INTRODUCTION

Technical Field

The field of this invention concerns compositions 10 for plasminogen activation.

Background

Recombinant technology has provided opportunities to produce proteins which previously were not available.

15 The great diversity of physiologically active proteins produced by genetic engineering, which in many cases were not previously accessible due to economic consid¬ erations, have now become accessible. Proteins such as human growth hormone, tissue plasminogen activator,

20 Factor VIIIc, erythropoietin, Interleukin-2, and G-CSF are only a few of the proteins which are already commercially available or will be commercially available shortly. All of these proteins have been shown to have important physiological activities and

25 many of them have already been designed for specific therapies.

The naturally occurring products are provided by natural intravascular processes, different from administration as a drug of the same product. In many

30 instances, the physiological process localizes admin¬ istration at the site requiring the particular product. Thus, rather than having the physiologically active product generally disseminated throughout the body, a high localized concentration is provided and the „ 35 protein is rapidly degraded or deactivated as it moves from the site where it is needed. Nature, by providing _t for specific ligand signals, provides that a particular activity be localized.

When administering drugs in many instances, one

40 cannot direct a drug to a specific site, nor control

the localized concentration to a desired degree. Therefore, one is forced to use relatively large amounts of the drug to ensure that there is sufficient amount of the drug at the site where it is needed. This need to administer drugs syste ically has many disadvantages.

In a number of cases, even though recombinant technology has provided for great economies as compared to extraction from natural sources, nevertheless the cost of the drug remains high. Secondly, in a number of cases, the drugs may have numerous effects, desirable as well as undesirable, so that there is an interest in providing the lowest amount of the drug to achieve the desired purpose. Also, there is interest in being able to enhance or diminish the lifetime of the activity provided by the drug, depending upon the nature of the drug.

Relevant Literature

Descriptions of laminin and its activities may be found in Woodley et al. , (1983) Biochem. Biophys. Acta. 761:278-83; Terranova et al. , (1980) Cell 22;712-26; Dziadek et al. , (1985) EMBO J. i:2515-18; Yurchenco et al. , (1985) J. Biol. Chem. 260t7636-44; Tharonis et al.,

(1988) J. Cell Biol. 107;1253-60; Sephel et al., (1989) Biochem. Biophys. Commun. 162;821-29; Liesi et al. ,

(1989) FEBS Lett. 24_4:141-48; Kleinman et al. , (1989) Arch. Biochem. Biophys. 272:39-45; and Iwamoto, et al. , (1987) Science 238:1132-34.

A pentapeptide sequence IKVAV from the A chain of laminin has been identified which stimulates neurite outgrowth, cell adhesion and cell migration (Tashiro (1989) J. Biol. Chem. 264:16167-182). This pentapeptide is contained within an 18-amino acid fragment comprising residues 2091-2108 of the laminin A chain (Sasaki et al., (1988) J. Biol. Chem. 263:16536-544). The fragment with an additional amino-terminal Cys residue (desig¬ nated PA22-2) has been demonstrated to stimulate collagenase IV activity in a culture medium of B16-F10

melanoma cells and increase the incidence of lung colonization by B16-F10 cells in mice (Kanemoto et al^ , (1990) Proc. Natl, Acad. Sci. USA 87:2279-83). Intact laminin has been shown to stimulate tissue plasminogen activator catalyzed activation of plasminogen to plasmin (Stack et al., (1990) Biochemistry 29:4966-70).

SUMMARY OF THE INVENTION Novel compositions are provided combining plasminogen activator and a laminin fragment or analog thereof, whereby the properties of the plasminogen activator are modified. The composition may be administered to a mammalian host for preventing the formation of or rapidly dissolving any clots or embolisms.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Novel compositions are provided comprising tissue plasminogen activator or analogs thereof with a peptide fragment of laminin or functional analogs thereof, where the resulting composition has enhanced plasminogen activation activity as compared to the same amount of plasminogen activator in the absence of the laminin associated peptide. These compositions may be formulated in accordance with conventional techniques employed for formulation of plasminogen activator and administered analogously to a host susceptible to the formation of an embolism or having an embolism.

