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WO1991019513A1 - METHODS OF MODULATING BLOOD PRESSURE USING TGF-β AND ANTAGONISTS THEREOF - Google Patents

METHODS OF MODULATING BLOOD PRESSURE USING TGF-β AND ANTAGONISTS THEREOF Download PDF

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Publication number
WO1991019513A1
WO1991019513A1 PCT/US1991/004449 US9104449W WO9119513A1 WO 1991019513 A1 WO1991019513 A1 WO 1991019513A1 US 9104449 W US9104449 W US 9104449W WO 9119513 A1 WO9119513 A1 WO 9119513A1
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Prior art keywords
tgf
antagonist
blood pressure
precursor
mature
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PCT/US1991/004449
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French (fr)
Inventor
Frederick B. Oleson, Jr.
Charles R. Comereski
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Bristol-Myers Squibb Company
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Publication of WO1991019513A1 publication Critical patent/WO1991019513A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TGF-9 transforming growth factor-beta
  • BP blood pressure
  • rTGF-j8 mature recombinant TGF- ⁇ l
  • the scope of the invention is not limited to the use of rTGF- l but rather encompasses the use of mature and precursor forms of all members of the TGF-9 family effective at modulating blood pressure, including natural and recombinant mature TGF- / 31, TGF-02, TGF- 3, TGF-04, etc., as well as TGF-y8 hybrids, analogs and latent TGF- ⁇ complexes.
  • the invention includes the use of any and all compositions effective at antagonizing TGF- activity, including but not limited to anti-TGF- ⁇ antibodies and TGF-y8 receptors.
  • TRANSFORMING GROWTH FACTOR-BETA TGF- ⁇ is a member of a recently described family of polypeptides that regulate cellular differentiation and proliferation. Other members of this family include Mullerian inhibitory substance (Cate et al., 1986, Cell 45:685-698), the inhibins (Mason et al., 1985, Nature 318:659-663) and a protein predicted from a transcript of the decapentaplegic gene complex of Drosophila (Padgett et al., 1987, Nature 325: 81-84.
  • TGF- ⁇ Four types of TGF- ⁇ have been identified and designated TGF- l, TGF- ⁇ 2, TGF- ⁇ l .2 , and TGF- ⁇ 3 .
  • the first described type, TGF-91 consists of two identical disulfide linked subunits having molecular weights of 13,000 (Assoian et al., 1983, J. Biol. Chem. 258:7155-7160; Frolik et al., 1983, Proc. Natl. Acad. Sci. USA 80:3676-3680; Frolik et al., 1984, J. Biol. Chem. 260:10995-11000).
  • tissue sources including placenta (Frolik et al., 1983, Nature 325:81-84), blood platelets (Childs et al., 1982, Proc. Natl. Acad. Sci. USA 79:5312- 5316; Assoian et al., 1983, J. Biol. Chem. 258:7155-7160) kidney (Roberts et al., 1983, Biochemistry 22:5692-5698), and demineralized bone (Seyedin et al., 1985, Proc. Natl. Acad. Sci. USA 82:119-123).
  • TGF- ⁇ l cDNA clones coding for human (Deryn ⁇ k et al., 1985, Nature 316:701-705), mouse (Derynck et al., 1986, J. Biol. Chem. 261:4377-4379) and simian (Sharpies et al., 1987, DNA 6:239-244) TGF- ⁇ l have been isolated. DNA sequence analysis of these clones indicates that TGF- ⁇ l is synthesized as a large precursor polypeptide, the carboxy terminus of which is cleaved to yield the mature TGF- ⁇ monomer. Strong sequence homology has been found throughout the TGF- ⁇ l precursor protein from all of the above sources.
  • TGF- ⁇ l promotes the anchorage independent growth of normal rat kidney fibroblasts (Roberts et al., 1981, Proc. Natl. Acad. Sci. USA 78:5339-5343; Roberts et al., 1982, Nature 295:417-419; Twardzik et al., 1985, J. Cell. Biochem. 28:289-297) ; in the presence of 10% serum alone, it is able to induce colony formation of AKR-2B fibroblasts (Tucker et al., 1983, Cancer Res. 43:1518-1586). TGF- ⁇ l has also been shown to cause fetal rat muscle mesenchymal cells to differentiate and produce cartilage specific macromolecules (Seyedin et al., 1986, J. Biol. Chem. 261:5693-5695).
  • TGF- ⁇ l purified from human platelets has been shown to inhibit the growth of certain cells in culture (Tucker et al., 1984, Science 226:705-707). TGF- ⁇ l has also been shown to inhibit the growth of several human cancer cell lines (Roberts et al., 1985, Proc. Natl. Acad. Sci. USA 82:119- 123) . This inhibitory/stimulatory effect of TGF- ⁇ l may depend on several factors including cell type and the physiological state of the cells (for review see Sporn et al., 1986, Science 233:532-534).
  • TGF- ⁇ 2 is a polypeptide of molecular weight 26,000 composed of two identical 13,000-dalton subunits which are disulfide like (Chiefetz et al., 1987, Cell 48:409-415; Ikeda et al., 1987, Biochemistry 26:2406- 2410) and has been isolated from bovine demineralized bone (Seydin et al., 1987, J. Biol. Chem.
  • TGF- ⁇ l and TGF- ⁇ 2 share 71% amino acid sequence identity in their mature regions, and 41% identity in their precursor structures.
  • TGF- ⁇ 3 the amino acid sequence of which has very recently been deduced from cDNA clones, appears to contains C-terminal 112 amino acid sequence with about 80% homology to the mature monomers of TGF- ⁇ l and TGF- ⁇ 2 (Dijke et al., 1988, Proc. Natl. Acad. Sci. USA 85:4715-4719).
  • TGF- ⁇ l.2 is a heterodimeric form comprising a ⁇ l and ⁇ 2 subunit linked by disulfide bonds (Chiefetz et al., 1987, Cell 48:409-415).
  • the invention relates to the use of TGF- ⁇ s as antihypertensive agents capable of rapidly and significantly lowering blood pressure.
  • This aspect of the invention encompasses the use of any and all TGF- ⁇ polypeptides having a hypotensive activity, including mature and precursor forms of TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3, hybrid TGF- ⁇ s, latent TGF- ⁇ complexes, TGF- ⁇ analogs, etc.
  • simian recombinant TGF- ⁇ l is administered parenterally to induce rapid significant, and sustained decreases of arterial blood pressure in cynomoigus monkeys.
  • TGF- ⁇ s may be used to rapidly lower blood pressure to normal levels in patients facing acute hypertension and emergency conditions associated with extreme hypertension.
  • the invention relates to the use of TGF- ⁇ antagonists to elevate blood pressure through the inhibition of hypotension induced by TGF- ⁇ and/or related factors.
  • Any composition which antagonizes TGF- ⁇ activity may be useful in this regard, including for example, anti-TGF- ⁇ antibodies and TGF- ⁇ receptors.
  • methods which lower and/or maintain the level of circulating TGF- ⁇ in an individual may result in a similar pressor effect.
  • anti-TGF- ⁇ antisense RNA molecules may inhibit synthesis and release of bioactive TGF- ⁇ s, thereby preventing excessive hypotensive signal generation and resulting hypotension. 4. DESCRIPTION OF THE FIGURES FIG. 1.
  • Nucleotide sequence of simian TGF- ⁇ l cDNA and deduced amino acid sequence The 1600 bp insert of pTGF- ