English:
[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 detect VEGF/KDR binding complexes would be extremely beneficial in detecting or monitoring angiogenesis.
[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 using such monomeric peptides.
[0018] 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
[0025] FIGURE 9 illustrates another representative monomeric peptide (32) having a high binding affinity for KDR.
[0026] FIGURE 10 illustrates another monomeric peptide-phospholipid conjugate
[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 (FlG. 12A); after 25 minutes (FlG. 12B); and after subtraction of the baseline and free circulating bubbles (FIG. 12C).
[0029] Applicants have unexpectedly discovered peptide phospholipid conjugates, which are useful in producing targeted ultrasound contrast agents and which have exceptional KDR binding efficiency. Two of these compounds are monomeric peptide phospholipid conjugates which include a linear peptide monomer which binds with high affinity to KDR while the other is a dimeric peptide phospholipid conjugate which includes two distinct monomer subunits, each binding to KDR. In addition, highly efficient methods for large scale production of purified forms of these conjugates and precursor materials have been discovered. Such methods include the production of dimeric peptide phospholipid conjugates having minimimal levels of TFA.
[0044] The reaction mixture may be diluted with a 1 : 1 mixture of water (0.1 %TF A) and CH 3 CN-CH 3 OH (1:1, v/v) (0.1%TFA) (-100 mL), applied to a reversed phase C2 column (Kromasil ® Prep C2, 10 μ, 300A, 50 x 250 mm, flow rate 100 rnL/niin) and the column may be eluted with a 3:1 mixture of water (0.1%TFA) and CH 3 CN-CH 3 OH (1 :1, v/v) (0.1%TFA) to remove hydrophilic impurities. Then the product may be eluted using a gradient OfCH 3 CN-CH 3 OH (1:1) (0.1% TFA) into water (0.1% TFA) (see Experimental Section for details). The collected fractions may be analyzed by reversed phase HPLC using an ELS detector which allows the detection of the desired product and the often difficult-to- separate DSPE-PEG2000-NH 2 phospholipid which has very little UV absorbance. This indicates the clear separation of the monomeric peptide phospholipid conjugates and DSPE- PEG2000-NH 2 phospholipid. The pure product-containing fractions may be collected, concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried to provide monomeric peptide phospholipid conjugates as a colorless solid. In order to prepare the required quantity of the monomeric peptide phospholipid conjugates, several runs may be conducted employing 0.5 g to 1.0 g of linear peptide monomer. In all cases the target monomeric peptide phospholipid conjugates may be were isolated in high yield and purity (e.g., 57-60% yield and >99% purity). Dimer Conjugate Generally
[0054] Analysis of the impurities obtained from nonoptimized syntheses of the monomer peptides indicates that the major problems arc raccmization, incomplete couplings and piperidine amide formation (presumably via an intermediate aspartimide or glutarimide intermediate), each of which contributes to suboptimal yield and purity. A dramatic decrease in formation of the piperidine amide may be attained by the use of 25% piperidine in DMF containing HOBt (0. IM) as the reagent for fmoc removal. Racemization may be considerably reduced by using DIC/HOBt as the activator for most couplings; and a 3 h coupling time using a four-fold excess of pre-activated Fmoc-amino acid with an intervening wash with anhydrous DMF (6x). N- α 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 the amino acid vessels of the instrument and then programming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution after each addition.
[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 the monomer and dimer peptides and their subsequent functionalization, which also may be optimized. The ivDde group on the e-amine of the C-terminal lysine of each of the peptide monomers may be removed using 10% hydrazine in DMF. Then Fmoc-Adoa, for monomer (13) or Lys(ivDde) for monomer (12) may be appended to the exposed lysine e-amino group using 4 equivalents of the Fmoc amino acid and 4 equivalents each of DTC and HOBt in DMF for 10 h. After completion of the synthesis, the peptide sequence may be cleaved 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 reaction was complete the peptide may be precipitated, washed with diethyl ether and dried.
[0062] Conjugation of the DSPE-PEG2000-NH 2 to the precursor dimer peptide may be accomplished by preparation of a glutaric acid monoamide mono NHS ester of the precursor dimer and reaction of this with the free amino group of the phospholipid ammonium salt. Thus the ivDde bearing precursor dimer peptide (16) may be reacted with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. As in the preparation of the precursor dimer peptide the solution may be diluted with ethyl acetate to precipitate the glutaric acid monoamide mono-NHS ester of the dimer (17), as a solid. The supernatant may be decanted to remove the un-reacted DSG. The solid glutaric acid monoamide mono-NHS ester of the dimer peptide (17) may then be washed several times with ethyl acetate to remove traces of DSG. Mass spectral results confirm the formation of the glutaric acid monoamide mono-NHS ester of the peptide dimer as a clean product.
[0067] Table 3 provides a description for the identification labels shown in Figures 6,
[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 details are 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 the phospholipid-peptide conjugate (21) and TFA ions. In this embodiment 21* nTFA adduct may be eluted down a size exclusion column in the presence of ammonium bicarbonate buffer. The crude 21« nTFA initially may be freed of the lyso- compound by preparative HPLC on a Zorbax C-3 column using a linear gradient of acetonitrile into water. Both phases may be buffered with 10 mM ammonium acetate. This provides separation of the lyso- compound as indicated by analytical HPLC.
[0075] Both the monomeric and dimeric peptide phospholipid conjugates described herein may be incorporated into ultrasound contrast agents such as, for example, gas filled micro vesicles. Such gas filled microvcsiclcs include, for example, gas filled microbubblcs, gas filled microballoons, gas filled microcapsules, etc. In a preferred embodiment, the peptide phospholipid conjugates may be incorporated into ultrasound contrast agents comprising gas filled microbubbles. Methods of preparation of gas filled microbubbles from phospholipids and phospholipid conjugates are known to those skilled in the art. For example, microbubbles according to the present invention can be prepared by methods described 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, which are incorporated by reference herein in their entirety. The methods disclosed in WO 04/069284 are particularly preferred.
