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WO1999020300A1 - Inhibition de l'adherence des cellules tumorales au collagene de type iv - Google Patents

Inhibition de l'adherence des cellules tumorales au collagene de type iv Download PDF

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Publication number
WO1999020300A1
WO1999020300A1 PCT/US1998/022405 US9822405W WO9920300A1 WO 1999020300 A1 WO1999020300 A1 WO 1999020300A1 US 9822405 W US9822405 W US 9822405W WO 9920300 A1 WO9920300 A1 WO 9920300A1
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peptide
polypeptide
collagen
gly
conjugate
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PCT/US1998/022405
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English (en)
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Gregg B. Fields
James B. Mccarthy
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The Regents Of The University Of Minnesota
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Publication of WO1999020300A1 publication Critical patent/WO1999020300A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Definitions

  • Type IV collagen is a distinctive glycoprotein which occurs almost exclusively in basement membranes, structures which are found in the basal surface of many cell types, including vascular endothelial cells, epithelial cells, etc.
  • Type IV collagen has a molecular weight (MW) of about 500,000 and consists commonly of two ⁇ l (MW 185,000) chains and one ⁇ 2 (MW 170,000) chain.
  • Type IV collagen has two major proteolytic domains: a large, globular, non-collagenous, NCI domain and another major triple-helical collagenous domain. The latter domain is interrupted by non-collagenous sequences of variable length. It is a complex and multidomain protein with different biological activities residing in different domains.
  • Type IV collagen self-assembles to polymeric structures which constitute the supportive frame of basement membranes.
  • Various macromolecular components bind to type IV collagen, such as laminin, entactin/nidogen, and heparin sulfate proteoglycan.
  • An additional function of type IV collagen is to mediate cell binding.
  • a variety of cell types specifically adhere and spread onto type IV collagen-coated substrata.
  • Various cell surface proteins, a 47 kD protein, a 70 kD protein, and members of the superfamily of integrins have been reported to mediate cell binding to type IV collagen.
  • IV-H1 also supports melanoma cell motility and selectively inhibits cell adhesion to type IV collagen (M.K. Chelberg et al., J. Cell. Biol., Ill, 261-270 (1990)). Melanoma cell motility is mediated by a chondroitin sulfate proteoglycan (D.J. Mickelson et al, J. Cell. Biol., 115, 287a (1991)) and dependent upon IV-H1 conformation (M.K. Chelberg et al, J. Cell. Biol, 111, 261-270 (1990); K.
  • LAM-L laminin derived synthetic peptide LAM-L (A chain residues 2097-2108) and its all D-enantiomer had near identical concentration-dependent activities for promotion of rat pheochromocytoma cell (PC 12) attachment, inhibition of PC 12 adhesion to laminin, and promotion of murine melanoma cell growth in mice (M. Nomizu et al., J. Biol. Chem., 267, 14118-14121 (1992)).
  • the cell surface receptor for LAM-L or LAM-D was not identified.
  • a synthetic combinatorial library has been used to select an all-D peptide (acetyl-Arg-Phe-Trp-Ile-Asn-Lys-NH 2 ) as a potent ligand for the ⁇ opioid receptor (C.T. Dooley, Science, 266, 2019-2022 (1994)).
  • the peptide was shown to be a full agonist, binding to the ⁇ receptor and inducing a conformational change which allowed for signal transduction. In this case, the all-L peptide was not active.
  • Schnolzer and Kent M. Schnolzer et al., Science, 256, 221-225
  • polypeptides which represent an all-D form of a fragment of the ⁇ l chain of human type IV collagen derived from the continuous collagenous region of the major triple helical domain.
  • These polypeptides can be prepared by conventional solid phase synthesis and preferably include 15 amino acid residues.
  • an all-D polypeptide may include amino acid residues that are not chiral and therefore are in neither the D or the L form (e.g., glycine).
  • the formula of the polypeptide is: gly-val- lys-gly-asp-lys-gly-asn-pro-gly-trp-pro-gly-ala-pro.
