, the ultrasoundcompositions may include standard pharmaceutically acceptable additives, including, forexample, emulsifying agents, viscosity modifiers, cryoprotectants, lyoprotectants, bulkingagents etc.

[0088] Any biocompatible gas may be used in the ultrasound contrast agents of the invention. The term “gas” as used herein includes any substances (including mixtures)substantially in gaseous form at the normal human body temperature. The gas may thusinclude, for example, air, nitrogen, oxygen, CO2, argon, xenon or krypton, fluorinated gases(including for example, perfluorocarbons, SFe, SeFg) a low molecular weight hydrocarbon(e.g., containing from 1 to 7 carbon atoms), for example, an alkane such as methane, ethane,a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane orcyclopentene, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne suchas acetylene or propyne and/or mixtures thereof. However, fluorinated gases are preferred.Fluorinated gases include materials that contain at least one fluorine atom such as SF6, freons(organic compounds containing one or more carbon atoms and fluorine, i.c., CF4, C2F6, C3F8, 34 WO 2007/067979 PCT/US2006/061793 C4FB, C4F10, CBrF3, CCI2F2, C2CIF5, and CBrClF2) and perfluorocarbons. The term perfluorocarbon refers to compounds containing only carbon and fluorine atoms and includes, in particular, saturated, unsaturated, and cyclic perfluorocarbons. The saturated perfluorocarbons, which are usually preferred, have the formula C„Fj,+2, where n is from 1 to12, preferably from 2 to 10, most preferably from 3 to 8 and even more preferably from 3 to6. Suitable perfluorocarbons include, for example, CF4, C2F6, C3F8C4F8, C4F10, C5F12, C6F2,C7F14, CgFig, and C9F20. Most preferably the gas or gas mixture comprises SF6 or aperfluorocarbon selected from the group consisting of C3Fs C4F8, C4F10, C5F2, CsFn, C7F14,CeFiB, with C4F10 being particularly preferred. See also WO 97/29783, WO 98/53857, WO98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501, WO 98/05364, WO98/17324. In a preferred embodiment the gas comprises C4Fioor SFc, optionally in admixturewith air, nitrogen, oxygen or carbon dioxide.

[0089] In certain circumstances it may be desirable to include a precursor to a gaseous substance (e.g., a material that is capable of being converted to a gas in vivo, oftenreferred to as a “gas precursor”). Preferably the gas precursor and the gas it produces arephysiologically acceptable. The gas precursor may be pH-activated, photo-activated,temperature activated, etc. For example, certain perfluorocarbons may be used astemperature activated gas precursors. These perfluorocarbons, such as perfluoropentane,have a liquid/gas phase transition temperature above room temperature (or the temperature atwhich the agents are produced and/or stored) but below body temperature; thus they undergoa phase shift and are converted to a gas within the human body. 10090] As discussed above, the gas can comprise a mixture of gases. The following combinations are particularly preferred gas mixtures: a mixture of gases (A) and (B) inwhich, at least one of the gases (B), present in an amount of between 0.5-41% by vol., has amolecular weight greater than 80 daltons and is a fluorinated gas and (A) is selected from the 35 WO 2007/067979 PCT/US2006/061793 group consisting of air, oxygen, nitrogen, carbon dioxide and mixtures thereof, the balance ofthe mixture being gas A.

[0091] Unless it contains a hypeipolarized gas, known to require special storage conditions, the lyophilized product may be stored and transported without need oftemperature control of its environment and in particular it may be supplied to hospitals andphysicians for on site formulation into a ready-to-use administrable suspension withoutrequiring such users to have special storage facilities. Preferably in such a case it can besupplied in the form of a two-component kit, which can include two separate containers or adual-chamber container. In the former case preferably the container is a conventionalseptum-sealed vial, wherein the vial containing the lyophilized residue of step b) is sealedwith a septum through which the carrier liquid may be injected using an optionally prefilledsyringe. In such a case the syringe used as the container of the second component is alsoused then for injecting the contrast agent. In the latter case, preferably the dual-chambercontainer is a dual-chamber syringe and once the lyophilizate has been reconstituted and. thensuitably mixed or gently shaken, the container can be used directly for injecting the contrastagent. In both cases means for directing or permitting application of sufficient bubbleforming energy into the contents of the container are provided. However, as noted above, inthe stabilised contrast agents according to the invention the size of the gas microbubbles issubstantially independent of the amount of agitation energy applied to the reconstituted driedproduct. Accordingly, no more than gentle hand shaking is generally required to givereproducible products with consistent microbubble size.

[0092J It can be appreciated by one of ordinary skilled in the art that other two- chamber reconstitution systems capable of combining the dried powder with the aqueoussolution in a sterile manner are also within the scope of the present invention. In suchsystems, it is particularly advantageous if the aqueous phase can be intciposcd between the 36 WO 2007/067979 PCT/US2006/061793 water-insoluble gas and the environment, to . increase shelf life of the product. Where a material necessary for forming the contrast agent is not already present in the container (e.g. a targeting ligand to be linked to the phospholipid during reconstitution), it can be packaged with the other components of the kit, preferably in a form or container adapted to facilitate ready combination with the other components of the kit.

[0093] No specific containers, vial or connection systems are required; the present invention may use conventional containers, vials and adapters. The only requirement is agood seal between the stopper and the container. The quality of the seal, therefore, becomesa matter of primary concern; any degradation of seal integrity could allow undesirablesubstances to enter the vial. In addition to assuring sterility, vacuum retention is essential forproducts stoppered at ambient or reduced pressures to assure safe and proper reconstitution.The stopper may be a compound or multicomponent formulation based on an elastomer, suchas poly(isobutylene) or butyl rubber.

[0094] In ultrasound applications the contrast agents formed by phospholipid stabilized microbubbles can be administered, for example, in doses such that the amount ofphospholipid injected is in the range 0.1 to 200 pg/kg body weight, preferably from about 0.1to 30 pg/kg.

[0095] Ultrasound imaging techniques that can be used in accordance with the present invention include known techniques, such as color Doppler, power Doppler, Doppleramplitude, stimulated acoustic imaging, and two- or three-dimensional imaging techniques.Imaging may be done in harmonic (resonant frequency) or fundamental modes, with thesecond harmonic preferred.

[009(5] The ultrasound contrast agents of the present invention may further be used in a variety of therapeutic imaging methods. The term therapeutic imaging includes within itsmeaning any method for the treatment of a disease in a patient which comprises the use of a 37 WO 2007/067979 PCT/US2006/061793 contrast imaging agent (e.g. for the delivery of a therapeutic agent to a selected receptor ortissue), and which is capable of exerting or is responsible to exert a biological effect in vitroand/or in vivo. Therapeutic imaging may advantageously be associated with the controlledlocalized destruction of the gas-filled microvesicles, e.g. by means of an ultrasound burst athigh acoustic pressure (typically higher than the one generally employed in non-destructivediagnostic imaging methods). This controlled destruction may be used, for instance, for thetreatment of blood clots (a technique also known as sonothrombolysis), optionally incombination with the localized release of a suitable therapeutic agent. Alternatively, saidtherapeutic imaging may include the delivery of a therapeutic agent into cells, as a result of atransient membrane permeabilization at the cellular level induced by the localized burst of themicrovesicles. This technique' can be used, for instance, for an effective delivery of geneticmaterial into the cells; optionally, a drug can be locally delivered in combination with geneticmaterial, thus allowing a combined pharmaceutical/genetic therapy of the patient (e.g. in caseof tumor treatment).

[0097] The term “therapeutic agent” includes within its meaning any substance, composition or particle which may be used in any therapeutic application, such as in methodsfor the treatment of a disease in a patient, as well as any substance which is capable ofexerting or responsible to exert a biological effect in vitro and/or in vivo. Therapeutic agentsthus include any compound or material capable of being used in the treatment (includingdiagnosis, prevention, alleviation, pain relief or cure) of any pathological status in a patient(including malady, affliction, disease lesion or injury). Examples of therapeutic agents aredrugs, pharmaceuticals, bioactive agents, cytotoxic agents, chemotherapy agents,radiotherapeutic agents, proteins, natural or synthetic peptides, including oligopeptides andpolypeptides, vitamins, steroids and genetic material, including nucleosides, nucleotides,oligonucleotides, polynucleotides and plasmids. 38 WO 2007/067979 PCT/US2006/061793

Materials and Analytical Methods [0098] Solvents for reactions, chromatographic purification and HPLC analyses were E. Merck Omni grade solvents from VWR Corporation (West Chester, PA). N-Methylpyrrolidinone (NMP) and Ν,Ν-dimethylformamide (DMF) were obtained fromPharmco Products Inc. (Brookfield, CT), and were peptide synthesis grade or lowwater/amine-free Biotech grade quality. Piperidine (sequencing grade, redistilled 99+%) andtrifiuoroacetic acid (TFA) (spectrophotometric grade or sequencing grade) were obtainedfrom Sigma-Aldrich Corporation (Milwaukee, WI) or from the Fluka Chemical Division ofSigma-Alrich Corporation. Ν,Ν’-Diisopropylcarbodiimide (DIC), phenol (99%), N,N-diisopropylethylamine (DIEA) and triisopropylsilane (TIS) were purchased from Sigma-Aldrich Corporation. Fmoc-protected amino acids, pseudoproline dipeptides, Fmoc-Asp(O-tBu)-Ser(v|rMe,Mepro)-OH and Fmoc-Gly-Thr(vMe’Mepro)-OH and N-hydroxybenzotriazole(HOBt) were obtained from Novabiochem (San Diego, CA). Fmoc-8-amino-3,6-dioxaoctanoic acid (Adoa) was obtained from NeoMPS Corp (San Diego, CA) or Suven LifeSciences (Hyderabad, India). Disuccinimidyl glutarate (DSG) and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino (polyethyleneglycol)2000] ammonium salt, [DSPE-PEG2OOO-NH2] were obtained from Pierce Chemical Co. (Rockford, TL.) and Avanti® PolarLipids (Alabaster, AL), respectively. Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH wereprepared in-house from the corresponding triglycine or diglycine by the reaction with Fmoc-OSu. An AG MP-50 ion-exchange resin was obtained from Bio-Rad (Hercules, CA).