[0081] Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer thereto. Examples of modified phospholipids are phosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), in brief "PE- PEGs", i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g. from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG, DMPE-PEG or DAPE-PEG (where DAPE is 1,2-diarachidoyl-sn- glycero-3-phosphoethanolamine). The compositions also may contain other amphiphilic compounds including, for instance, fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; sterols, such as cholesterol, or esters of sterols with fatty acids or with sugar acids; glycerol or glycerol esters including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate, glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate; tertiary or quaternary alkyl-ammonium salts, such as 1 ,2- distearoyl-3-trimethylamrnonium-propane (DSTAP), 1 ,2-dipalmitoyl-3-trimethylammonium- propane (DPTAP),and mixtures or combinations thereof.
[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 methods for the treatment of a disease in a patient, as well as any substance which is capable of exerting or responsible to exert a biological effect in vitro and/or in vivo. Therapeutic agents thus include any compound or material capable of being used in the treatment (including diagnosis, prevention, alleviation, pain relief or cure) of any pathological status in a patient (including malady, affliction, disease lesion or injury). Examples of therapeutic agents are drugs, pharmaceuticals, bioactive agents, cytotoxic agents, chemotherapy agents, radiotherapeutic agents, proteins, natural or synthetic peptides, including oligopeptides and polypeptides, vitamins, steroids and genetic material, including nucleosides, nucleotides, oligonucleotides, polynucleotides and plasmids.
[00103] Examples 1-2 below refer to the monomeric peptide phospholipid conjugate shown 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 the compound shown in Figure 2, a similar process may used to prepare the monomeric peptide phospholipid conjugate shown in Figure 10 and the linear peptide monomer (32) shown in Figure 9 as well as other monomer peptide-phospholipid conjugates. Additionally, co- pending U.S. Application No. 10/661,156, filed September 11, 2003, sets forth methods for the preparation of the peptide monomers and is incorporated by reference herein in its entirety.
[00107] A ~0.5 g portion of the crude linear peptide monomer (2) was dissolved in a minimum amount of CH3CN (~20 mL). The volume of the solution was adjusted to -100 mL with water and employing a third pump the solution was loaded onto a reversed phase C 18 preparative column (Waters, XTerra ® Prep MS Cl 8, 10 μ, 3OθA, 50 x 250 mm, flow rate 100 rnL/min) which had been pre-equilibrated with 10% CH 3 CN in water (0.1% TFA). The column 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 was ramped to 20% CH 3 CN-water (0.1%TFA) over 1 min, and a linear gradient at a rate of 0.6%/min Of CH 3 CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The collected fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (5μ particle, 120 A pore) and product-containing fractions of >95% purity were pooled and freeze-dried to afford the corresponding pure linear peptide monomer (2). Typically the purification of 0.5 g of crude (2) afforded 0.12 g (24% yield) of the desired product (>95% purity). EXAMPLE 2
[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, was allocated among amino acid, HOBt and DIC for solution preparation. For example, for the synthesis involving 5O g of resin, the calculated volume of 250 mL of DMF was distributed among amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vessel 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 chosen volume of DMF and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF were delivered successively and after each delivery mixing by nitrogen bubbling was conducted. After the last reagent was delivered mixing by nitrogen bubbling was initiated and conducted for 4 min. This served to preactivate 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 was agitated for 3 h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g, 6x) and the cleavage of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2 x 10 min). The resin was thoroughly washed with DMF (5 mL/g, 6x) to assure complete removal of piperidine from the 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 not conducted in order to minimize the formation of diketopiperazine during the activation time as discussed in the text. Therefore, in these two cases, the solution of the amino acid, HOBt and DIC were added to the reaction vessel sequentially and the coupling process was conducted with 'in situ' activation. After chain elongation was completed, the fmoc group of the N-terminal amino acid was removed in the standard manner 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 - 6 mL/g of resin), 2 x 20 min.
[00116] Functionalization of the e-amino group of C-terminal Lysine moieties of the monomer peptides (with Fmoc-Adoa or with Fmoc-Lys(ivDde) as required) was accomplished by first removing the ivDde group of the e-amino group with freshly prepared 10% hydrazine in DMF (5 mL/g of resin - 2 x 10 min). For appending of Fmoc-Adoa or Fmoc-Lys(ivDde) the coupling time was increased to 1 O h. After completion of the peptide synthesis, the resin was treated with 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. After evaporation of the volatiles under vacuum, the paste was triturated with ether to provide a solid which was collected by filtration washed with diethyl ether and dried. A lO mmol scale synthesis of (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH 2 gave 30 g (103% of theory) of the crude peptide. In the case of (13) Ac- VC* WEDSWGGEVC *FRYDPGGGK(Adoa- Adoa)-NH 2 , a 10 mmol scale synthesis gave 28 g (107% of theory) of crude peptide. The greater than theoretical yields are most likely due to moisture and residual solvents.
[00118] A ~0.5 g portion of the crude cyclic disulfide peptide monomer (12) was dissolved in a minimum amount of DMSO (~3 mL). The volume of the solution was adjusted to ~100 mL with 20% CHaCN-water and employing a third pump, the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra ® Prep MS C18, 10 μ, 3OθA, 50 x 250 mm, flow rate 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 the flow of the equilibrating eluent from the preparative 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 the column was eluted with 10% CHsCN-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 CH 3 CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The collected fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (5μ particle, 12θApore) and product-containing fractions of >95% purity were pooled and freeze-dried to afford the corresponding cyclic disulfide peptide monomer (12). Typically the purification of 0.5 g of crude peptide monomer (12) afforded 0.1 g (20% yield) of the desired product (>95% purity).
[00120] As shown in Figure 3, disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added in one portion. Then solid peptide monomer (12) Ac- AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH 2 (0.50 g, 0.17 mmol) was added in portions to the stirred solution of DSG over a period of two min. After stirring for 30 min at room temperature, the solution was diluted with anhydrous ethyl acetate to ~ 50 mL, (this served to precipitate intermediate mono-NHS ester (14)). The entire mixture was centrifuged and the supernatant was decanted leaving intermediate mono-NHS ester (14) as a colorless solid. The solid was resuspended with ethyl acetate; the solution containing the suspended solid mono-NHS ester (14) was centrifuged to separate the solid and the supernatant was again decanted. This washing process was repeated twice to remove completely the excess DSG.
[00121] The solid mono-NHS ester (14) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added. Then solid peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)- NH 2 , (0.50 g, 0.19 mmol, 1.12 eq.) was added in portions to the stirred solution over a three min. period and the resulting mixture was stirred for 18 h. The reaction was monitored by mass spectrometry; after the complete consumption of the peptide monomer glutaric acid monoamide mono-NHS ester (14) was confirmed, neat hydrazine (0.1 mL) was added to remove the ivDde protecting group of the ivDde-bearing dimer (15) and the mixture was stirred for 20 min at room temperature.
[00124] Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was added portionwise to a solution of disuccinimidyl glutarate (DSG, 0.15 g, 0.46 mmol), and diisopropylethylamine (0.06 g, 0.47 mmol) in anhydrous DMF (3.0 mL) with stirring over a period of 3 min. Then the solution was stirred at ambient temperature for 30 min. The reaction mixture was diluted to ~ 50 mL with anhydrous ethyl acetate; this resulted in precipitation of the dimer glutaric acid monoamide mono-NHS ester (17), the glutaric acid monoamide mono-NHS ester of the precursor dimer peptide (16). The solution was centrifuged 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 from the compacted solid dimer glutaric acid monoamide mono-NHS ester (17) which was again resuspended in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG. The solid intermediate glutaric acid monoamide mono-NHS ester dimer derivative (17) thus obtained was dissolved in anhydrous DMF/CH2CI2 (8:2, v/v) (3.0 mL); diisopropylethylamine (0.06 g, 0.47 mmol) was added and the solution was stirred. [00125] Meanwhile, DSPE-PEG2000-NH 2 phospholipid ammonium salt (18) (0.235 g,
[00134] Additional parameters and results were as follows: HPJLC: Ret. time: 5.2 min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6 x 50 mm, 5 μ particle, 12θ Apore; Eluent: A: H 2 O (0.1% TFA), B: CH 3 CN (0.1 %TF A); 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; 1816.3 [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE analysis (counter-ion %wt./wt): TFA estimated to be 0.2%; acetate 0.15%.
[00136] Before purification of the dimer peptide (27) acetate, caution was taken to carefully wash the whole preparative HPLC system including the column with TFA-free eluents, CH 3 CN/H 2 O/IO mM NH 4 OAc. The crude reaction mixture was then applied to a reverse phase C-18 preparative column (Atlantis C-18, 5 μm particle, 100 A pore, 30 x 150 mm, flow rate 30 mL/min), pre-equilibrated with 15% B (A: 10 mM IMH 4 OAc in H 2 O; B: 10 mM NH 4 OAc in CH3CN/H 2 O, 9/1, v/v). The column was washed with the same eluent until the DMF plug was eluted. The eluent composition was increased to 25% B over 2 min. and then ramped to 65% B over 40 min. The fractions were analyzed on an analytical reverse phase C-18 column (Waters MS C-18, 4.6 x 50 mm, 5 μm particle, 100 A pore, flow rate 3 mL/min) and the product-containing fractions of >95% purity were pooled and freeze-dried to afford 25 mg of the dimer peptide (27) as its acetate salt as a fluffy white solid. The yield was 21.8%.
[00140] Additional parameters and results were as follows: HPLC: Ret. Time: 13.3 min; Assay >99% (area %); Column: Zorbax 300SB-C3, 3 x 150 mm, 3.5 μm, 300 A pore; Eluent: A: H 2 O (0.1 % TFA), B: CH 3 CN/MeOH 1/1 (0.1 %TFA); Elution: Tnitial condition: 60% B, linear gradient 60-90% B over 3 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm and ELSD; CE analysis (counter-ion %wt./wt): % wt. TFA: 0.3 %; % wt acetate 0.4%.
[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 μm particle; Eluent A : H 2 O (HPLC Grade with 0.1% TFA by volume); Eluent B: CH 3 CN (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.
[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 PEG2000 mass distribution curve was observed. HPLC: System A: Column: Zorbax 300SB C-3 ; 3 mm i.d. x 150 mm; 3.5 μm 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, hold at 90% B for 11 min; Flow rate: 0.5 mL/min; Detection: UV at 220 ran. Ret time: 6.77 min; Area %: 99.6%. System B: Column: Zorbax 300SB C-3; 3 mm i.d. x 150 mm; 3.5 μm 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. Flow rate: 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 sources of materials referred to in Examples 9-12.
[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, 176.4 and 19.8 mg of the three components, respectively) and PEG-4000 (22.6 g) were solubilized in 120 g of t-butyl alcohol at 60 0 C, in a water bath. The solution was filled in vials with 0.8 mL of solution each. The samples were frozen at -45°C and lyophilized. The air in the headspace was replaced with a mixture of C 4 Fio/Nitrogen (50/50) and vials capped and crimped. The lyophilized samples were reconstituted with 5 mL OfH 2 O per vial.
[00152] Example 9A was repeated using a mixture of DSPC/DPPG/ and the monorncric 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
[00161] An aqueous suspension of DSPE-PEG1000 (2.5 mg - 1.4 μmole) and dimeric peptide conjugate (11) shown in Figure 5 (7.0 mg - 0.84 μmole) was prepared in 1 mL of distilled water at 60 0 C to obtain a micellar suspension.
[00166] DSPC (16.3 mg - 20.6 μmoles), DSPA (3.7 mg - 5.15 μmoles) and monomeric peptide phospholipid conjugate (1) shown in Figure 1 (1.6 mg - 0.26 μmole) were dissolved in cyclooctane (1.6 mL) at 80 0 C. This organic phase was emulsified in a PEG4000 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 0 C for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was diluted with 20ml of a PEG4000 10% aqueous solution.
[00173] Example 12A was repeated by replacing the dimeric peptide phospholipid conjugate (11) shown in Figure 5 with the same relative molar amount of the monomeric peptide phospholipid conjugate (1) shown in Figure 2.
[00181] A surface of dimensions 14 x 25 mm was delimited on the glass coverslip using a special marker (Dako Pen) and 400 μL of Fc-VEGF-R2 solution at 4 μg/mL in PBS was deposited on this surface. After an overnight incubation at 4°C, the solution was aspirated, replaced by 0.5 mL of a solution of BSA 1% w/v in PBS-0.05% Tween 80, pH 7.4 and incubated for 3 hours at RT. Then the coverslip was washed three times with 5 ml of PBS-0.05% Tween 80.
[00186] In the following tables 8 and 9, the binding activity of the microbubbles prepared according to Examples 11 and 12 above were illustrated.
[00188] The ability of ultrasound contrast agents containing KlDR binding lipopeptides of the invention to bind to KDR-expressing tissue in vivo was assessed using a known model of angiogenesis: the rabbit VX2 tumor model.
[00191] Rabbits received first 0.3 mL of Vetranquil (Acepromazine, Sanofi, injected intramuscularly) and were then anesthetized with an intramuscular injection of Ketaminol®5/Xylazine (Veterinaria AG/Sigma) mixture (50/10 mg/mL, 0.7 mL/kg). One hundred microliters of VX2 tumor homogenate was injected intramuscularly. Fifteen days after implantation of VX2 tumors, animals were anesthetized again with the same mixture, plus subcutaneous injection of 50% Urethane (2mL/kg 5 s.c.) (Sigma) for imaging experiments.
[00195] Example 16 was repeated by replacing the dimeric peptide phospholipid conjugate with the same molar amount of monomeric phospholipid conjugate (1) shown in