  • This specific polypeptide formally substantially corresponds to isolated type IV collagen residuesl263- 1277 from the major triple helical region of the ⁇ l chain of type IV collagen, although all the amino acids are in the D-form where appropriate (gly is in neither the L nor the D form).
  • the single letter amino acid code for this polypeptide is GVKGDKGNPGWPGAP.
  • this specific polypeptide is designated "D-IVH1". The all-D polypeptide D-IV HI was assayed for biological activity.
  • the all-D form efficiently inhibits tumor cell binding to type IV collagen, tumor cell invasion of basement membranes, and tumor cell metastasis in vivo. Also, like the all-L form, the all-D form is highly specific in its cell binding properties. Therefore, it is believed that polypeptides such as D-IVH1 may be useful to (a) inhibit the metastasis and invasion of tumor cells, and (b) target cytotoxic agents to tumor cells. Since it is expected that further hydrolysis of the peptide D-IVH1 in vitro or in vivo will yield some fragments of substantially equivalent bioactivity, such lower molecular weight peptides are also considered to be within the scope of the present invention.
  • the present invention also provides peptide-conjugates wherein the all-D form, or the all-L form, of the polypeptides described herein, particularly the IV-H1 peptide (e.g., a peptide incorporating ⁇ l(IV) residues 1263-1277), is attached (covalently bonded) to a non-peptide moiety, such as a lipophilic C 10 alkyl "tail” and polyethylene glycol (PEG).
  • a non-peptide moiety such as a lipophilic C 10 alkyl "tail” and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • polypeptides and peptide-conjugates described herein can also include a cytotoxic agent for selective targeting of tumor cells for therapeutic effect.
  • the cytotoxic agent is covalently bonded to a peptide portion, although it could be covalently bonded to a non-peptide moiety.
  • the present invention also provides therapeutic methods.
  • the present invention provides a method of inhibiting tumor cell binding (adhesion) to type IV collagen comprising contacting the tumor cell with a polypeptide or peptide-conjugate as described herein.
  • Another method of the present invention involves inhibiting tumor cell invasion of a basement membrane.
  • the method includes modulating the tumor cell with a polypeptide or peptide-conjugate as described herein.
  • the present invention also provides a method of inhibiting tumor cell metastasis comprising modulating the tumor cell with a polypeptide or peptide-conjugate as described herein.
  • each of these methods is carried out in vivo.
  • inhibiting does not necessarily mean complete elimination of the activity, rather it means that the level of the activity (tumor cell binding, invasion, or metastasis) is decreased relative to the level of that activity in the absence of the polypeptide or peptide- conjugate.
  • modulating means bringing the polypeptide or peptide- conjugate in close proximity to, and preferably so close that it is in contact with, the tumor cell.
  • Figures 1 A and IB show the relative inhibition of M14#5 human melanoma cell adhesion to 10 ⁇ g/mL type IV collagen (TIV), fibronectin (FN), laminin (LM), or bovine serum albumin (BSA) by 100 ⁇ g/mL of L-IVH1, D-IVH1, or RI-IVH1 (a polypeptide having the sequence pro-ala-gly-pro-trp-gly- pro-asn-gly-lys-asp-gly-lys-val-gly, which is the all-D form synthesized in the reverse order and referred to as "Retro-Inverso").
  • TIV type IV collagen
  • FN fibronectin
  • LM laminin
  • BSA bovine serum albumin
  • Figure 2 A and B show the inhibition of M14#5 human melanoma cell invasion through MATRIGEL by 500 ⁇ g/mL (A) or 1 mg/mL (B) of L-IVH1, D-IVH1, or RI-IVH1 (a polypeptide having the sequence pro-ala-gly- pro-trp-gly-pro-asn-gly-lys-asp-gly-lys-val-gly, which is the all-D form synthesized in the reverse order and referred to as "Retro-Inverso").
  • Cells were mixed with the peptides and then tested for their ability to invade through MATRIGEL basement membrane (obtained from Collaborative Biomedical Products).
  • the data represents the means of triplicate points plus or minus the standard errors of the means.