10099] Analytical HPLC data were generally obtained using a Shimadzu LG-1 OAT VP dual pump gradient system employing a Waters XTerra MS-C18 4.6 x 50 mm column,(particle size: 5μ; 12θΑ pore size) and gradient or isocratic elution systems using water (0.1 %TFA) as eluent A and CH3CN (0.1% TFA) or CH3CN-CH3OH (1:1, v/v) (0.1% TFA) aseluent B. Detection of compounds was accomplished using UV at 220 and 254 nm. The 39 WO 2007/067979 PCT/US2006/061793 purity of the phospholipid-PEG-peptide derivatives was determined on a YMC C-4 (5μΜ, 300A, 4.6 x 250 mm) column or on a Zorbax 300 SB-C3 (3.5μΜ; 300A, 3 x 150 mm) column using a SEDEX 55 Light Scattering Detector (LSD) and with a UV detector.

[00100] Preparative HPLC was conducted on a Shimadzu LC-8A dual pump gradientsystem equipped with a SPD-10AV UV detector fitted with a preparative flow cell.

Generally the solution containing the crude peptide was loaded onto a reversed phase 08,C4 or C3 column, depending on the compound characteristics, using a third pump attached tothe preparative Shimadzu LC-8A dual pump gradient system. After the solution of the crudeproduct mixture was applied to the preparative HPLC column the reaction solvents andsolvents employed as diluents, such as DMF or DMSO, were eluted from the column at loworganic phase composition. Then the desired product was eluted using a gradient elution ofeluent B into eluent A. Product-containing fractions were combined based on their purity asdetermined by analytical HPLC and mass spectral analysis. The combined fractions werefreeze-dried to provide the desired product.

[00101] Amino acid composition analyses were performed at the Keck BiotechnologyResource Laboratory at Yale University, New Haven, CT. Mass spectral data were obtainedfrom MScan Inc. (606 Brandywine Parkway, West Chester PA 19380) or obtained in-houseon an Agilent LC-MSD 1100 Mass Spectrometer. For the purposes of fraction selection andcharacterization of the products mass spectral values were usually obtained using APT-ES innegative ion mode. Generally the molecular weight of the target peptides was -3000; themass spectra usually exhibited doubly or triply negatively charged ion mass values ratherthan [M-H]". These were generally employed for selection of fractions for collection andcombination to obtain the pure peptide during HPLC purification. In some cases fractionsexhibited dominant peaks attributable to [M-2H]/2 + 57 or [M-2H]/2 + 114 in the massspectrum. These peaks arc due to the formation of adducts of one or two molecules of 40 191850/2 trifluoroacetic acid per molecule of the peptide. After careful collection of fractions bycomparing MS results and HPLC purities and freeze-drying process, a small amount of theisolated fluffy solid was dissolved in water (0.5 mg/mL) and treated with a drop of aqueousN-methyl-D-glucamine (~ 0.5 M). This solution was analyzed by HPLC and MS for finalpurity results of the purified peptide. Peptide solutions in the presence of N-methyl-D-glucamine did not exhibit [M-2H]/2 + 57 or [M-2H]/2 + 114 mass value peaks in the massspectrum, instead the expected [M-2H]/2 or [M-3H]/3 peaks were observed.

[00102] The following non-limiting Examples provide additional detail on efficient processes used for obtaining large quantities of highly purified forms of the monomeric anddimeric peptide phospholipid conjugates. These non-limiting Examples also describe thepreparation of representative targeted microbubbles which include these monomeric anddimeric peptide phospholipid conjugates. These Examples also describe the use of suchtargeted microbubbles in static binding tests on KDR-transfected cells and dynamic bindingtests on rh VEGF-R2/Fc chimeric protein. The Examples further describe the evaluation ofultrasound contrast agents containing KDR binding lipopeptides in a rabbit VX2 tumor model.

EXAMPLES

[00103] Examples 1-2 below refer to the monomeric peptide phospholipid conjugateshown in Figure 2. A process for synthesizing this compound is shown in Figure 1.

Although these Examples refer more specifically to the process for synthesizing thecompound shown in Figure 2, a similar process may used to prepare the monomeric peptidephospholipid conjugate shown in Figure 10 and the linear peptide monomer (32) shown inFigure 9 as well as other monomer peptide-phospholipid conjugates. Additionally,published U.S. Application No. 2005-0100963 A1, filed September 11,2003, sets forth methods for 41 191850/2 the preparation of the peptide monomers. EXAMPLE 1

Solid Phase Synthesis (SPPS) and Purification of Linear Peptide Monomer (2) Ac- RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2, (SEQ ID NO. 2) Ac-Arg-AIa-Gln-

Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-

Gly-Gly-Gly-Gly-Gly-Lys-NH2,; N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl- L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L- alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L- leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide [00104] The linear peptide monomer (2) was synthesized by an established automated protocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2mmol/g), Fmoc-protected amino acids and DIC-mediated HOBt ester activation in DMF.

The peptide sequence was synthesized in stepwise fashion by SPPS methods on the Fmoc-Pal-Peg-PS resin, typically on a 10 mmol scale. The amino acid couplings were carried outwith a 4-fold excess each of amino acid and the DIC-HOBt reagent pair in DMF.

[00105] In a typical coupling of an amino acid, 5 mL of dry DMF per gram of resinwas used. The total volume of DMF, calculated on the basis of resin used, was allocatedamong amino acid, HOBt and DIC for solution preparation. For example, for the synthesisinvolving 50 g (10 mmol scale) of resin, the calculated volume of 250 mL of DMF wasdistributed among amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acidvessel on the Sonata Pilot Peptide Synthesizer was charged with the solid dry amino acid (4-fold. excess with respect to the resin). At inception of the coupling step, the software of theinstrument was employed to deliver successively the chosen volume of DMF (for dilution ofthe amino acid) and HOBt (4 eq.) in DMF and DTC (4 eq.) in DMF and mixing by nitrogenbubbling was initiated and conducted for 4 min. This served to pre-activate the amino acidand to insure complete dissolution of all components of the mixture. After activation, thesoftware mediated the transfer of the solution of the activated Fmoc-amino acid to the 42 WO 2007/067979 PCT/US2006/061793 reaction vessel containing the resin. After transfer was complete the vessel was agitated for 3h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin was washedthoroughly with DMF (5 mL/g, 6x) and the cleavage of the Fmoc-group was performed with25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2 xlO min). The resin wasthoroughly washed with DMF (5 mL/g, 6x) to assure complete removal of piperidine fromthe resin in preparation for the ensuing amino acid coupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, the pre-activation in the amino acid bottle was notconducted in order to minimize the formation of diketopiperazine during the activation timeas discussed in the text. Therefore, in these two cases, the solutions of amino acid, HOBt andDIC were added to the reaction vessel sequentially and the coupling process was conducted with ‘in situ’ activation.

[00106] After chain elongation was completed, the Fmoc group of the N-terminal amino acid was removed in the standard maimer followed by the standard wash with DMF(vide supra). The N-terminal amino acid was then capped by treatment with freshly prepared,acetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF/6mL/g of resin), 2 x 20 min. After completion of the peptide synthesis, the resin was treatedwith the cleavage cocktail, ‘Reagent B’ (TFA:water:phenol:triisopropylsilane, 88:5:5:2,v/v/w/v) (10 mL/g of resin) for 4 h. The volatiles were removed and the paste thus obtainedwas triturated with ether to provide a solid which was washed with ether (3x) withintervening centrifugation (to compact the suspended solids in order to allow decantation ofthe supernatant) and then dried under vacuum to provide the required peptide as an off-whitesolid. A 10 mmol scale synthesis of the linear peptide monomer (2) gave 33.82 g (103% oftheory) of the crude peptide. The greater than theoretical yield was most likely due tomoisture and residual solvents. 43 WO 2007/067979 PCT/US2006/061793

Purification of the Linear Peptide Monomer (2) Ac- RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 2); Ac-Arg-Ala-Gln-

Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-

Gly-Gly-Gly-Gly-Gly-Lys-NH2; N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl- L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L- alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L- leucyl-L-seiyl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide [00107] A ~0.5 g portion of the crude linear peptide monomer (2) was dissolved in aminimum amount of CH3CN (-20 mL). The volume of the solution was adjusted to -100 mLwith water and employing a third pump the solution was loaded onto a reversed phase C18preparative column (Waters, XTerra® Prep MS Cl 8, 10 μ, 300A, 50 x 250 mm, flow rate 100mL/min) which had been pre-equilibrated with 10% CH3CN in water (0.1% TFA). Thecolumn was not eluted with the equilibrating eluent during application of the sample solution.After the sample solution was applied to the column, the composition of the eluent wasramped to 20% CHsCN-water (0.1%TFA) over 1 min, and a linear gradient at a rate of0.6%/min of CH3CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50min. Fractions (15 mL) were manually collected, using UV at 220 iim as an indicator ofproduct elution. The collected fractions were analyzed, on a Waters XTerra analyticalreversed phase C-18 column (5μ particle, 120A pore)'and product-containing fractions of>95% purity were pooled and freeze-dried to afford the corresponding pure linear peptidemonomer (2). Typically the purification of 0.5 g of crude (2) afforded 0.12 g (24% yield) ofthe desired product (>95% purity). EXAMPLE 2

Preparation of Monomeric Peptide Phospholipid Conjugate (1) Ac-

RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO. 1); Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ue-Leu-Ser-Met-Ala-Asp-Gln-Leu-

Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-(DSPE-PEG2000-NH-Glut)-NH2; N- acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L- isoleucyl-L-glutamyl-L-leucyl-l-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L- leucyI-L-arginyl-L-histidyl-L-aIanyl-L-phenylalan.yl-L-leucyI-L-serinyl-glycyl-glycyl-glycl- glycyl-glycyl-L-lysinamide 44 WO 2007/067979 PCT/US2006/061793 [00108] The monomeric peptide phospholipid conjugate (1), Ac- RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ IDNO. 1), was prepared by conjugation of (3), the glutaric acid monoamide mono-NHS ester ofpeptide monomer (2), with DSPE-PEG2000-NH2 phospholipid ammonium salt (4),.