中文:
[0007]因此,不仅是KDR的VEGF结合活性在体内对血管生成很关键,而且检测内皮细胞上的KDR上调的能力或检测VEGF/KDR结合复合物的能力对检测或监测血管生成应当极为有益。
[0015]本发明还提供以高亲和力结合KDR或VEGF/KDR复合物的单体肽,以及合成和使用这些单体肽的方法。
[0018]图2图示了单体肽磷脂缀合物(1),包括对KDR具有高结合亲和力的肽。
[0019]图3图示了一种用于由肽单体生产前体二聚体肽(16)的方法。
[0025]图9图示了另一种对KDR具有高结合亲和力的代表性单体肽(32)。
[0026]图10图示了另一种包含示于图9的单体肽的单体肽-磷脂缀合物(31)。
[0028]图12A-C显示了通过在造影剂中使用单体磷脂肽缀合物(1)(示于图2)在下列情况下所获得的图像:基线(图12A)、在25分钟后(图12B)以及在扣除基线和游离循环气泡之后(图12C)。
[0029]申请人出乎意料地发现了可用于生产靶向超声造影剂并具有优越的KDR结合效力的肽磷脂缀合物。这些化合物中有两种为含有以高亲和力结合KDR的线性肽单体的单体肽磷脂缀合物,而另一种为含有两个不同的单体亚单位的二聚体肽磷脂缀合物,每个单体均结合KDR。另外,业已发现了用于大规模生产这些缀合物和前体物质的纯化形式的高效方法。这些方法包括生产具有TFA水平最低的二聚体肽磷脂缀合物。
[0044]反应混合物可用水(0.1%TFA)和CH3CN-CH3OH(1:1,体积/体积)(0.1%TFA)的1:1混合物(约100mL)稀释,再加到反相C2柱(PrepC2,10μ,50×250mm,流速100mL/分钟)上,该柱可用水(0.1%TFA)和CH3CN-CH3OH(1:1,体积/体积)(0.1%TFA)的3:1混合物洗脱,以除去亲水杂质。然后,产物可使用CH3CN-CH3OH(1:1)(0.1%TFA)至水(0.1%TFA)的梯度洗脱(细节参见实验部分)。可使用ELS检测器,通过反相HPLC对所收集的流分进行分析,ELS检测器允许检测需要的产物以及经常难以分离的DSPE-PEG2000-NH2磷脂,其UV吸光度非常小。这表明单体肽磷脂缀合物和DSPE-PEG2000-NH2磷脂完全分离。可收集含有纯产物的流分,在旋转蒸发器上浓缩(以减少甲醇含量),冻干,得到单体肽磷脂缀合物(无色固体)。为了制备所需量的单体肽磷脂缀合物,可使用0.5g至1.0g的线性肽单体实施几个轮次。在所有情况下,靶单体肽磷脂缀合物都可以以高得率和纯度(例如得率57-60%和纯度>99%)分离出来。
[0054]从非优化合成单体肽获得的杂质分析表明,主要问题是外消旋、不完全偶联和哌啶酰胺形成(假定经由中间体天冬氨酰亚胺或戊二酰亚胺中间体),其中每个问题都导致未达到最优得率和纯度。哌啶酰胺形成的急剧下降可通过使用含HOBt(0.1M)的25%哌啶的DMF溶液作为用于脱去Fmoc的试剂来实现。可使用DIC/HOBt作为大部分偶联的活化剂、3小时偶联时间、使用4倍过量的预活化Fmoc-氨基酸、用无水DMF(6次)间插洗涤显著降低外消旋。Nα-Fmoc氨基酸可恰好在其偶联转变之前被溶解,用DIC/HOBt的DMF溶液预活化4分钟,并转移至反应容器。这可以在Sonata设备上如下实现:将固体Fmoc-氨基酸加到该设备的氨基酸容器中,然后给该设备设定好程序,以序贯加入DMF、HOBt/DMF和DIC/DMF,并在每次加入后使溶液鼓泡。
[0056]假正交保护的衍生物Fmoc-Lys(ivDde)-OH可用于使单体和二聚体肽的C-末端赖氨酸ε-胺选择性无掩蔽,并使它们随后官能化,这也可以被优化。可使用10%肼的DMF溶液,来脱去每个肽单体C-末端赖氨酸ε-胺上的ivDde基团。然后,可使用4当量的Fmoc氨基酸以及各4当量的DIC和HOBt的DMF溶液,使Fmoc-Adoa(用于单体(13))或Lys(ivDde)(用于单体(12))接在暴露的赖氨酸ε-氨基上,时间长达10小时。在完成合成后,可从树脂上切下肽序列,并使用“试剂B”(TFA:水:苯酚:三异丙基硅烷,88:5:5:2,体积/体积/重量/体积)脱保护4小时。在完成切割反应后,可使肽沉淀出来,用乙醚洗涤,并干燥。
[0062]DSPE-PEG2000-NH2与前体二聚体肽的缀合可通过制备前体二聚体的戊二酸单酰胺单NHS酯并使其与磷脂铵盐的游离氨基反应而完成。因此,具有ivDde的前体二聚体肽(16)可在DIEA(5当量)存在下与DSG(4当量)的DMF溶液反应30分钟。和在制备前体二聚体肽时一样,溶液可用乙酸乙酯稀释,以将二聚体戊二酸单酰胺单NHS酯(17)沉淀出来成为固体。上清液可被倾析出去,以除去未反应的DSG。然后,可用乙酸乙酯洗涤固体二聚体肽的戊二酸单酰胺单NHS酯(17)几次,以除去痕量DSG。质谱结果证实形成的肽二聚体戊二酸单酰胺单NHS酯为纯净产物。
[0067]表3提供了对图6、7和8中显示的识别标记的描述。
[0072]尽管上述方法以及图6和7中的方法提供了极佳的结果,但是,第二种方法的优势是需要较少的步骤。其它细节见下面的实施例部分。
[0073]转到图8,另一个实施方案利用磷脂-肽缀合物(21)和TFA离子之间的大小差异提供了用于生产TFA含量最少的二聚体缀合物的方法。在该实施方案中,在碳酸氢铵缓冲液存在下,21·nTFA加合物可从大小排阻柱上洗脱下来。起初,可在ZorbaxC-3柱上,使用乙腈至水的线性梯度,通过制备型HPLC使粗21·nTFA不含溶解型化合物。两相都可以用10mM乙酸铵缓冲。如分析型HPLC所示,这提供了溶解型化合物的分离。
[0075]本文所述的单体和二聚体肽磷脂缀合物可掺入到超声造影剂如含气微囊泡中。所述含气微囊泡包括例如含气微泡、含气微球、含气微囊等。在一个优选实施方案中,肽磷脂缀合物可掺入到含有含气微泡的超声造影剂中。由磷脂和磷脂缀合物制备含气微泡的方法是本领域技术人员已知的。例如,本发明的微泡可通过任一个以下专利中所述方法制备:EP554213、WO04/069284、美国专利第5,413,774号、美国专利第5,578,292号、EP744962、EP682530、美国专利第5,556,610号、美国专利第5,846,518号、美国专利第6,183,725号、EP474833、美国专利第5,271,928号、美国专利第5,380,519号、美国专利第5,531,980号、美国专利第5,567,414号、美国专利第5,658,551号、美国专利第5,643,553号、美国专利第5,911,972号、美国专利第6,110,443号、美国专利第6,136,293号、EP619743、美国专利第5,445,813号、美国专利第5,597,549号、美国专利第5,686,060号、美国专利第6,187,288号和美国专利第5,908,610号,这些专利整体在此引入作为参考。特别优选在WO04/069284中公开的方法。
[0081]适宜的磷脂还包括通过使亲水聚合物与其连接而修饰的磷脂。经修饰的磷脂的实例为用聚乙二醇(PEG)修饰的磷脂酰乙醇胺(PE),简称“PE-PEG”,即磷脂酰乙醇胺中的亲水乙醇胺部分连接可变分子量(例如从300道尔顿到5000道尔顿)的PEG分子,例如DPPE-PEG、DSPE-PEG、DMPE-PEG或DAPE-PEG(其中DAPE为1,2-二花生四烯酰-sn-甘油-3-磷酸乙醇胺),其组成中还可以包含其它的两亲化合物,包括例如脂肪酸,例如棕榈酸、硬脂酸、花生四烯酸或油酸;甾醇,例如胆固醇,或甾醇与脂肪酸或与糖酸的酯;甘油或甘油酯,包括甘油三棕榈酸酯、甘油二硬脂酸酯、甘油三硬脂酸酯、甘油二肉豆蔻酸酯、甘油三肉豆蔻酸酯、甘油二月桂酸酯、甘油三月桂酸酯、甘油二棕榈酸酯;叔或季烷基铵盐,例如1,2-二硬脂酰-3-三甲基铵-丙烷(DSTAP)、1,2-二棕榈酰-3-三甲基铵-丙烷(DPTAP)及其混合物和组合。
[0097]术语“治疗剂”在其含义中包括可用于任何治疗用途(例如用于治疗患者的疾病的方法)的任何物质、组合物或颗粒,以及在体外和/或体内能够发挥或负责发挥生物学作用的任何物质。治疗剂因此包括能够用于治疗(包括诊断、预防、缓解、疼痛减轻或治愈)患者的任何病理状况(包括疾病、不适、病灶或损伤)的任何化合物或物质。治疗剂的实例为药物、药剂、生物活性剂、细胞毒性剂、化疗剂、放疗剂、蛋白质、天然或合成肽(包括寡肽和多肽)、维生素、类固醇和遗传物质,包括核苷、核苷酸、寡核苷酸、多核苷酸和质粒。
[00103]下面的实施例1-2涉及示于图2的单体肽磷脂缀合物。此化合物的合成方法见图1。尽管这些实施例更具体地涉及合成示于图2的化合物的方法,但可使用类似方法制备示于图10的单体肽磷脂缀合物和示于图9的线性肽单体(32)以及其它单体肽-磷脂缀合物。另外,同时待审的美国申请第10/661,156号(2003年9月11日申请)记载了肽单体的制备方法,该申请整体在此引入作为参考。
[00107]将约0.5g部分的粗线性肽单体(2)溶解在最少量CH3CN(约20mL)中。用水将溶液体积调整至约100mL,使用第三个泵将溶液加到反相C18制备型柱(Waters,PrepMSC18,10μ,50×250mm,流速100mL/分钟)上,该柱已用10%CH3CN的水溶液(0.1%TFA)预平衡。该柱在样品溶液上样的过程中不用平衡洗脱液洗脱。在样品溶液加到该柱后,在1分钟内将洗脱液的组成阶次升至20%CH3CN-水(0.1%TFA),以0.6%/分钟CH3CN(0.1%TFA)至水(0.1%TFA)的速率开始线性梯度,并保持50分钟。使用220nm的UV作为产物洗脱液的指示器手动收集流分(15mL)。所收集的流分在WatersXTerra分析型反相C-18柱(5μ颗粒,孔径)上进行分析,合并纯度>95%的含产物流分后冻干,得到相应的纯线性肽单体(2)。通常,0.5g粗产物(2)的纯化得到0.12g(得率24%)所需产物(纯度>95%)。
[00114]在典型的顺序氨基酸偶联中,每克树脂使用5mL无水DMF。基于使用的树脂计算的DMF总体积在用于溶液制备的氨基酸、HOBt和DIC之间分配。例如,对于包含50g树脂的合成,计算的250mLDMF体积在氨基酸(150mL)、HOBt(50mL)和DIC(50mL)之间分配。在Pilot肽合成仪上的氨基酸容器中装入固体无水氨基酸(相对于树脂4倍过量)。在偶联步骤开始时,连续地传递选定体积的DMF和HOBt(4当量)的DMF溶液以及DIC(4当量)的DMF溶液,在每次传递后通过通入氮气进行混合。在最后一次试剂传递后,通过通入氮气启动混合,进行4分钟。这用于预活化氨基酸,并确保混合物的所有组分完全溶解。
[00115]在活化后,将活化的Fmoc-氨基酸溶液转移至装有树脂的反应容器。在转移完成后,搅拌容器3小时,同时周期性通入氮气。在3小时偶联时间后,用DMF(5mL/g,6次)彻底洗涤树脂,用含有HOBt(0.1M)的25%哌啶的DMF溶液(5mL/g)进行Fmoc基团的切割(2×10分钟)。用DMF(5mL/g,6次)彻底洗涤树脂,以确保在制备中从树脂中完全除去哌啶,用以保证氨基酸偶联。就Fmoc-Gly-Gly-Gly-OH和Fmoc-Gly-Gly-OH而言,在氨基酸瓶中不进行预活化,以便在如本文论述的活化时间当中使二酮哌嗪的形成减至最低。因此,在这两种情况下,将氨基酸、HOBt和DIC的溶液序贯加入反应容器,并以“原位”活化实施偶联方法。在完成链延伸之后,按标准方式脱去N-末端氨基酸的Fmoc基团,接着用DMF进行标准洗涤(参见上文)。然后,通过用新鲜制备的乙酰化混合物(0.5M乙酸酐、0.125MDIEA和0.015MHOBt的DMF溶液,6mL/g树脂)处理2×20分钟给N-末端氨基酸加帽。
[00116]首先用新鲜制备的10%肼的DMF溶液(5mL/g树脂-2×10分钟)脱去ε-氨基上的ivDde基团,而完成单体肽(根据需要具有Fmoc-Adoa或Fmoc-Lys(ivDde))的C-末端赖氨酸部分ε-氨基的官能化。为添加Fmoc-Adoa或Fmoc-Lys(ivDde),把偶联时间增加至10小时。在完成肽合成后,用切割混合物‘试剂B’(TFA:水:苯酚:三异丙基硅烷,88:5:5:2,体积/体积/重量/体积)(10mL/g树脂)处理树脂4小时。真空蒸发挥发物之后,用乙醚研磨膏状物,得到固体,通过过滤收集,再用乙醚洗涤,干燥。10mmol规模的(12)Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH2合成得到30g(理论值的103%)粗肽。就(13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2而言,10mmol规模的合成得到28g(理论值的107%)粗肽。超过理论得率最有可能是源于潮湿和残余的溶剂。
[00118]将约0.5g部分的粗环状二硫化物肽单体(12)溶解在最少量DMSO(约3mL)中。用20%CH3CN-水将溶液体积调整至约100mL,使用第三个泵将溶液加到反相C18制备型柱(Waters,PrepMSC18,10μ,50×250mm,流速100mL/分钟)上,该柱已用10%CH3CN的水溶液(0.1%TFA)预平衡。在样品溶液上样的过程中,停止制备型HPLC系统中的平衡洗脱液流动。在样品溶液上柱后,再启动梯度HPLC系统中的平衡洗脱液流动,用10%CH3CN-水(0.1%TFA)洗脱该柱,直至洗脱出DMSO。然后,在1分钟内将洗脱液的组成阶次升至35%CH3CN-水(0.1%TFA),此后,启动0.5%/分钟速率的CH3CN(0.1%TFA)至水(0.1%TFA)的线性梯度,并保持50分钟。使用220nm的UV作为产物洗脱的指示器手动收集流分(15mL)。所收集的流分在WatersXTerra分析型反相C-18柱(5μ颗粒,孔径120)上进行分析,合并纯度>95%的含产物流分后冻干,得到相应的环状二硫化物肽单体(12)。通常,0.5g粗肽单体(12)的纯化得到0.1g(得率20%)所需产物(纯度>95%)。
[00120]如图3所示,将戊二酸二琥珀酰亚胺酯(DSG,0.28g,0.86mmol)溶解在搅拌的无水二甲基甲酰胺(2.0mL)中,一次性加入二异丙基乙胺(0.11g,0.85mmol)。然后,在2分钟的时间段内将固体肽单体(12)Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH2(0.50g,0.17mmol)分批加入搅拌的DSG溶液中。在室温搅拌30分钟后,用无水乙酸乙酯将该溶液稀释至约50mL,(其用于沉淀中间体单NHS酯(14))。离心整个混合物,倾析上清液,留下为无色固体的中间体单NHS酯(14)。用乙酸乙酯重悬浮固体;离心含有悬浮的固体单NHS酯(14)的溶液,以分离固体,再倾析上清液。重复此清洗方法2次,以完全除去过量的DSG。
[00121]将固体单NHS酯(14)溶解在搅拌的无水二甲基甲酰胺(2.0mL)中,加入二异丙基乙胺(0.11g,0.85mmol)。然后,在3分钟时间段内,向搅拌的溶液中分批加入固体肽单体(13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2(0.50g,0.19mmol,1.12当量),对所得混合物搅拌18小时。通过质谱监测反应;在证实完全消耗了肽单体戊二酸单酰胺单NHS酯(14)之后,加入纯净肼(0.1mL),以脱去具有ivDde的二聚体(15)的ivDde保护基,并于室温搅拌混合物20分钟。
[00124]在3分钟的时间段内,边搅拌边向戊二酸二琥珀酰亚胺酯(DSG,0.15g,0.46mmol)和二异丙基乙胺(0.06g,0.47mmol)的无水DMF(3.0mL)溶液中分批加入固体前体二聚体肽(16)(0.5g,0.092mmol)。然后,于室温搅拌该溶液30分钟。用无水乙酸乙酯稀释反应混合物至约50mL;这导致前体二聚体肽(16)的戊二酸单酰胺单NHS酯—二聚体戊二酸单酰胺单NHS酯(17)沉淀。离心该溶液,得到为无色固体的沉淀6(m/z,负离子,1887.3(M-3H)/3,1415.1(M-4H)/4,1131.9(M-5H)/5)。含有过量DSG的上清液乙酸乙酯层从压紧的固体二聚体戊二酸单酰胺单NHS酯(17)倾析出去,该固体再重悬浮在乙酸乙酯中,离心,再多洗涤2次,以除去余下的痕量DSG。将由此获得的固体中间体戊二酸单酰胺单NHS酯二聚体衍生物(17)溶解在无水DMF/CH2Cl2(8:2,体积/体积)(3.0mL)中;加入二异丙基乙胺(0.06g,0.47mmol),并搅拌溶液。
[00134]其它的参数和结果如下:HPLC:保留时间:5.2分钟;测定>99%(面积%);柱子:WatersXTerraMS-C18,4.6×50mm,5μ颗粒,孔径;洗脱液:A:H2O(0.1%TFA),B:CH3CN(0.1%TFA);洗脱:初始条件:20%B,在8分钟内20-60%B的线性梯度;流速:3mL/分钟;检测:220nm的UV;质谱:API-ES;模式:负离子;1816.3[M-3H]/3,1362.0[M-4H]/4,1089.2[M-5H]/5;CE分析(抗衡离子%重量/重量):TFA估计为0.2%;乙酸盐0.15%。
[00136]在纯化二聚体肽(27)乙酸盐之前,要注意小心地用无TFA的洗脱液、CH3CN/H2O/10mMNH4OAc洗涤整个制备型HPLC系统,包括柱子。然后,将粗反应混合物加到反相C-18制备型柱(AtlantisC-18,5μm颗粒,孔径30×150mm,流速30mL/分钟)上,用15%B(A:10mMNH4OAc在H2O中的溶液;B:10mMNH4OAc在CH3CN/H2O中的溶液,9/1,体积/体积)预平衡。用同样的洗脱液洗柱子,直至洗脱出DMF塞流。在2分钟内将洗脱液组成增加至25%B,然后在40分钟内阶次升至65%B。流分在分析型反向C-18柱(WatersMSC-18,4.6×50mm,5μm颗粒,孔径流速3mL/分钟)上进行分析,合并纯度>95%的含产物流分后冻干,得到25mg为其乙酸盐的二聚体肽(27),为白色蓬松固体。得率为21.8%。
[00140]其它的参数和结果如下:HPLC:保留时间:13.3分钟;测定>99%(面积%);柱子:Zorbax300SB-C3,3×150mm,3.5μm,孔径洗脱液:A:H2O(0.1%TFA),B:CH3CN/MeOH(0.1%TFA);洗脱:初始条件:60%B,3分钟内60-90%B的线性梯度;流速:0.5mL/分钟;检测:220nm的UV和ELSD;CE分析(抗衡离子%重量/重量):%重量TFA:0.3%;%重量乙酸盐0.4%。
[00141]使用的材料和用于分析型HPLC系统的条件包括:柱子:Zorbax300SBC-3;3mm内径×150mm;3.5μm颗粒;洗脱液A:H2O(HPLC级,以体积计含有0.1%TFA);洗脱液B:CH3CN(以体积计0.1%TFA)。洗脱:初始条件:50%B,然后在3分钟内50-90%B的线性梯度,于90%B保持11分钟;流速0.5mL/分钟;检测:220nm的UV。保留时间:(化合物(21)):6.77分钟,保留时间(溶解型的):4.06分钟。
[00149]用于收集分析数据的材料和条件包括:氟分析(通过QTI的IC):751ppm(0.15%TFA重量/重量);质谱:方法:MALDI-TOF;模式:正离子;检测的平均分子量为8461,观察到典型的PEG2000质量分布曲线。HPLC:系统A:柱子:Zorbax300SBC-3;3mm内径×150mm;3.5μm颗粒;洗脱液A:水(HPLC级,以体积计含有0.1%TFA);洗脱液B:乙腈(以体积计含0.1%TFA)。初始条件:50%B;洗脱:在3分钟内50-90%B的线性梯度,于90%B保持11分钟;流速:0.5mL/分钟;检测:于220nm的UV。保留时间:6.77分钟;面积%:99.6%。系统B:柱子:Zorbax300SBC-3;3mm内径×150mm;3.5μm颗粒;洗脱液A:水(HPLC级,以体积计含0.1%TFA);洗脱液B:乙腈(以体积计含0.1%TFA)。初始条件:50%B;洗脱:在3分钟内50-90%B的线性梯度,然后在12分钟内阶次升至100%B。流速:0.5mL/分钟;检测:LSD;保留时间:13.98分钟。面积%:99.3%。
[00151]在60°C水浴中,将383mgDSPC/DPPG/和示于图5的二聚体肽磷脂缀合物(11)的混合物(摩尔比49.75/49.75/0.5,三种组分分别相当于187.1mg、176.4mg和19.8mg)和PEG-4000(22.6g)溶解在120g叔丁醇中。将溶液装入小瓶中,每瓶0.8ml溶液。样品在-45°C冷冻,再冻干。顶部空间的空气用C4F10/氮气(50/50)混合气体置换,小瓶加盖卷边。每瓶冻干的样品用5mLH2O重配。
[00152]重复实施例9A,只是使用DSPC/DPPG/和示于图10的单体肽磷脂缀合物(31)(摩尔比49.5/49.5/1,三种组分分别相当于182.8mg、172.3mg和28.2mg)的混合物。
[00161]于60°C,用1mL蒸馏水制备DSPE-PEG1000(2.5mg-1.4μmol)和示于图5的二聚体肽缀合物(11)(7.0mg-0.84μmol)的水性混悬液,得到胶束混悬液。
[00166]于80°C,将DSPC(16.3mg-20.6μmol)、DSPA(3.7mg-5.15μmol)和示于图1的单体肽磷脂缀合物(1)(1.6mg-0.26μmol)溶解在环辛烷(1.6mL)中。使用高速匀浆器(PolytronPT3000,轴直径3cm),将该有机相在10%PEG4000水相(20mL)中以8000rpm乳化1分钟,得到乳状液。
[00173]重复实施例12A,只是用相同相对摩尔量的示于图2的单体肽磷脂缀合物(1)替代示于图5的二聚体肽磷脂缀合物(11)。
[00181]使用特定标记(DakoPen),在玻璃盖玻片上划分14×25mm尺寸的表面,将在PBS中为4μg/mL的400μLFc-VEGF-R2溶液沉积在该表面上。于4°C过夜温育后,吸出溶液,更换为0.5mL1%重量/体积BSA的PBS-0.05%吐温(Tween)80溶液(pH7.4),并于室温温育3小时。然后,用5mlPBS-0.05%吐温(Tween)80洗涤盖玻片3次。
[00186]在下表8和9中,描述了按照以上实施例11和12制备的微泡的结合活性。
[00188]使用已知的血管生成模型:兔VX2肿瘤模型,在体内评价含有本发明的KDR结合脂肽的超声造影剂结合表达KDR的组织的能力。
[00191]兔子首先接受0.3mLVetranquil(Acepromazine,Sanofi,肌内注射),然后用肌内注射5/噻拉嗪(VeterinariaAG/Sigma)混合物(50/10mg/mL,0.7mL/kg)麻醉。肌内注射100μl的VX2肿瘤匀浆物。在移植VX2肿瘤后15天,再用相同混合物麻醉动物,外加皮下注射50%乌拉坦(2mL/kg,皮下)(Sigma),供成像试验用。
[00195]重复实施例16,只是用相同摩尔量的示于图2的单体磷脂缀合物(1)替换二聚体肽磷脂缀合物。