The tissue plasminogen activator which is employed may be from any mammalian source, particularly primate, more particularly human, and may be the naturally occurring tissue plasminogen activator, alleles thereof, mutants thereof, including deletions, insertions and substitutions, or active fragments thereof, where such active fragments are capable of activating plasminogen. Various modifications of tissue plasminogen activator (tPA) have been reported, see, for example, EPA

0 357 296; EPA 0 178 105; EPA 0 373 896; EPA 117 981; and WO 90/03 436.

The laminin associated sequence will, for the most part, include the sequence R-K-Q-A-A-S-I-K-V-A-V, more preferably include the sequence (C-)S-R-A-R-K-Q-A-A-S- I-K-V-A-V-S-A-D-R, where the ( ) indicates optional presence. Usually, within the indicated sequences, there will be not more than 2 single mutations, including deletions, insertions or substitutions. For the most part, substitutions will be conservative, where amino acids having substantially the same conformation and polarity as the naturally present amino acid will be used for the substitution.

Illustrative conservative substitutions include groups such as G,A; V,I,L; S,G,M,C; D,E; K,R; N,Q; and F,W,Y,H. Conservative substitutions may be extended, where N,Q may be used for substitution of D,E and vice versa or S,T,M and vice versa.

The above sequences are sequences from the human laminin A chain and may be modified in a number of ways. Thus, one or more different groups may be attached at an end for convenience of isolation and purification, to stabilize the peptide, or the like. For example, one or more of the amino acids may be substituted by the unnatural D-stereoisomer. Particularly, one or more alanines may be substituted. Alternatively, terminal amino acids may be employed having the unnatural chirality. In addition, one may provide for a terminal amide or a terminal acylated amino, particularly acetylated, or alkylated, particularly methylated, amino acids. Where a cysteine is provided at the N-terminus, the cysteine may be alkylated or unsubstituted on the mercaptan group. Substituents may be aliphatic, alicyclic, aromatic, heterocyclic or substituted derivatives thereof, generally from 1 to 12 carbon atoms, usually 1 to 6, and 0 to 5, usually 0 to 3 heteroatoms, such as 0, N, S, P or the like.

For the most part, the peptides of this invention will be fewer than 60 amino acids, preferably fewer than 30 amino acids, generally ranging from about 10-30, more usually from about 15-25 amino acids. Usually, not more than 10% of the amino acids present in the group will be modified by mutation, particularly substitutions.

The subject peptides may be prepared in a variety of conventional ways, including synthesis on a support, recombinant technology, or any other convenient means. For the most part, synthesis will be used, particularly where the amino acid sequence is fewer than about 30 amino acids. By employing synthesis, one may introduce various unnatural amino acids along the chain, more readily modify the N- or C-terminus or the like. In this way, great flexibility is introduced to allow for modifications which will enhance the lifetime of the peptide, modulate its activating effect, improve stability and formulation, or the like.

The subject peptide will usually be combined with tPA at a ratio of at least about 5 μg, more usually at least about 10 μg and up to about 500 μg, more usually not more than about 200 μg per 0.1 pmole of tPA or its analog.

The formulations may be prepared in various ways. The tPA or analog thereof and laminin associated peptide may be combined in an appropriate aqueous medium comprising buffer, stabilizers, saline, or the like, such as phosphate buffered saline, where the various components are physiologically acceptable, and the mixture lyophilized to provide for an intimate mixture of the tPA and laminin associated peptide. Alter¬ natively, each of the components may be separately formulated in physiologically acceptable solutions and mixed prior to use. In the powdered formulation, various physiologically acceptable excipients may be employed, where the tPA may be formulated in conventional manners.

The level of administration of the tPA can be substantially diminished, depending upon the amount of laminin associated peptide included. Thus, increases in activity of the tPA of 10-fold or more may be achieved, so that the amount of tPA which is administered will be at least about 2-fold less than presently prescribed, preferably at least about 5-fold less, and usually not more than about 30-fold less, more usually not more than about 20-fold less. The amount of tPA which is administered will also vary depending upon the manner of administration, the length of time administered, the desired rapidity of response, and the like. Thus, substantial reductions in the amounts as to each of these methods of administration can be achieved. The amount of tPA administered will generally be in the range of about 20,000 to 200,000 IU/kg hr.