[00109] A round-bottomed flask equipped with magnetic stir bar and septum cap wascharged sequentially with anhydrous dimethylformamide (7.5 mL), disuccinimidyl glutarate(DSG, 0.25 g, 0.75 mmol) and diisopropylethylamine (0.10 g, 0.78 mmol) with stirring.

Solid linear peptide monomer (2) (0.5 g, 0.152 mmol) was added portionwise to the abovesolution over a period of 2 min; then the solution was stirred for 30 min at ambienttemperature. The reaction mixture was diluted to ~ 50 mL with anhydrous ethyl acetate; thisresulted in precipitation of the intermediate mono-NHS ester (3), the glutaric acidmonoamide mono-NHS ester of peptide monomer (2). The solution was centrifuged to bringdown mono-NHS ester (3)) as a colorless solid. The supernatant containing excess DSG wasdecanted from the compacted solid. mono-NHS ester (3) which was again dispersed in ethylacetate, centrifuged, and washed twice more to remove the remaining traces of DSG. Thesolid intermediate mono-NHS ester (3) thus obtained was dissolved in anhydrous DMF (10.0mL); diisopropylethylamine (0.10 g, 0.78 mmol) was added; and the mixture was stirred.[00110] Meanwhile, DSPE-PEG2000-NH2 phospholipid ammonium salt (4) (0.38 g,0.14 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask andtrifluoroacetic acid (2 drops) was added to protonate the phosphodiester oxygen facilitatingsolubilization of phospholipid ammonium salt in dichloromethane. The clear solution wasthen evaporated on a rotary evaporator to remove the volatiles and dried further under vacuum.

[00111] The solid phospholipid ammonium salt (4) was dissolved in DMF (5 mL) andtransferred to the stirred solution of mono-NHS ester (3) and the resulting mixture was stirred 45 WO 2007/067979 PCT/US2006/061793 for 24 h at ambient temperature. The reaction mixture was diluted to 100 mL with a 1:1mixture of CH3OH and CbLCN-water (1:1, v/v) and the insolubles were filtered. Half of thefiltered solution was loaded onto a reversed phase C2 preparative column (Kromasil® PrepC2, 10 μ, 300 A, 50 x 250 mm) which had been pre-equilibrated with 3:1 (v/v) mixture ofwater (0.1%TFA) and CH3OH-CH3CN (1:1, v/v, 0.1%TFA) at a flow rate of 100 mL/min.Note that the column was not eluted with the equilibrating eluent during loading of thesample. After the sample solution was loaded the column was washed with the equilibration

I eluent until the plug of DMF was eluted. The composition of the eluent was ramped to 70%CH3OH-CH3CN (1:1, 0.1%TFA) over 9 min and a linear gradient of 0.75%/min of CH3OH-CH3CN (1:1, 0.1%TFA) into water (0.1% TFA) was initiated and run for 40 min. Fractions(15 mL) were collected using UV (220 nm) as an indicator of product elution. Fractions werechecked for purity on an analytical HPLC system (column: YMC C-4, 5 μ, 300 A, 4.6 x 250mm) using UV at 220 nm and an evaporative light scattering detector (ELSD). The latterdetector (ELSD) was employed to detect DSPE-PEG2OOO-NH2 phospholipid ammonium salt(4) which has very little U V absorbance at 220 nm. Product-containing fractions of >98%purity, and devoid of DSPE-PEG2OOO-NH2 phospholipid ammonium salt (4) were combinedand concentrated on a rotary evaporator to reduce the content of CH3OH. The concentratedsolution was then diluted with 10% CH3CN in water until a faint flocculent precipitateformed. The resulting solution was freeze-dried to provide monomeric peptide phospholipidconjugate (1) as a colorless solid. The second portion of crude monomeric peptidephospholipid conjugate (1) was purified as described above. The combined yield of thetarget monomeric peptide phospholipid conjugate (1) was 0.40 g (47% yield).

[00112] Examples 3-5 below refer to the dimeric peptide phospholipid conjugateshown in Figure 5. Representative methods of synthesizing the dimeric conjugate are shownin Figures 3,4, 6, 7 and 8. 46 WO 2007/067979 PCT/US2006/061793 EXAMPLE 3

Solid Phase Synthesis (SPPS), Cyclization and Purification of Monomer Peptides (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH2 and (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 [00113] The linear peptides were synthesized by an established automated protocol ona SONATA® /Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and DCI-mediated HOBt ester activation in DMF. The peptidesequence on the Fmoc-Pal-Peg-PS resin was synthesized in stepwise fashion by SPPSmethods typically on a 10 mmol scale. The amino acid coupling was carried out with a 4-fold excess each of amino acid and DIC-HOBt reagent in DMF.

[00114] In a typical coupling of an amino acid in the sequence, 5 mL of dry DMF per gram of resin was used. The total volume of DMF, calculated on the basis of resin used, wasallocated among amino acid, HOBt and DIC for solution preparation. For example, for thesynthesis involving 50 g of resin, the calculated volume of 250 mL of DMF was distributedamong amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vessel on theSonata® Pilot Peptide Synthesizer was charged with the solid dry amino acid (4-fold excesswith respect to the resin). At inception of the coupling step, the chosen volume of DMF andHOBt (4 eq.) in DMF and DIC (4 eq.) in DMF were delivered successively and after eachdelivery mixing by nitrogen bubbling was conducted. After the last reagent was deliveredmixing by nitrogen bubbling was initiated and conducted for 4 min. This served topreactivate the amino acid and. to insure complete dissolution of all components of the mixture.

[00115] After activation, the solution of the activated Fmoc-amino acid was transferred to the reaction vessel containing the resin. After transfer was complete the vessel wasagitated for 3 h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin waswashed thoroughly with DMF (5 mL/g, 6x) and the cleavage of the Fmoc-group was 47 WO 2007/067979 PCT/VS2006/061793 performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2x10 min). Theresin was thoroughly washed with DMF (5 mL/g, 6x) to assure complete removal ofpiperidine from the resin in preparation for the ensuing amino acid coupling. In the case ofFmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, the pre-activation in the amino acid bottlewas not conducted in order to minimize the formation of diketopiperazine during theactivation time as discussed in the text. Therefore, in these two cases, the solution of theamino acid, HOBt and DIC were added to the reaction vessel sequentially and the couplingprocess was conducted with ‘in situ’ activation. After chain elongation was completed, thefmoc group of the N-terminal amino acid was removed in the standard manner followed bythe standard wash with DMF (vide supra). The N-terminal amino acid was then capped bytreatment with freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M DIEAand 0.015M HOBt in DMF - 6 mL/g of resin), 2 x 20 min.

[00116] Functionalization of the e-amino group of C-terminal Lysine moieties of themonomer peptides (with Fmoc-Adoa or with Fmoc-Lys(ivDde) as required) wasaccomplished by first removing the ivDde group of the e-amino group with freshly prepared10% hydrazine in DMF (5 mL/g of resin -2x10 min). For appending of Fmoc-Adoa orFmoc-Lys(ivDde) the coupling time was increased to 10 h. After completion of the peptidesynthesis, the resin was treated with the cleavage cocktail, ‘Reagent B’(TFA:water:phenol:triisopropylsilaTie, 88:5:5:2, v/v/w/v) (10 mL/g of resin) for 4 h. Afterevaporation of the volatiles under vacuum, the paste was triturated with ether to provide asolid which was collected by filtration washed with diethyl ether and dried. A 10 mmol scalesynthesis of (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)']-NH2 gave 30 g(103% of theory) of the crude peptide. In the case of (13) Ac- VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2, a 10 mmol scale synthesis gave 28 48 WO 2007/067979 PCT/US2006/061793 g (107% of theory) of crude peptide. The greater than theoretical yields are most likely due to moisture and residual solvents.

Cyclization of the Linear Di-Cysteine Peptides to Cyclic Disulfide Peptides[00117] Cyclic disulfide peptides were prepared from the corresponding linear di-cysteine peptides by DMSO-assisted oxidation using DMSO/water (95/5, v/v). The crudelinear peptide was dissolved in the solvent mixture (5 mL/g) in a wide mouth beaker, and thepH of the solution was adjusted to'8.5 by the addition of solid N-methyl-D-glucamine inportions. The resulting mixture was stirred for 36 h at ambient temperature. The solutionwas then diluted with acetonitrile (50 mL/g) and the mixture was stirred for 2 min. The solidcyclic disulfide peptide was collected by filtration, washed with diethyl ether and dried.