  • Figure 3 shows the inhibition of M14#5 human melanoma cell adhesion to 10 ⁇ g/mL type IV collagen by D-IVHl(-Y) (closed squares), D-IVH1' (closed circles), D-IVH1(-Y)C10 (open squares), D-IVHIC'IO (open circles), or D-IVHl'PEG (starred circles).
  • Cells were preincubated with the peptides for 15 minutes and then added to the wells in the presence of the peptides for a 60-minute incubation period at 37°C.
  • the data represent the means of triplicate points plus or minus the standard errors of the means.
  • Figure 4 is a graph showing the inhibition of M14#5 human melanoma cell adhesion to 10 ⁇ g/mL type IV collagen by D-IVHT (closed squares), D-IVHI'CIO (open squares), or D-IVHl'PEG (closed circles).
  • D-IVHT closed squares
  • D-IVHI'CIO open squares
  • D-IVHl'PEG closed circles
  • Cells were preincubated with the peptides for 15 minutes and then added to the wells in the presence of the peptides for a 60-minute incubation period at 37°C.
  • the data represent the means of triplicate points plus or minus the standard errors of the means.
  • the structure of the two ⁇ l chains and the single ⁇ 2 chain of type IV collagen has been the subject of much study.
  • the sequence of the l chain is shown in Figure 2 of U.S. Patent No. 5,082,926 (Chelberg et al.).
  • the total number of amino acids per collagen molecule is approximately 4,550, with each 1(IV) chain containing approximately 1,390 amino acids.
  • IV-H1 The inhibitory activities of IV-H1 synthesized with all-L amino acids (designation L-IVH1), all-D amino acids (designated D-IVH1), and IV-H1 synthesized in reverse sequence order with all-D amino acids (retro-inverso; designated RI-IVH1) were analyzed.
  • the all-D IV-H1 inhibits melanoma cell adhesion to type IV collagen (Figure 1) and invasion of MATRIGEL basement membrane ( Figure 2) at least as well as does the all-L form.
  • the retro-inverso form of IV-H1 has only weak inhibitory properties at best.
  • the present invention provides polypeptides which represent an all-D form of a fragment of the ⁇ l chain of human type IV collagen derived from continuous collagenous region of the major triple helical domain.
  • both the all-L and all-D versions of IV-H1 inhibit melanoma cell metastasis in vivo (Table 1).
  • the all-D version inhibits spontaneous Lewis lung tumor metastasis.
  • the present invention also provides peptide-conjugates, i.e., where a non-peptide moiety is incorporated onto a polypeptide as described above, particularly onto the peptide IV-H1, for the all-D form as well as the all-L form of the polypeptide.
  • Peptide-conjugates are typically created to improve the bioavailability and subsequent half-life of peptide-based drugs in vivo.
  • the peptide-conjugates of the present invention have been shown to inhibit adhesion of tumor cells to type-IV collagen, and are believed to provide inhibitory activity with respect to tumor cell invasion of basement membranes and tumor cell metastasis.
  • the non-peptide moieties are typically those that impart some hydrophobic character to the peptide and are not readily hydrolyzed.
  • Preferred non-peptide moieties include alkyl chains (typically, C 6 -C 18 alkyls to provide, e.g., monoalkyl tails and dialkyl tails), phospholipids, and polyalkylene glycols. Specific examples include, for example, a lipophilic C, 0 alkyl "tail” and polyethylene glycol (PEG).
  • Such conjugates can be synthesized by methods known in the art, particularly solid phase methods.
  • the non-peptide moiety can be any organic group having a long alkyl group (preferably, a linear chain).
  • the organic group can include at least two long alkyl groups (preferably, linear chains) that are capable of forming lipid-like structures.
  • This organic group also includes suitable functional groups for attachment to the peptide portion.
  • the organic group is attached to the peptide portion through a linker group having suitable functionality such as ester groups, amide groups, and combinations thereof.
  • Suitable non-peptide moieties can be derived from compounds such as, for example, alkylamines, alkylesters, and phospholipids.
  • bilayer membrane systems can be formed, where the lipid moiety serves as an anchor for the functional head group to the lipid assembly.
  • peptide- conjugates may form a great variety of structures in solution including micelles and vesicles. They can also be mixed with vesicle-forming lipids, such as dilauryl phosphatidylcholine, to form stable mixed vesicles with peptide head groups. These can be used as delivery vesicles for the peptide and optionally a cytotoxic agent.
  • a drug targeting system against melanoma cells can be designed using such complexes.
  • non-peptide moieties were added to one of two forms of all-D IV-H1; one containing just the IV-H1 sequence [designated D-IVHl(-Y)], and one containing the IV-H1 sequence and a C-terminal Tyr residue (designated D-IVH1').
  • C 10 -D-IV-H1 [designated either D-IVH1(-Y)C10 or D-IVHI'CIO] and PEG 1900 -D-IV-H1 (designated D-rVHl'PEG) were tested for inhibition of Ml 4 human melanoma cell adhesion to type IV collagen.
  • D-IVHl'PEG (designated D-IVHl'PEG) were subsequently retested for inhibition of Ml 4 human melanoma cell adhesion to type IV collagen.
  • adding a conjugate to the D-IV-H1 sequence does not compromise the inhibitory properties of D-IV-H1 , and may improve the in vivo half-life of this potential therapeutic.
  • the present invention also provides complexes and methods wherein a cytotoxic agent can be delivered to a cell. That is, the polypeptides or peptide-conjugates described herein can be used to target specific tumor cells, bind thereto, optionally invade the cellular structure, and deliver a cytotoxic agent.
  • cytotoxic agents include DNA intercalators, metal chelators, alkylating agents, and membrane disrupting agents. Examples of specific such agents include risin A, dioxorubicin, and mitomycin C.
  • the complexes (polypeptides and conjugates with or without cytotoxic agents attached thereto) of the present invention can be made by a variety of solid-phase or solution techniques.
  • the polypeptides can be prepared by other methods (e.g., solution methods) and then attached to a support material for subsequent coupling with a non-peptide moiety, it is preferred that standard solid-phase organic synthesis techniques, such as solid- phase peptide synthesis (SPPS) techniques be used for preparation of the peptides as well as the conjugates.
  • SPPS solid- phase peptide synthesis
  • solid-phase peptide synthesis involves a covalent attachment step (i.e., anchoring) that links the nascent peptide chain to a support material (typically, an insoluble polymeric support) containing appropriate functional groups for attachment.
  • a covalent attachment step i.e., anchoring
  • the anchored peptide is extended by a series of addition (deprotection/coupling) cycles that involve adding N ⁇ -protected and side-chain-protected amino acids stepwise in the C to N direction.
  • chain assembly has been accomplished, protecting groups are removed and the peptide is cleaved from the support.
  • the non-peptide moiety and/or the cytotoxic agent is added to the peptide before the protecting groups are removed.
  • SPPS begins by using a handle to attach the initial amino acid residue to a functionalized support material.
  • a handle i.e., linker
  • a handle is a bifunctional spacer that, on one end, incorporates features of a smoothly cleavable protecting group, and on the other end, a functional group, often a carboxyl group, that can be activated to allow coupling to the functionalized support material.
  • Known handles include acid-labile p-alkoxybenzyl (PAB) handles, photolabile o-nitrobenzyl ester handles, and handles such as those described by Albericio et al., J. Org. Chem., 55, 3730-3743 (1990) and references cited therein, and in U.S. Patent Nos.
  • the synthesis cycle generally consists of deprotection of the N ⁇ -amino group of the amino acid or peptide on the support material, washing, and, if necessary, a neutralization step, followed by reaction with a carboxyl-activated form of the next N ⁇ -protected amino acid. The cycle is repeated to form the peptide of interest.
  • Solid-phase peptide synthesis methods using functionalized insoluble support materials are well known. See, for example, Merrifield, J. Am. Chem. Soc, 85, 2149 (1963); Barany and Merrifield, In Peptides, Vol. 2, pp. 1-284 (1979); Barany et al, Int. J. Peptide Protein Res.
  • Fmoc methodologies are preferably used. This involves the use of mild orthogonal techniques using the base-labile N ⁇ -9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.
  • Fmoc amino acids can be prepared using fluorenylmethyl succinimidyl carbonate (Fmoc-OSu), Fmoc chloride, or [4-(9- fluorenylmethyloxycarbonyloxy)phenyl] dimethylsulfonium methyl sulfate (Fmoc-ODSP).
  • Fmoc-OSu fluorenylmethyl succinimidyl carbonate
  • Fmoc-ODSP [4-(9- fluorenylmethyloxycarbonyloxy)phenyl] dimethylsulfonium methyl sulfate
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene
  • the immobilized conjugate can then be removed, for example, using trifluoroacetic acid (TFA) at room temperature.
  • TFA trifluoroacetic acid
  • Fmoc soiid-phase peptide synthesis methodologies are well known to one of skill in the art and are discussed in Fields et al., In Synthetic Peptides: A User '_. Guide (G.A. Grant, Ed.), Chapter 3, pp. 77-183, W.H. Freeman and Co., NY (1992); and Fields et al., Int. J. Peptide Protein Res., 35, 161-214 (1990).
  • a variety of support materials for preparation of the complexes of the present invention can be used.
  • They can be of inorganic or organic materials and can be in a variety of forms (e.g., membranes, particles, spherical beads, fibers, gels, glasses, etc.). Examples include, porous glass, silica, polystyrene, polyethylene terephthalate, polydimethylacrylamides, cotton, paper, and the like. Examples of suitable support materials are described by Fields et al., Int. J. Peptide Protein Res., 35, 161-214 (1990); and Synthetic Peptides: A User's Guide (G.A. Grant, Ed.), Chapter 3, pp. 77-183, W.H. Freeman and Co., NY (1992).
  • Functionalized polystyrene such as amino-functionalized polystyrene, aminomethyl polystyrene, aminoacyl polystyrene, p-methylbenzhydrylamine polystyrene, or polyethylene glycol-polystyrene resins can be used for this purpose.
  • the elution gradient was either 0-60% B or 0-100% B in 60 minutes, where A was 0.1% TFA in water and B was 0.1 % TFA in acetonitrile. Detection was at 229 nm. Peptide-conjugate purification was achieved using either the method described above or a Vydac 214TP 152022 C 4 column (15-20 ⁇ m particle size, 300 Angstrom pore size, 250 x 22 mm) at a flow rate of 10 ml/minute. The elution gradient was 55-90% B in 20 minutes, where A was 0.05% TFA in water and B was 0.05% TFA in acetonitrile.
  • Detection was at 229 nm.
  • Analytical RP- ⁇ PLC was performed on a Hewlett- Packard 1090 Liquid Chromatograph equipped with a Hypersil Cig column (5 ⁇ m particle size, 120 Angstrom pore size, 200 x 2.1 mm) at a flow rate of 0.3 ml/minute.
  • the elution gradient was 0-60% B in 45 minutes, where A and B were the same as for peptide purification.
  • Diode array detection was at 220, 254, and 280 nm.
  • the toxin such as the risin A chain
  • the risin A chain sequence (Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu) is assembled onto the N-terminus of the resin-bound IV-H1 sequence by standard solid-phase methods (G.
  • the all-D IV-H1 is synthesized, an Fmoc-Lys(Dde) residue is incorporated (where Dde is l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-ethyl), the Fmoc group is removed, and the risin A chain sequence is added to the resin-bound peptide.
  • the Dde group is removed with hydrazine (C. Fields et al., Biopolymers, 33, 1695-1707 (1993) and the conjugate (alkyl tail or PEG) is added to the N- ⁇ -amino group of the resin-bound peptide.
  • the peptide or peptide-conjugate is then purified and characterized as described above.
  • Human melanoma cells were cultured in Eagle's minimum essential media supplemented with 10% fetal bovine sera, 1 mM sodium pyruvate, 0.1 mg/mL gentamicin (Boehringer Mannheim, Indianapolis, IN), 50 units/mL penicillin, and 0.05 mg/mL streptomycin. Cells were passaged 8 times and then replaced from frozen stocks of early passage cells to minimize phenotypic drift. All cells were maintained at 37°C in a humidified incubator containing 5% CO 2 . All media reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Labeling of Peptides
  • Assays are first performed to quantitate the amount of each peptide adsorbed to the wells after adsorption and rinsing.
  • Synthetic peptides are radiolabeled by reductive methylation using sodium cyanoborohydride and 3H- formaldehyde. By this technique, the ⁇ -amino groups of Lys and the ⁇ -amino terminus become labeled.
  • the radiolabeled substrate is added to microtiter wells and incubated overnight. Wells are blocked, then rinsed. Lysis buffer (0.5 M NaOH, 1% SDS) is then used to remove the radioactivity for quantitation.
  • Adhesion Assay Adhesion of cells was determined as described previously (C.
  • Immulon 1 plates (Dynatech Laboratories Inc., Chantilly, VA) overnight at 37°C. Nonspecific binding sites were blocked with 2 mg/mL ovalbumin in phosphate buffered saline (PBS) for 2 hours at 37°C. Cells were radiolabeled overnight with 20 ⁇ Ci/mL Tran 35 S-LabelTM (>1000 Ci/mmol specific activity; ICN, Costa Mesa, CA). Cells were released from tissue culture flasks with 37°C PBS containing 0.05% trypsin and 0.53 mM EDTA, then washed several times with PBS.
  • PBS phosphate buffered saline
  • Cells were added to the wells at a density of 50,000 cells/mL in a total volume of 100 ⁇ L of the respective cell media containing 2 mg/mL ovalbumin and incubated for 2 hours at 37°C.
  • Wells were washed several times with PBS containing 2 mg/mL ovalbumin and the remaining adherent cells were lysed and radioactivity determined as described (C. Fields et al., J. Biol. Chem., 268, 14153-14160 (1993); A. Miles et al., J. Biol. Chem., 269, 30939-30945 (1994); C. Li et al., Biochemistry, 36, 15404-15410 (1997); J. Lauer et al., J. Med.
  • Adhesion percentages were based on total counts of radioactivity added to each well. Competition of cell adhesion assays were performed as described previously (A. Miles et al., J. Biol. Chem., 269, 30939-30945 (1994); C. Li et al., Biochemistry, 36, 15404-15410 (1997)) using substrate at concentrations which provide >50% initial cell adhesion. Cells were preincubated for 30 minutes at 37°C with various concentrations of the inhibitory peptide, then the cells, in the continued presence of the inhibitor, are added to the wells and allowed to adhere for 30 minutes at 37°C.
  • L-IVH1, D-IVHl, and RI-IVH1 were tested for their ability to inhibit metastasis in vivo as described previously (I. Saiki et al., Jpn. J. Cancer Res., 84, 326-335 (1993)).
  • Highly metastatic A375SM human melanoma cells (A375SM melanoma cells supplied by Dr. James B. McCarthy, University of Minnesota, who had originally obtained them from Dr. I.J. Fidler, M.D. Anderson Hospital, Houston, TX) were pre-incubated several different concentrations of L-IVH1, D-IVHl, and RI-IVH1 (Table 1).
  • mice were then injected into the lateral tail veins of immunocompromised (KSN nude female) mice (Shizuoka Laboratory Animal Center, Hamamatsu, Japan), which had 24 hours prior to this been injected with 20 ⁇ L of anti-asialo GM1 antisera (Shizuoda Lavoratory Animal Center, Hamamatsu, Japan). After 50 days, the mice were sacrificed and the number of lung metastatic nodules was quantified in a blinded fashion. The data represent the means of 5 animals/group, plus or minus the standard deviations (SD) of the means. The all-L and all-D versions of IV-H1 were found to inhibit melanoma cell metastasis in vivo (Table 1).
  • L-IVH1 100 50 + 12 (36-62)
  • L-rvm 1000 16 + 13 (4-34)
  • RI-IVH1 10 86 + 12 (72-102)
  • RI-IVHl 100 84 + 8 (76-96)
  • D-IVHl The inhibitory behaviors of D-IVHl have also been examined by synthesizing several peptide-conjugates, i.e., where a non-peptide moiety is incorporated onto IV-Hl. Peptide-conjugates are created to improve the bioavailability and subsequent half-life of peptide-based drugs in vivo. Two conjugates have been studied: a lipophilic C, 0 alkyl "tail” and polyethylene glycol (PEG). Conjugates were added to one of two forms of all-D IV-Hl; one containing just the IV-Hl sequence [designated D-IVHl(-Y)], and one containing the IV-Hl sequence and a C-terminal Tyr residue (designated D-IVHl').
  • the C 10 alkyl tail was coupled to resin-bound all-D IV-Hl and the product purified and characterized using methods described previously (P. Berndt et al, J. Am. Chem. Soc, 117, 95159-9522 (1995); and Y.C. Yu, J. Am. Chem. Soc, 118, 12515-12520 (1996)).
  • PEG of MW 1900 was coupled to resin-bound all-D IV-Hl and the product purified and characterized as described previously (P. Berndt et al., J. Am. Chem. Soc, 117, 9515-9522 (1995); Y.C. Yu et al, J. Am. Chem.
  • Co-D-IV-Hl [designated either D-IVH1(-Y)C10 or D-IVH1'C10] and PEG 19oo -D-rV-Hl (designated D-IVHl'PEG) were tested for inhibition of M14 human melanoma cell adhesion to type IV collagen using an assay previously described (A.J. Miles et al, J. Biol.
  • D-IV-H1 C. o -D-IV-Hl [designated D-IVHI'CIO] and PEG 1900 -D-IV-H1 (designated D-IVHl'PEG) were subsequently retested for inhibition of Ml 4 human melanoma cell adhesion to type IV collagen.
  • D-IV-H1, C 10 -D-IV-H1, and PEGj ⁇ oo -D-IV-Hl all inhibited melanoma cell adhesion to type IV collagen in similar dose-dependent fashions ( Figure 4).
  • adding a conjugate to the D-IV-H1 sequence does not compromise the inhibitory properties of D-IV-H1, and may improve the in vivo half-life of this potential therapeutic.

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Abstract

L'invention concerne des conjugués de peptides et de polypeptides et des procédés de leur utilisation. Le polypeptide possède une séquence d'acides aminés qui se présente comme un fragment de la région collagène continue du domaine majeur à triple hélice de la chaîne α1 du collagène de type IV, dans lequel le polypeptide est sous une forme entièrement D. Le conjugué de peptides comprend un fragment de polypeptidique provenant de la région collagène continue du domaine majeur à triple hélice de la chaîne α1 du collagène de type IV, lié par covalence à une fraction non peptidique.
PCT/US1998/022405 1997-10-22 1998-10-22 Inhibition de l'adherence des cellules tumorales au collagene de type iv WO1999020300A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106632616A (zh) * 2016-07-18 2017-05-10 复旦大学附属妇产科医院 具有卵巢肿瘤靶向d构型多肽及其基因递释系统

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991008755A1 (fr) * 1989-12-14 1991-06-27 Regents Of The University Of Minnesota Polypeptide presentant une activite d'adherence, de propagation et de motilite de cellules de collagene de type iv
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WO1991008755A1 (fr) * 1989-12-14 1991-06-27 Regents Of The University Of Minnesota Polypeptide presentant une activite d'adherence, de propagation et de motilite de cellules de collagene de type iv
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CN106632616A (zh) * 2016-07-18 2017-05-10 复旦大学附属妇产科医院 具有卵巢肿瘤靶向d构型多肽及其基因递释系统
CN106632616B (zh) * 2016-07-18 2020-12-25 复旦大学附属妇产科医院 具有卵巢肿瘤靶向d构型多肽的基因递释系统

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