The peptide can also be administered in the absence of exogenous tPA to stimulate the activity of endogenous tPA. For example, the peptide may be coated or bound covalently to the surface of devices, such as stents, catheters and other such devices, which are inserted in human blood vessels or tissue to prevent fibrin deposition on these objects. The peptide may also be administered intraperitoneally at the time of abdominal surgery to prevent adhesions which form as a consequence of fibrin formation on serosal surfaces.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

MATERIALS AND METHODS

Materials. Synthetic peptides IKVAV-NH2 and SRARK-NH2 were purchased from the University of Louisville Peptide Synthesis Facility. All other peptides were from Multiple Peptide Systems, San Diego, CA. The composition and sequence of all peptides were verified using an Applied Biosysterns 420A derivatizer/analyzer amino acid analysis system

and an Applied Biosystems 477A pulse liquid phase sequencer with "on-line" 120A PTH amino acid analysis. Peptides with biological activity were further puri¬ fied by reverse-phase HPLC. The synthetic substrates D-Val-Leu-Lys-p-nitroanilide (VLK-pNA) and D-Ile-Pro- Arg-p-nitroanilide (IPR-pNA) and human fibrinogen were purchased from Helena Laboratories, Beaumont, TX. Fibrinogen was rendered plasminogen-free by chroma- tography on L-Lysine-Sepharose (Deutsch and Mertz, (1970) Science 170:1095-1096) and CNBr fragments of fibrinogen were generated according to the method of Blomback et al., (1968) Nature, 218:130-134. Urokinase (u-PA) was purchased from Calbiochem, San Diego, CA and coupled to CNBr activated Sepharose according to the method of Cuatrecasas et al. , (1968) Proc. Natl. Acad. Sci. USA, 6J.:636-643. Two chain recombinant tissue plasminogen activator (t-PA) was supplied by Dr. Henry Berger at Wellcome Research Laboratories, Research Triangle Park, NC. Recombinant human fibroblast collagenase (type I) was kindly supplied by Dr. Jerry McGeehan at Glaxo Research Laboratories, Research Triangle Park, NC.

Proteins. Plasminogen (Pg) was purified from human plasma by affinity chromatography on L-Lys- Sepharose (Deutsch and Mertz, (1970) Science 170:1095- 1096) and separated into isoforms 1 and 2 by affinity chromatography on concanavalin A-Sepharose (Gonzalez- Gronow and Robbins, (1984) Biochemistry 23:190-194) . Affinity chromatography purified form 2 was utilized for all experiments. Plasmin (Pm) was generated by incubating 100 μg of Pg with 100 μl of urokinase- Sepharose in 20 mM Hepes, pH 7.4 for 1 h at 25°C followed by centrifugation to remove the resin. Lys77-Pg was prepared by limited proteolysis of Glu-Pg with Pm in a molar ratio of 10:1 for 30 min at 25°C followed by chromatography on pancreatic trypsin inhibitor-Sepharose to remove Pm (Castellino and Power, (1981) Meth. Enzymology 80:365-378). Protein

concentrations were determined spectrophotometrically at 280 nm using an Aι%/icm value of 16.8 and molecular weights of 92,000, 83,000 and 81,000 for Glu-Pg, Lys77-Pg and Pm, respectively (Castellino, (1981) Chem. Rev. 81:431-436).

Type IV collagenase/gelatinase was purified from the serum free conditioned medium of porcine synovial membranes stimulated with phorbol 12-myristate 13- acetate by inhibitor-affinity chromatography (Stack and Gray, (1988) FASEB J. A1006). Briefly, pooled conditioned medium was concentrated by ultrafiltration and the metalloproteinase zy ogens activated by incubation with 0.7 /*M p-aminophenylmercuric acetate for 4 h at 35°C. Type I collagenase and type IV collagenase were separated from other proteins using an affinity matrix consisting of N-[l(R,S)-carboxy-n- butyl]-Leu-Phe-Ala coupled through the C-terminus to EAH-Sepharose 4B (Pharmacia) . Chromatography on DEAE- Sephacel was used to separate type I collagenase from type IV collagenase.

Effect of synthetic peptides on Pg activation. Coupled assays were used to evaluate the initial rate of Pg activation by t-PA or u-PA by monitoring the amidolytic activity of generated Pm (Wohl et al. , (1980) J. Biol. Chem. 255:2005-2013). Glu-Pg or

Lys77-Pg was incubated in 96-well microtitre plates at 37°C in 20 mM Hepes, pH 7.4 in a total volume of 175 μl with the Pm substrate VLK-pNA (0.3 mM final concentration) . Pg activation was initiated by the addition of 0.11 nM (4 IU/ l) t-PA or 0.8 nM (2 IU/ml) u-PA and the Pm hydrolysis of VLK-pNA was monitored by measuring the change in absorbance at a wavelength of 405 nm at timed intervals using an Anthos Labtech Instruments model 2001 plate reader. Initial veloci- ties (v.) were calculated from the slope (b) of plots of A4. _n 05 _c ¹ vs t2 using the eq^uation vl. = b( ^x l+κm/S o)'/Eke

(Wohl et al., 1980) J. Biol. Chem. 255_:2005-2013 where K _m is the apparent Michaelis constant for VLK-pNA

hydrolysis by Pm (0.3 mK) , k _e is the empirically determined catalytic rate constant for Pm hydrolysis (3.2 x 10 M min-1 mol Pm-1) and E is the molar extinction coefficient of pNA at 405 nm (8800 M-l cm-1, Erlanger et al. , (1965) Arch. Biochem. Biophys.

95:271-278).

2091 2108 The effect of the peptide LamA and control peptides on Pg activation was determined by pre-incubating Pg with the peptide (0-500 /jg/ml) at 37°C for 10 in. in 155 μl of 20 mM Hepes containing

0.3 mM VLK-pNA. After incubation, plasminogen activator was added and the reaction monitored as described above. Effect of peptide LamA 2091—2108 on amidolytic activity of plasminogen activators. The amidolytic activities of t-PA and u-PA in the presence of the peptide LamA 2091—2108 were determined by incubating t-PA (0.11 nM) or u-PA (0.8 nM) with peptide

(0-500 /xg/ml) at 37°C in 155 μl 20 mM Hepes pH 7.4. The reaction was initiated by addition of VLK-pNA

(0.3 mM) and substrate hydrolysis was monitored at a wavelength of 405 nm as described above. activity. Activity of collagenases was assayed using the synthetic collagenase substrate Dnp-Pro-Leu-Gly-

Leu-Trp-Ala-D-Arg-NH2 (Dnp-PLGLWAR-NH2) (5 μK) in

0.05 M Tris-HCl, 5 mM CaCl2, 0.2 M NaCl, pH 7.7 at

37°C (Stack and Gray, J. Biol Chem. (1989) 264:42787-

4281). Fluorometric measurements were performed with an SLM-Aminco SPF-500C spectrofluorometer. The initial rate of substrate hydrolysis was determined by monitoring the increase in fluorescence emission at a wavelength of 346 nm using an excitation wavelength of

280 nm. The effect of LamA ^2091"2108 on two structurally and mechanistically related collagenases was determined by incubating human fibroblast type I collagenase (30 nM) or porcine synovial type IV collagenase/gelatinase (2.7 pM) with the synthetic

substrate followed by addition of increasing amounts of LamA ^2091"2108 (0.3-100/tM) . Kinetic data for collagenase activity in the presence of LamA 2091—2108 were fit by least squares regression analysis to the equation log (A /A. - 1) = log K = nlog [I], where A _Q and A. represent the activity determined in the absenc ^~~ "e and presence of LamA2091—2108, respectively and n is the number of binding sites/enzyme molecule

(Ambrose et al. , (1950) J. Am. Chem. Soc. 72, 317-321. ^Ic so concentrations were estimated from the slope and intercept values.

RESULTS Kinetics of Pg activation in the presence of LamA 2091- 2108. _m Th,e synt.,het.i.c pept.i.d,e _LamA,2091-2108 caused. a dose-dependent increase in the velocity of t-PA catalyzed Pg activation, resulting in a 15-fold increase in activation velocity at a peptide concentration of 100 g/ml. In contrast, no stimulation of u-PA catalyzed Pg activation was observed in the presence of up to 500 g/ml LamA 2091-

2108

(Table I) . The amidolytic activities of t-PA and

Pm were also unaffected by the addition of

LamA

Table I

Effect of LamA2091-2108 on Fibrinolytic Enzymes

Activity (mol substrate hydrolyzed/min)

Enzymes were incubated with peptide (500 ig/ml) for 10 min at 37° followed by addition of the indicated substrate as described in Methods.

A variety of synthetic peptides similar in size to nnq -_ ino LamA ^< - ^j -- ^> - ^> _t j-, _{ut 0} f unrelated amino acid sequence were tested for the ability to stimulate Pg activation by t-

2091 108 PA. As shown in Table II (E-G) only LamA had a stimulatory effect on Pg activation. An unrelated laminin-derived peptide YIGSR also had no effect on Pg activation (Table II H) .

Table II Effect of Synthetic Peptides on Plasminogen Activation by

ith 0.3 μK Pg in the presence of 0.3 mM VLK-pNA for 10 min at 37°C followed by addition of t-PA (0.11 nM) .

To determine whether the unpaired amino-terminal Cys residue added to the peptide fragment contributed to the stimulatory effect, the Cys was alkylated with iodoacetamide (Glazer et al. , (1975) in Chemical Modification of Proteins, Elsevier Biomedical Press,

Amsterdam, pp. 101-112). The stimulatory properties of the alkylated peptide were identical to that of the parent compound. Two pentapeptide fragments of

2091—2108 LamA did not alter the rate of Pg activation (Table II C, D) , suggesting that more than these fragments are necessary for stimulation. In addition, treatment of the peptide with Staphlococcus aureus V8

proteinase, which introduces a single cleavage in the peptide (verified by HPLC), removed the stimulatory effect, providing further evidence that a substantial portion of the peptide is necessary for stimulation of

9ΩQ1 —91 Ωfi Pg activation. The effect of LamA ^uy (100 /jg/ml) on the initial rate of Pg activation was determined.

The k . /K of t-PA catalyzed Pg activation calculated cat m ι . from the data increases from 3.2 μK~ sec~ in the absence of peptide to 69.2 μK -1sec-1 in the presence of the peptide, resulting in an overall 22-fold increase in activation efficiency.

Fibrinogen fragments potentiate t-PA dependent Pg activation, with maximum stimulation at a fragment concentration of approximately 20 g/ml (Nieuwenhuizen et al., (1983) Biochem. Biophys. Acta. 755:531-533). The effect of LamA 2091—2108 on fibrinogen stimulation of Pg activation was determined. Although fibrinogen fragments result in a greater overall stimulation of Pg activation, simultaneous addition of LamA 2091—2108 and fibrinogen fragments abolishes the additional stimulatory effect achieved with fibrinogen alone, indicating that LamA ^" and fibrinogen may compete for similar binding sites on the Pg molecule.

Effect of LamA 2091—2108 on collagenase activity. The effect of LamA ^2091"2108 on the activity of purified interstitial and type IV collagenase was determined.

Addition of LamA ^2091"2108 had an inhibitory effect on collagenase activity with an IC _go of 3 μK for type I collagenase and 43 μK for type IV collagenase. Since free Cys is a weak inhibitor of collagenase activity (IC ₅₀ of 3 mM, Darlak et al. , (1990) J. Biol. Chem. 2^5:5199-5205), the amino-terminal Cys of LamA ^2091"2108 was alkylated with N-ethylmaleimide (Glazer et al. , 1975). The alkylated peptide retained slight inhibitory activity with an IC _5Q of approximately 100 μK for both collagenases.

It is evident from the above results, that by using a small oligopeptide, which can be inexpensively and efficiently synthesized, dramatically reduced amounts of tPA or its analogs may be employed in protecting against clots. Thus, novel compositions can be provided where the subject fragment and tPA may be combined together in a formulation and administered at substantially lower levels of tPA, without adverse effects on the patient. In this manner, substantial economies can be achieved in the amounts of tPA which must be administered, while assuring the desired level of protection afforded by tPA.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.