Purification of Monomer Peptides

Peptide Monomer (12) Ac-AGPTWC*EDDWYYC* WLFGTGGGK[IC(ivDde)]-NH2; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Lys(ivDde)]-NH2 cyclic (6-13) disulfide [00118] A -0.5 g portion of the crude cyclic disulfide peptide monomer (12) wasdissolved in a minimum amount of DMSO (—3 mL). The volume of the solution wasadjusted to -100 mL with 20% CH3CN-water and employing a third pump, the solution wasloaded onto a reversed phase Cl 8 preparative column (Waters, XTerra® Prep MS Cl 8, 10 μ,300A, 50 x 250 mm, flowrate 100 mL/min), which had been pre-equilibrated with 10%CH3CN in water (0.1% TFA). During application of the sample solution to the column theflow of the equilibrating eluent from the preparative HPLC system was stopped.. After thesample solution was applied to the column, the flow of equilibrating eluent from the gradientHPLC system was reinitiated and the column was eluted with 10% CH3CN-water (0.1% TFA) until the DMSO was eluted. Then the eluent composition was ramped to 35% CH3CN-water (0.1 % TFA) over 1 min after which a linear gradient at a rate of 0.5%/min CH3CN(0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 49 WO 2007/067979 PCT/US2006/061793 mL) were manually collected using UV at 220 nm as an indicator of product elution. Thecollected fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column(5μ particle, 120A pore) and product-containing fractions of >95% purity were pooled andfreeze-dried to afford the corresponding cyclic disulfide peptide monomer (12). Typicallythe purification of 0.5 g of crude peptide monomer (12) afforded 0.1 g (20% yield) of thedesired product (>95% purity).

Peptide Monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 ; Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide [00119] Following the procedure employed for the HPLC purification of peptide monomer (2), the crude cyclic disulfide peptide monomer (13) Ac- VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 (0.5 g) dissolved in 20% CH3CN-water mixture (100 mL) was loaded onto a reversed phase Cl 8 preparative column (Waters,XTerra® Prep MS C18,50x250 mm, 10 μ particle, 300A pore, flow rate 100 mL/min),which had been pre-equilibrated with 10% CH3CN (0.1% TFA) in water (0.1% TFA).

During application of the sample solution to the column the flow of the equilibrating eluentfrom the preparative HPLC system was stopped. After the sample solution was applied to thecolumn, the flow of equilibrating eluent from the gradient HPLC system was reinitiated andthe column was eluted with 10% CH3CN-water (0.1% TFA) for 5 min. Then the eluentcomposition was ramped to 30% CH3CN (0.1% TFA)-water (0.1%TFA) over 1 min and alinear gradient elution at arate of 0.5%/min of CH3CN (0.1% TFA) into water (0.1% TFA)was initiated and maintained for 50 min. Fractions (15 mL) were manually collected usingUV at 220 nm as an indicator of product elution. The fractions were analyzed on a WatersXTerra analytical reversed phase C-18 column (4.6 mm i.d. x 50 mm, 5μ particle, 120A pore)and product-containing fractions of >95% purity were pooled and freeze-dried to afford the 50 WO 2007/067979 PCT/US2006/061793 corresponding cyclic disulfide peptide monomer (13). Typically the purification of 0.5 g of crude peptide monomer (3) afforded 0.12 g (24% yield) of the desired product (>95% purity). EXAMPLE 4

Preparation and Purification of Precursor Dimer Peptide (16) Ac- AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH2 cyclic (2-12) disulfideJ-NEL cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Tip-Tyr-Tyr-Cys-Tip-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(-Adoa-Adoa-Glut-Lys)]-NH2 cyclic (2-12) disulfide ]-NH2 cyclic (6-13) disulfide[00120] As shown in Figure 3, disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol) wasdissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11g, 0.85 mmol) was added in one portion. Then solid peptide monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH2 (0.50 g, 0.17 mmol) was added inportions to the stirred solution of DSG over a period of two min. After stirring for 30 min atroom temperature, the solution was diluted with anhydrous ethyl acetate to ~ 50 mL, (thisserved to precipitate intermediate mono-NHS ester (14)). The entire mixture was centrifugedand the supernatant was decanted leaving intermediate mono-NHS ester (14) as a colorlesssolid. The solid was resuspended with ethyl acetate; the solution containing the suspendedsolid mono-NHS ester (14) was centrifuged to separate the solid and the supernatant wasagain decanted. This washing process was repeated twice to remove completely the excess DSG.

[00121] The solid mono-NHS ester (14) was dissolved in stirred anhydrousdimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added.

Then solid peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2, (0.50 g, 0.19 mmol, 1.12 eq.) was added in portions to the stirred solution over a threemin. period and the resulting mixture was stirred for 18 h. The reaction was monitored bymass spectrometry; after the complete consumption of the peptide monomer glutaric acidmonoamide mono-NHS ester (14) was confirmed, neat hydrazine (0.1 mL) was added to 51 WO 2007/067979 PCT/US2006/061793 remove the ivDde protecting group of the ivDde-bearing dimer (15) and the mixture was stirred for 20 min at room temperature.

[00122] The solution was then acidified by dropwise addition of TFA and the mixturewas diluted to 100 mL with 10% CH3CN (0.1% TFA) in water (0.1% TFA). The solutionwas filtered to remove particulates and half of the clarified solution was loaded onto areversed phase Cl 8 preparative column (Waters, XTqrra® Prep MS Cl8, 10 μ, 50 x 250 mm,flowrate 100 mL/min) pre-equilibrated with 10% CH3CN in water (0.1% TFA). Duringapplication of the sample solution to the column the flow of the equilibrating eluent from thepreparative HPLC system was stopped. After the sample solution was applied to the column,the flow of equilibrating eluent from the gradient HPLC system was reinitiated and thecolumn was eluted with 10% CH3CN-water (0.1% TFA) in order to flush DMF from thecolumn. After elution of the DMF plug was completed the eluent composition was increasedto 20% CH3CN over one min. and the elution was continued with a linear gradient rate of0.6%/min of CH3CN (0.1% TFA) into water (0.1% TFA). Fractions (15 mL) were collectedusing UV (220 nm) as an indicator of product elution. The fractions were analyzed, on areversed phased C18 column (Waters MS C18,-4.6nnni.d. x 50 mm, 5μ particle, 120Apore)and the product-containing fractions of >95% purity were pooled and freeze-dried to provideprecursor dimer peptide (16) as a colorless, fluffy solid. The remaining crude precursordimer peptide (16) was purified in the same manner. From 0.5 g each of monomer peptides(12) and (13), 320 mg (overall yield 33%) of the desired dimer (16) was obtained (> 95%purity). EXAMPLE 5

Preparation of KDR-Binding Dimeric Peptide Phospholipid Conjugate (11) Acetyl-L-alanyl- glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L- tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-l-lcucyl-L-phcnylalanyl-glycyl-L- 52 WO 2007/067979 PCT/US2006/061793 threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L- aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl- L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L- lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-anuno-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide; Ac-AGPTWCEDDWYYCWLFGTGGGK {Ac-VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH2 cyclic (2-12) disulfide}-NH2cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys{Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys[-Adoa-Adoa-Glut-Lys(DSPE-PEG2000-NH~Glut)-]-NH2 cyclic (2-12) disulfide}-NH2 cyclic (6-13) disulfide.

[00123] The KDR-binding dimer (11) may be prepared by conjugation of precursordimer peptide (16), Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac- VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH2 cyclic (2-12) disnlfide]-NH2cyclic (6-13) disulfide, with DSPE-PEG2000-NH2 phospholipid, ammonium salt (18) asshown in Figure 4.

[00124] Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was added portionwise to a solution of disuccinimidyl glntarate (DSG, 0.15 g, 0.46 mmol), anddiisopropylethylamine (0.06 g, 0.47 mmol) in anhydrous DMF (3.0 mL) with stirring over aperiod of 3 min. Then the solution was stirred at ambient temperature for 30 min. Thereaction mixture was diluted to ~ 50 mL with anhydrous ethyl acetate; this resulted inprecipitation of the dimer glutaric acid monoamide mono-NHS ester (17), the glutaric acidmonoamide mono-NHS ester of the precursor dimer peptide (16). The solution wascentrifuged to pellet 6 (m/z, neg. ion, 1887.3 (M-3H)/3, 1415.1 (M-4H)/4, 1131.9 (M-5H)/5)as a colorless solid. The supernatant ethyl acetate layer containing excess DSG was decanted 53 WO 2007/067979 PCT/US2006/061793 from the compacted solid dimer glutaric acid monoamide mono-NHS ester (17) which wasagain resuspended in ethyl acetate, centrifuged and washed twice more to remove theremaining traces of DSG. The solid intermediate glutaric acid monoamide mono-NHS esterdimer derivative (17) thus obtained was dissolved in anhydrous DMF/CH2CI2 (8:2, v/v) (3.0mL); diisopropylethylamine (0.06 g, 0.47 mmol) was added and the solution was stirred.[00125] Meanwhile, DSPE-PEG2OOO-NH2 phospholipid ammonium salt (18) (0.235 g,0.084 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask andTFA (2 drops) was added to protonate the phosphodiester oxygen, facilitating solubilizationof phospholipid ammonium salt (18) in dichloromethane. The clear solution was concentrated to remove the volatiles and dried further under vacuum.

[00126] The solid phospholipid ammonium salt (18) was dissolved in DMF (2 mL) andtransferred to the stirred solution of glutaric acid monoamide mono-NHS ester dimerderivative (17) and the resulting mixture was stirred for 24 h at ambient temperature. Thereaction mixture was diluted with a solution of 50% CH3OH, 25% CH3CN and 25% water(1:1) to— 100 mL and the insolubles were filtered. Half of the filtered solution was loadedonto a reverse phased C4 preparative column (Rromasil® Prep C4, 10μ, 300A, 50 x 250 mm)which had been pre-equilibrated with 1:1 mixture of CH3OH and CH3CN (1:1,0.1 %TFA)and water (0.1 %TFA) at a flow rate of 100 mL/min. During application of the samplesolution to the column the flow of the equilibrating eluent from the preparative HPLC systemwas stopped. After the sample solution was loaded the flow of the equilibrating eluent wasreinitiated and the column was washed until the plug of DMF was eluted.

[00127] The composition of the eluent was then ramped to 70% CH3OH-CH3CN (1:1,0.1%TFA)-water (0.1%TFA) over 1 min and a linear gradient of 0.75%/min of CH3OH-CH3CN (1:1, 0.1%TFA) into water (0.1% TFA) was initiated. The elution was continuedafter reaching 100% B in order to achieve complete elution of the product from the column. 54 WO 2007/067979 PCT/US2006/061793

Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution and after the main product was eluted fraction collection was continued for several minutes in order to insure elution of trace amounts of starting phospholipid ammonium salt (18).

Fractions were checked for purity on an analytical HPLC system (column: YMC C4, 5μΜ,300A, 4.6 x 250 mm) using UV at 220 nm and an evaporative light scattering detector(ELSD). The latter detector is employed to detect DSPE-PEG2000-NH2 which has a weakchromophore at 220 nm. Product-containing fractions of >98% purity, and devoid of DSPE-PEG2OOO-NH2 phospholipid ammonium salt (8) were combined and concentrated to reducethe content of CH3OH. The solution was then diluted with, 10% CH3CN in water until a faintflocculent precipitate formed. The resulting solution was freeze-dried to afford the dimericpeptide phospholipid conjugate (11) as a colorless solid. The second portion of crude dimericpeptide phospholipid conjugate (11) was purified as described above. The combined yield ofthe target dimeric peptide phospholipid conjugate (11) was 0.39 g (57% yield). The samplesof the dimeric peptide phospholipid conjugate (11) made from different sample purificationruns were pooled together, dissolved, in tert-butanol-acetonitrile-water mixture and re-lyophilized to provide the dimeric peptide phospholipid, conjugate (11) as a colorless, fluffysolid which was further dried under vacuum.

[00128] Examples 6-8 below refer to the preparation of the dimer peptide-phospholipid - conjugate shown in Figure 5, wherein the dimeric conjugate contains very low levels of TFA.Figures 6-8 illustrate the methods described in the Examples below. EXAMPLE 6

Preparation of Dimeric Conjugate Having Low TFA Levels Via the Use of a Glutaryl Linker

Preparation of (23), (26) and dimer peptide (27) acetate salt by conversion of (22), (25) and dimer peptide 27 · nTFA salts to acetates by AG MP-50 ion-exchange resin [00129] For compound (23) an AG MP-50 ion-exchange resin (1.5 meq/mL resin bed)was suspended in 20% of CH3CN/H2O. The suspension was packed in a 3 x 30 cm glass 55 WO 2007/067979 PCT/US2006/061793 column and the final volume was 150 mL. The column was connected to a pump and aconductivity meter. It was washed with 20% of CH3CN/H2O at 17 mL/min flow rate untilthe conductivity was below 1 ps/cm. Compound (22) (210 mg) was dissolved in 20% ofCH3CN/H2O (80 mL) and the resulting solution was loaded to the column. The column waswashed again with the same eluent until its conductivity was below 1 ps/cm. A gradient ofNELiOAc in 20% of CH3CN/H2O was applied at 200 mM, 400 mM, 600 mM and 800 mM,250 mL each. The compound came out at 600 mM NH4OAC. The fractions were analyzedby HPLC and the ones containing the compound were combined and lyophilized severaltimes until the weight of the material was constant. 176 mg of the pure material (23) wasobtained as a white fluffy solid. The yield was 83.8%.

[00130] Additional parameters and results were as follows: HPLC : Ret. Time: 5.6min; Assay > 98% (area %); Column: Waters XTerra MS-C18, 4.6 x 50 mm, 5 μ particle, 120 A pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (0.1 %TFA); Elution: Initialcondition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3 mL/min; Detection: . UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1441.7 [M-2HJ/2, 960.9[M-3H]/3. CE analysis (counter-ion %wt./wt.): TFA estimated to he 0.3%; acetate 1.1%.[00131] For compound (26), following the same procedure for compound (23), 300 mgof the peptide TFA salt (25) in 80 mL of water was loaded at 17 ihL/min. to a 150 mL of AGMP-50 column, which was washed with H2O to conductivity of 1 ps/cm. The column wasthen washed with H2O again after loading, and the same step gradient of aqueous NH4OACinto H2O as employed for the ion exchange of compound (23) was applied. Lyophilization ofthe combined fractions to a constant weight afforded 200 mg of the acetate (26) as a whitefluffy solid. The yield was 66.7%.

[00132] Additional parameters and results were as follows: HPLC: Ret. Time: 5.6min; Assay 97.0% (area %); Column: Waters XTerra MS-C18, 4.6 x 50 mm, 5 μ particle, 56 WO 2007/067979 PCT/US2006/061793 120 A pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (0.1 %TFA); Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1336.9 [M-2H]/2, 890.8 [M-

3H]/3; CE analysis (counter-ion %wt./wt.): TFA estimated to be 0.4%; acetate 4.2%; IC analysis (F%): 0.26.

[00133] For the dimer peptide (27) acetate salt, similar to the procedure for compound(23), an AG MP-50 column (100 mL wet volume) was washed with 30% CH3CN/H2O untilthe conductivity was below 1 ps/cm. Compound (27) as the TFA salt, (120 mg in 80 mL of30% of CH3CN/H2O) was loaded onto the column and the column was washed with the sameeluent until the conductivity was stable at 1 ps/cm. A step gradient of NIL O Ac 30% ofCH3CN/H2O into 30% of CH3CN/H2O was run as for compound (23) and the compound waseluted at ca 600 mM NH&lt;)OAc. The combined fractions were lyophilized and thenrelyophilized several times until the material displayed a constant weight to provide 104 mgof the pure material (27) as an acetate salt. The yield was 86.7%.

[00134] Additional parameters and results were as follows: HPLC: Ret. time: 5.2min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6 x 50 mm, 5 p particle, 120 A pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (0.1%TFA); Elution: Initialcondition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3 tnL/min; Detection:UV at 220 nm;Mass Spectrum: API-ES; Mode: Negative ion; 1816.3 [M-3H]/3, 1362.0[M-4H]/4, 1089.2 [M-5H]/5; CE analysis (counter-ion %wt./wt.): TFA estimated to be0.2%; acetate 0.15%.

Preparation and purification of the dimer peptide (27) acetate salt from compound(23) and compound (26) [00135] To a solution of disuccinimidyl glutarate (18 mg, 0.055 mmol) in anhydrousDMF (0.1 mL) was added a solution of compound (23) (61mg, 0.021 mmol) in 0.2 mL of 57 WO 2007/067979 PCT/US2006/061793 anhydrous DMF dropwise (pH 8, neutralized by DIEA). The clear solution was stirred at RTfor 0.5h. HPLC and MS showed the completion of the reaction. Solvent was removed invacuo and EtOAc (8 mL) was added to precipitate the intermediate (24). The mixture wascentrifuged and decanted to remove excess glutarate. This EtOAc washing was repeated 3more times and the resulting solid was dried using a stream of dry nitrogen. It was thendissolved in 0.3 mL of anhydrous DMF. Compound (26), (56 mg, 0.021 mmol) was addedand the pH of the solution was adjusted to 8 by addition of DIEA. The solution was stirredfor 16 h at room temperature after which by HPLC and MS analysis indicated completion ofthe reaction. A 30 pL aliquot of NH2NH2 was added and the mixture was stirred for 5 min tocleave the ivDde group. The reaction mixture was analyzed by HPLC and MS, whichindicated complete removal of the ivDde group.

[00136] Before purification of the dimer peptide (27) acetate, caution was taken tocarefully wash the whole preparative HPLC system including the column with TFA-freeeluents, CH3CN/H2O/10 mM NH4OAC. The crude reaction mixture was then applied to areverse phase C-18 preparative column (Atlantis C-18, 5 pm particle, 100 A pore, 30 x 150mm, flow rate 30 mL/min), pre-equilibrated with 15% B (A: 10 mM NH4OAc in H2O; Β: 10mM NH4OAC in CH3CN/H2O, 9/1, v/v). The column was washed with the same eluent untilthe DMF plug was eluted. The eluent composition was increased to 25% B over 2 min. andthen ramped to 65% B over 40 min. The fractions were analyzed on an analytical reversephase C-18 column (Waters MS C-18, 4.6 x 50 mm, 5 pm particle, 100 A pore, flow rate 3mL/min) and the product-containing fractions of >95% purity were pooled and freeze-driedto afford 25 mg of the dimer peptide (27) as its acetate salt as a fluffy white solid. The yieldwas 21.8%.

[00137] Additional parameters and results were as follows: HPLC: Ret. time: 5.2min; Assay > 99% (area %); Column: Waters XTerra MS-C18,4.6 x 50 mm, 5 μ particle, 58 ' WO 2007/067979 PCT/US2006/061793 120 A pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (0.1%TFA); Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; [M-3H]/3,1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE analysis (counter-ion %wt./wt.): TFA estimated to be less than 0.2%; acetate 1.1%. EXAMPLE 7-Figure 7

Preparation of Dimer Peptide-Phospholipid Conjugates Having Low TFA Levels Via IonExchange Resin

Preparation and purification of the phospholipid peptide conjugate (21) as its acetatesalt from dimer peptide (27) acetate salt [00138] To a solution of disuccinimidyl glutarate-DSG (3.7 mg, 11.3 μτηοΐ) in anhydrous DMF (0.1 mL) was added a solution of neutralized dimer peptide (27) acetate salt,(15 mg, 2.75 pmol) in anhydrous DMF (0.2 mL), dropwise. The reaction solution was stirredat RT for 0.5 h. HPLC analysis with a Waters Xterra C-18 column and MS showed thecompletion of the reaction. The solvent was evaporated and EtOAc (8 mL) was added toprecipitate the intermediate (28). The vessel containing the precipitated intermediate (28)was centrifuged and the liquid layer was decanted. This procedure was repeated 3 times toremove the excess of DSG. The solid was dried with a stream of dry nitrogen and thendissolved in 0.3 mL of anhydrous DMF. DSPE-PEG2000-NH2 ammonium salt (29) (6.5 mg,2.33 μτηοΐ) was added in solid form and the pH of the mixture was adjusted to (28). Thereaction mixture was stirred at RT for 16 h. The mixture was analyzed by MS and HPLCwith a Zorbax 300 SB-C3 column and this indicated that the reaction was complete. 100139] To minimize the potential contamination of the product with TFA, the crudereaction mixture was purified by preparative HPLC equipped using a new Zorbax 300SB-C3column (21.2 x 150 mm, 5 μ particle) which had never been exposed to TFA. The HPLCsystem was pre-washed by CH3CN/H2O/NH4OAC extensively to remove traces of TFA. The 59 WO 2007/067979 PCT/US2006/061793 reaction mixture was loaded onto the column which was pre-equilibrated with 20% B (A: 10mM NH4OAc in H2O; B: 10 mM NKUOAc in CH3CN/H2O, 9/1 v/v) at a flow rate of 30mL/min. The column was eluted at 30 mL/min with the same eluent until the plug of DMFwas eluted. The eluent composition was then increased to 40% B over 3 min and thenramped to 90% B over 50 min. The collected fractions were analyzed on an analyticalreverse phase C-3 column (Zorbax 300SB-C3, 3 x 150 mm, 3.5 pm particle, 300 A pore, flowrate: 0.5 mL/min), where detection was accomplished using UV at 220 nm and anevaporative light.scattering detector (ELSD). The fractions containing the pure product werepooled and lyophilized. A 6.5 mg portion of the final product (21) acetate salt was obtained.The yield was 33.0%.

[00140] Additional parameters and results were as follows: HPLC: Ret. Time: 13.3min; Assay >99% (area %); Column: Zorbax 300SB-C3, 3 x 150 mm, 3.5 pm, 300 A pore;Eluent: A: H2O (0.1 % TFA), B: CH3CN/MeOH 1/1 (0.1 %TFA); Elution: Initial condition:60% B, linear gradient 60-90% B over 3 min; Flow rate: 0.5 mL/min; Detection: UV at 220nm and ELSD; CE analysis (counter-ion %wt./wt.): % wt. TFA: 0.3 %; % wt acetate 0.4%. EXAMPLE 8-Figure 8

Preparation of Dimeric Conjugate Having Low TFA Levels Via Sequential PurificationUsing Zorbax C-3 RP Preparative HPLC and Sephadex G-25 Gel PermeationChromatography [00141] - Materials used and conditions for the analytical HPLC system include the following: Column: Zorbax 300SB C-3 ; 3 mm i.d. x 150 mm; 3.5 pm particle; Eluent A :H2O (HPLC Grade with 0.1% TFA by volume); Eluent B: ,CH3CN (0.1% TFA by volume).Elution: Initial condition: 50% B then a linear gradient of 50-90% B over 3 min, hold at 90%B for 11 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm. Ret. time: (Compound (21)):6.77 min, Rt (lyso): 4.06 min. 60 WO 2007/067979 PCT/US2006/061793

Preparative HPLC using preparative Zorbax C-3 column to remove the lyso-compound from (21) [00142] The crude compound was loaded at a concentration of 30% eluent B.

Materials used and conditions include: Conditions: Column: Waters Zorbax 300SB C-3; 21.2mm i.d. x 150 mm; 3.5 pm particle; Eluents: Eluent A: H2O (HPLC Grade with 10 mMNHtOAc); Eluent B: CH3CN/H2O, 9/1 (final NH4OAC concentration: 10 mM ).

[00143] The composition of the eluent was then changed to 45% B over 2 min, thenthe column was eluted with a linear gradient of 45-100 % B over 40 min; Flow rate: 30mL/min; Detection: UV at 220 nm.

[00144] The crude compound (100 mg) was dissolved in 25 mL of a solution of 30% B. The preparative HPLC system was equilibrated at 30% B. The compound was loaded onto the Zorbax C-3 column. The mobile phase composition was ramped to 45% B over 2 min.A linear gradient from 45-100% B over 40 min was used for the elution of (21). The product eluted between 26.5-33 min.

[00145] The fractions that contained (21) were combined and lyophilized to give awhite fluffy sohd. This was dissolved in water-acetonitrile, then lyophilized again. Thisprovided 70 mg product devoid, of the lyso- compound. The recovery was about 70%. Afterchromatography was completed, the system was washed with 95% B for 15 min at a flow rateof 30 mL/min. The column was then washed with CH3CN/H2O (50/50, without TFA orbuffer) for 30 min at a flow rate of 15 mL/min. The column was then stored at roomtemperature for future use. Analytical HPLC confirmed the absence of the lyso- compoundin the isolated material. Further analysis confirmed that no lyso- compound formed after 5days at room temperature. The material still contained significant amounts (4.2 wt %) of TFA.

Removal of TFA from (21) by gel permeation chromatography on Sephadex G-25 61 WO 2007/067979 PCT/US2006/061793 [00146] A Sephadex G-25 column (100 g resin, bead size 20-80 pm, total gel volume ~ 500 mL, column height: 27 cm) was equilibrated with 4L of 50 mM ammonium bicarbonate.

Then (21) (70 mg) was dissolved in 30 mL (final volume) of 60 mM ammonium bicarbonate in 10% aqueous acetonitrile. The solution was filtered and then loaded on to the Sephadex G- 25 column. The column was eluted with 50 mM ammonium bicarbonate buffer with collection of 10 mL fractions. The collected fractions were monitored by analytical HPLC(UV detection at 220 nm). The results are provided in Table 4 below.

Table 4

Fraction # Volume (mL) Compound present (by HPLC analysisof fraction) 1 10 No 3 10 No' 6 10 No 9 10 No 12 10 No 15 10 No 18 10 No 19 10 No 20 10 Yes 21 10 Yes 24 10 Yes 27 10 Yes 28 10 Yes 29 10 No [00147] Fractions 20-28 were pooled and lyophilized. The lyophilized materialobtained was rc-dissolvcd in a small volume of water and the solution was frozen andlyophilized to remove residual amounts of ammonium bicarbonate. The final weight of thedesired material was 58 mg. The recovery was 83%. 62 WO 2007/067979 PCT/US2006/061793

[00148] To ascertain the effective removal of TFA, the sample was subjected to CE analysis for TFA and acetate ions. The TFA is clearly present in the starting material (4.2%) according to the previous assay, while it is hardly detected (0.2 %) after the gel permeation procedure. No acetate ion was detected.

Analytical data for (21) obtained by serial Zorbax C-3 preparative HPLC andSephadex G-25 gel permeation chromatography [00149] Materials used and conditions for collecting analytical data include: Fluorine analysis (IC by QTI): 751 ppm (0.15% TFA wt/wt); Mass Spectrum: Method: MALDI-TOF; Mode: Positive Ion; Average molecular weight detected was 8461 the typical PEG2000mass distribution curve was observed. HPLC: System A: Column: Zorbax 300SB C-3 ;- 3nim i.d. x 150 mm; 3.5 pm particle; Eluent A : Water (HPLC Grade with 0.1% TFA byvolume); Eluent B: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution:linear gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate: 0.5 mL/min;Detection: UV at 220 nm. Ret time: 6.77 min; Area %: 99.6%. System B: Column: Zorbax300SB C-3; 3 mm i.d. x 150 mm; 3.5 pm particle; Eluent A : Water (HPLC Grade with 0.1%TFA by volume); Eluent B: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B;Elution: linear gradient of 50 - 90% B over 3 min then ramp to 100% B over 12 min. Flowrate: 0.5 mL/min; Detection: LSD; Ret: time: 13.98 min. Area %: 99.3%.

[00150] Table 5 below provides definitions for the abbreviations used, and the sourcesof materials referred to in Examples 9-12.

Table 5 DSPA.Na (Genzyme) IUPAC: l,2-Distearoyl-sn-glycero-3-phosphosphatidic acid, sodium salt DPPG.Na (Genzyme) IUPAC: l,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt DPPE (Genzyme) IUPAC: l,2-Dipalmitoyl-sn-glyceip-3-phosphoethanolamine DSPC Distearoyl-glycero-phosphatidylcholine (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3- phosphocholine 63 WO 2007/067979 PCT/US2006/061793 DSPG.Na (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3-phosphoglycerol, sodium salt DSPE-PEG1000 Distearoyl-glycero-phosphoethanolamine-N-methoxy(polyethylene glycol)1000 (Avanti Polar) DSPE-PEG2000 Distearoyl-glycero-phosphoethanolamine-N-methoxy(polyethylene glycol)2000 (Avanti Polar) Stearate* Sodium Stearate (Fluka) PEG4000 (polyethylene glycol) MW 4000 (Fluka) Mannitol (Fluka) *the acid form, i.e., stearic acid, can also be used in any of the microbubble preparations herein. EXAMPLE 9

Preparation of Targeted Microbubbles with DSPC/DPPG Envelope

Example 9A

[00151] 383 mg of a mixture of DSPC/DPPG/ and the dimeric peptide phospholipid conjugate (11) shown in Figure 5 (molar ratio 49.75/49.75/0.5, corresponding to 187.1, 176.4and 19.8 mg of the three components, respectively) and PEG-4000 (22.6 g) were solubilizedin 120 g of t-butyl alcohol at 60°C, in a water bath. The solution was filled in vials with 0.8mL of solution each. The samples were frozen at -45°C and lyophilized. The air in theheadspace was replaced with, a mixture of C4Fio/Nitrogen (50/50) and vials capped andcrimped. The lyophilized samples were reconstituted with 5 mL of H2O per vial.

Example 9B

[00152] Example 9A was repeated using a mixture of DSPC/DPPG/ and the monomeric peptide phospholipid conjugate (31) shown in Figure 10 (molar ratio 49.5/49.5/1,corresponding to 182.8, 172.3 and 28.2 mg of the three components, respectively) EXAMPLE 10

Preparation of Targeted Microbubblcs with DPPE/DPPG Envelope

Example 10A 64 WO 2007/067979 PCT/US2006/061793 [00153] An aqueous suspension of DSPE-PEG1000 (0.43 mg - 0.24 pmole) and the monomeric peptide phospholipid conjugate (31) shown in Figure 10 (3.0 mg — 0.5 pmole) was prepared in 500pL of distilled water at 60°C to obtain a micellar suspension.

[00154] Separately, DPPE (15.8 mg - 22.8 pmoles) and DPPG (4.2 mg - 5.7 gmoles)were dispersed in a solution of mannitol 10% in distilled water (20mL) at 70°C for 20minutes. The dispersion was then cooled to room temperature. Perfluoroheptane (1.6mL) wasemulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probediameter of 3 cm) for 1 minute at 10500 rpm to obtain an emulsion.

[00155] The micellar suspension was added to the emulsion and the resulting mixturewas heated at 60°C for 1 hour under stirring. After cooling to room temperature (1 hour), theobtained emulsion was divided in 4mL fractions in 50mL round bottom flasks. The emulsion was frozen at -45°C for 5 minutes and freeze-dried at 0.2mBar for 24 hours (Freeze-DrierChrist Beta 1-8K).

[00156] Before redispersion, the lyophilisate was exposed to an atmosphere containingC4F10/nitrogen (50/50 by volume). The lyophilized, product was then dispersed in a volumeof water twice the initial one by gentle hand shaking.

Example 10B

[00157] An aqueous suspension of DSPE-PEG1000 (0.5 mg — 0.27 pmole) anddimeric peptidephospholipid conjugate (11) shown in Figure 5 (5.3 mg - 0.63 μτηοίε) wasprepared in 500pL of distilled water at 60°C to obtain a micellar suspension. 100158] Separately, DPPE (15.8 mg - 22.8 pmoles) and DPPG (4.2 mg - 5.7 pmoles)were dispersed in a solution of PEG4000 10% in distilled water (20mL) at 70°C for 20minutes. The dispersion was then cooled to room temperature. Perfluoroheptane (1.6mL) wasemulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probediameter of 3 cm) for 1 minute at 10000 rpm to obtain an emulsion. . 65 WO 2007/067979 PCT/US2006/061793 [00159] The micellar suspension was added to the emulsion and the resulting mixture was heated at 80°C for 1 hour under stirring. After cooling to room temperature (1 hour), theobtained emulsion was washed once by centrifugation (200g/10min - Sigma centrifuge3K.10) to eliminate the excess of phospholipid. The separated pellet (containing emulsifiedmicrodroplets of solvent) was recovered and re-suspended with the initial volume of a 10%PEG4000 aqueous solution.

[00160] The obtained emulsion was sampled into DIN8R vials (ImL/vial). Then vialswere cooled at -50°C (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at -25°C and0.2 mBar for 12 hours with a final drying step at 30°C and 0.1 mBar for 7 hours.

Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) andsealed. The lyophilized product was redispersed in a volume of water twice the initial one bygentle hand shaking. EXAMPLE 11

Preparation of Targeted. Microbubbles with DSPC/DSPA Envelope

Example 11A

[00161] An aqueous suspension of DSPE-PEG1000 (2.5 mg -1.4 pmole) and dimeric peptide conjugate (11) shown in Figure 5 (7.0 mg - 0.84 pmole) was prepared in 1 mL ofdistilled water at 60°C to obtain a micellar suspension.

[00162] Separately, DSPC (16.3mg - 20.6 pmoles) and DSPA (3.7 mg - 5.15 gmoles)were dissolved in cyclooctane (1.6 mL) at 80°C. This organic phase was added to a PEG400010% solution in water (20 mL) using a high speed homogenizer (Polytron T3000, probediameter of 3 cm) for 1 minute at 8000rpm, to obtain an emulsion.

[00163] The micellar suspension was mixed with the emulsion and the resultingmixture was heated at 80°C for 1 hour under agitation. After cooling to room temperature (1hour), the obtained emulsion was washed once by centrifugation (1500g/10min - Sigma 66 WO 2007/067979 PCT/US2006/061793 centrifuge 3K.10) to eliminate the excess of the phospholipid. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution.

[00164] The obtained suspension was sampled into DIN8R vials (1 mL / vial). Thenvials were cooled to -50°C (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at -25°Cand 0.2 mbar for 12 hours, with a final drying step at 30°C and 0.1 mbar for 7 hours.

Vials were exposed to an atmosphere containing C4Fio/Nitrogen (35/65 by volume) and sealed.

The lyophilized product was then dispersed in a volume of water twice the initial one bygentle hand shaking.

Example 1 IB

[00165] Example 11A was repeated, but using 0.7mg of DSPE-PEG2000 (0.26pmoles) and 1.6 mg of monomeric peptide-phospholipid conjugate (1) shown in Figure ,2(0.26 pmole) to prepare the micellar suspension.

Example 11C

[00166] DSPC (16.3 mg - 20.6 pmoles), DSPA (3.7 mg - 5.15 pmoles) andmonomeric peptide phospholipid conjugate (1) shown in Figure 1 (1.6 mg - 0.26 pmole)were dissolved in cyclooctane (1.6 mL) at 80°C. This organic phase was emulsified in aPEG4000 10% aqueous phase (20 mL) using a high speed homogenizer (Polytron PT3000,probe diameter of 3 cm) for 1 minute at 8000 rpm to obtain an emulsion.

[00167] The resulting emulsion was heated at 80°C for 1 hour under stirring. Aftercooling to room temperature (1 hour), the obtained emulsion was diluted with 20ml of aPEG4000 10% aqueous solution. 67 WO 2007/067979 PCT/US2006/061793 [00168] The emulsion was sampled into DIN8R vials (ImL/vial). Then vials were cooled at -50°C (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at -25°C and 0.2 mBar for 12 hours with a final drying step at 30°C and 0.1 mBar for 7 hours.

Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) andsealed. The lyophilized product was redispersed in a volume of water twice the initial one by-gentle hand shaking. EXAMPLE 12

Preparation of Targeted Microbubbles with DSPC/S tearate Envelope

Example 12A

[00169] An aqueous suspension of DSPE-PEG2000 (2.5 mg - 0.9 gmoles) and thedimeric phospholipid conjugate (11) shown in Figure 5 (2.5 mg - 0.3 pmoles) was preparedin 660pL of distilled water at 60°C to obtain the micellar suspension.

[00170] Separately, DSPC (18.2 mg - 23.1 pmoles) and stearate (1.8 mg - 5.8 pmoles)were dissolved in cyclooctane (1.6 mL) at 80°C. This organic phase was added to aPEG4000 10% solution in water (20 mL) using a high speed homogenizer (Polytron T3000,probe diameter of 3 cm) for 1 minute at 9000ipm, to obtain an emulsion.

[00171 ] The micellar solution was mixed with the emulsion and the resulting mixture was heated at 80°C for 1 hour under agitation. After cooling to room temperature (1 hour),the obtained emulsion was washed once by centrifugation (1500g/l Omin - Sigma centrifuge3K10) to eliminate the excess of phospholipids. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended with twice the initialvolume of a 10% PEG 4000 aqueous solution.

[00172J The obtained suspension was sampled into DJLN8R vials (1 mL/vial). Thenvials were cooled to -50°C (Christ Epsilon 2-12DS Freeze Diyer) and freeze-dried at -25°Cand 0.2 mbar for 12 hours, with a final drying step at 30°C and 0.1 mbar for 7 hours. 68 WO 2007/067979 PCT/US2006/061793

Vials were exposed to an atmosphere containing C4Fio/Nitrogen (35/65 by volume) and sealed.

The lyophilized product was dispersed in a volume of water twice the initial one by gentlehand shaking.

Example 12B

[00173] Example 12A was repeated by replacing the dimeric peptide phospholipidconjugate (11) shown in Figure 5 with the same relative molar amount of the monomericpeptide phospholipid conjugate (1) shown in Figure 2.

Example 12C

[00174] Example 11C was repeated with DSPC (18.2mg-23.1 pmoles), sodiumstearate (1.8 mg- 5.8 pmoles) and the dimeric peptide phospholipid conjugate (11) shown inFigure 5 (2.2 mg - 0.26 pmole). The agitation speed for emulsification was fixed to9000rpm. After cooling to room temperature (1 hour), the obtained emulsion was washedonce by centrifugation (1500g/10min - Sigma centrifuge 3K10) to eliminate the excess of thephospholipid. The separated supernatant (containing emulsified microdroplets of solvent) wasrecovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution. EXAMPLE 13

Static Binding Test on KDR-Transfected Cells

Plasmid production and purification [00175] Full-length KDR was cloned into the pcDNA6 vector and the plasmid wasamplified in competent DH5ra E. coli. Plasmid amplification and purification was performedusing E. coli JM 109 and a kit from Quiagen.

Transfection of 293H cells on Thermanox® coverslips[00176] Cells were grown on poly-D-lysine-coated Thermanox® circular coverslips in24-well plate. Transfection was done as recommended in the lipofectamine 2000 protocol 69 WO 2007/067979 PCT/US2006/061793 (Invitrogen, cat# 11668-019) using 1 pg of DNA (pc-DNA6-fKDR)/per coverslip (1.3 cm2)in 0.1 mL. Transfection was done in serum-free media, the transfection reagent mix wasremoved from cells after 2 hours and replaced with regular serum-containing medium. Someof the cell-coated coverslips were mock-transfected (with no DNA). The next day, expressionof the KDR receptor was assessed by immunocytochemistry and the binding assay wasperformed.

Bubble binding assay [00177] The transfected cells were incubated with KDR-targeted microbubblesresuspended in 50% human plasma in PBS. For the incubation with the transfected cells asmall plastic cap was filled with a suspension containing a 1.3xlO8 bubbles and the cap wascovered with an inverted Thermanox® coverslip so as to put the transfected cells in contactwith the targeted microbubbles. After 30 min of incubation at RT, the coverslip was liftedwith tweezers, rinsed three times in PBS and examined under a microscope to assess bindingof the targeted microbubbles.

Determination of the % of surface covered by microbubbles[00178] Images were acquired with a digital camera DC300F (Leica) and the percentof surface covered by bound microbubbles in the imaged area was determined using thesoftware QWin version 3.1 (Leica Microsystem AG, Basel, Switzerland). Pictures were takenof each Thermanox®coverslip. For each preparation of Examples 9 and 10, the bindingassay was repeated a minimum of two times thus obtaining an average value of the surface covered.

In the following Tables 6 and 7, the binding activity of the microbubblcs prepared accordingto Examples 9 and 10 above arc recorded.

[00179] As indicated by the Tables, the same peptide may show different bindingactivities when included (as a lipopeptide) in different phospholipid formulations forming the 70 WO 2007/067979 PCT/US2006/061793 stabilizing envelope of the microbubhle. Microbubbles containing KDR binding lipopeptides of the invention bind specifically to KDR-expressing cells while they did not hind appreciably to mock transfected cells. EXAMPLE 14

Dynamic Binding test on rh VEGF-R2/Fc Chimeric Protein

Preparation of Fc-VEGF-R2-coated coverslips[00180] Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler, PA, USA) werecoated with recombinant human VEGF-R2/Fc Chimeric protein (R&amp;amp;D Systems) accordingthe following methodology.

[00181] A surface of dimensions 14 x 25 mm was delimited on the glass coverslipusing a special marker (Dako Pen) and 400 pL of Fc-VEGF-R2 solution at 4 pg/mL in PBSwas deposited on this surface. After an overnight incubation at 4°C, the solution wasaspirated, replaced by 0.5 mL of a solution of BSA 1% w/v in PBS-0.05% Tween 80, pH 7.4and incubated for 3 hours at RT. Then the coverslip was washed, three times with 5 ml of PBS-0.05% Tween 80.

Binding assay [00182] Binding studies of targeted bubbles were carried out using a parallel-plate flow chamber (FCS2, Bioptech Tnc., Butler, PA, USA) with a chamber gasket of 0.25 mm inthickness, with a customized adapter for upside-down chamber inversion. The coatedcoverslip was inserted as a plate of the flow chamber. Microbubbles (5 x 106 bubbles/mL in50% human plasma in PBS) were drawn through the flow chamber using an adjustableinfusion pump (Auto Syringe® AS50 Infusion Pump, Baxter, Deerfield, JLL, USA) with a 60mL syringe (Terumo). The pump flow rate was adjusted to 1 mL/min to obtain the desiredshear rate of about 114 s'1. After 10 minutes, the flow was stopped and pictures were takenrandomly at different positions on the covcrsEp (on areas of about 0.025 mm2) using a 40 x 71 WO 2007/067979 PCT/US2006/061793 objective and a CCD monochrome camera (F-View Π, Soft Imaging Systems, Germany) connected to an inverted Olympus IX 50 microscope.

[00183] The number of microbubbles on each picture was determined, averaged withrespect to the total number of pictures and the obtained value was then divided by ten (toobtain the “slope”, i.e. the average amount of bound microbubbles per minute).

[00184] For each preparation of Examples 11 and 12, the binding assay was repeated four times thus obtaining an average value of the slope.

[00185] The slope represents the bubble binding rate on the target substrate. Forinstance, a slope value of 8 indicates that an average of eighty (80) microbubbles was boundon the coated coverslip in ten minutes. A higher slope indicates a better capacity of bubblesto bind to the target under flow conditions.

[00186] In the following tables 8 and 9, the binding activity of the microbubblesprepared according to Examples 11 and 12 above were illustrated.

[00187] As inferable from the tables, the same peptide may show different bindingactivities when included (as a peptide-phospholipid conjugate or lipopeptide) in differentphospholipid formulations forming the stabilizing envelope of the microbubhle.

Table 6

Example KDR Mock KDR- Mock 9A 28.6% 0.4% 28.3% 9B 28.0% 0.3% 27.7%

Table 7

Example KDR Mock KDR- Mock 10A 23.6% 0.2% 23.5% 10B 28.0% 0.0% 28.0% 72 WO 2007/067979 PCT/US2006/061793

Table 8

Example Slope 11A 8.2 11B 8.1 11C 5.8

Table 9

Example Slope 12A 9.0 12B 8.0 12C 7.8 EXAMPLE 15

In Vivo Evaluation of Ultrasound Contrast Agents Targeted to KDR[00188] The ability of ultrasound contrast agents containing KDR binding lipopeptidesof the invention to bind to KDR-expressing tissue in vivo was assessed using a known modelof angiogenesis: the rabbit VX2 tumor model.

[00189] A known model of angiogenic tissue was used to examine the ability of theKDR-targeted ultrasound microbubbles to localize to and provide an image of angiogenic tissue.

The VX2 rabbit carcinoma was serially implanted in the dorsal muscle ofNew Zealandrabbits (Charles River Laboratories, France) weighting 2.5/3kg.

Preparation of tumor homogenate [00190] Tumor was surgically removed, placed into McCoy’s culture mediumcontaining 10% fetal calf serum, antibiotics, 1.5 mM Glutamax 1 and cut into small pieces 73 WO 2007/067979 PCT/US2006/061793 that were rinsed to remove blood and debris. Then tumor pieces (3 to 5 cm3) were placed in a 50 ml Falcon tube containing 5 mL of complete medium. The tumor tissue was ground (Polytron) until no more solid pieces were visible. The murky fluid was centrifuged for 5 minutes at 300g and the supernatant discarded. Seven mL of fresh medium was added per 5 mL of pellet.

Tumor implantation [00191] Rabbits received first 0.3 mL ofVetranquil (Acepromazine, Sanofi, injectedintramuscularly) and were then anesthetized with an intramuscular injection ofKetaminol®5/Xylazine (Veterinaria AG/Sigma) mixture (50/10 mg/mL, 0.7 mL/kg).

One hundred microliters of VX2 tumor homogenate was injected intramuscularly. Fifteendays after implantation of VX2 tumors, animals were anesthetized again with the samemixture, plus subcutaneous injection of 50% Urethane (2mL/kg, s.c.) (Sigma) for imagingexperiments.

In vivo ultrasound imaging [00192] VX2 tumor imaging was performed using an ultrasound imaging system ATLHDI5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversion at highacoustic power (MI=0.9) was used to evaluate accumulation of targeted microbubbles on theKDR receptor expressed on the endothelium of neovessels. The linear probe was fixed on theskin directly over the implanted tumors.

[00193] After bubble injection (0.1 gL/kg of gas) using the preparations of eitherExample 16 or Example 17, insonation was stopped allowing bubbles to accumulate for 25minutes. Then, insonation was reactivated at high acoustic power (MI 0.9) destroying all thebubbles present in the tumor. The amount of free circulating bubbles was then assessed byrecording the signal obtained after 20 sec accumulation without insonation.

Video frames from VX2 tumor imaging experiments were captured with video-capture and 74 WO 2007/067979 PCT/US2006/061793 analysed with Image-Pro Plus 2.0 software. The image representing free circulating bubbles was subtracted from the image obtained at 25 min, to provide an image representing bound bubbles.

Referring to Figure 11 (which shows the results with the preparation of Example 16) andFigure 12 (which shows the results with the preparation of Example 17) , Figures 11A and12A show an image before bubble injection (baseline); Figures 1 IB and 12B show retentionof bubble contrast in the tumor 25 minutes post injection; and Figures 11C and 12C show theresult obtained after subtraction of the baseline and free circulating bubbles and representbound microbubbles containing KDR lipopeptides according to the present invention.Examples 15-17 and Figures 11 and 12 confirm that ultrasound contrast agents bearing suchKDR binding moieties localize to KDR expressing (and thus angiogenic) tissue in animal models. EXAMPLE 16 [00194] Example 12A was repeated by replacing DSPE-PEG2000 with DSPE-PEGI000 (2.7 mg, 1.54 pmol) and. using 2.5 mg (0.31 pmol) of dimeric peptide phospholipidconjugate (11) shown in Figure 5. EXAMPLE 17 [00195] Example 16 was repeated by replacing the dimeric peptide phospholipidconjugate with the same molar amount of monomeric phospholipid conjugate (1) shown inFigure 2. 75 ΟΊΟϋΕΖΕΊ ΠΊΕΖΕ , οίίκεπ rwzn οϊό ίπίε^ο ρίομ pnszn irn πτ -|eoe,ρνιο ίχεπ ίεοεπε γώεήε ποεήίεε πρ’ίοο .□όοεζεπ πί&amp;amp;έο mpnon ίπίΛ οχηποοιηπη Ίϊζ

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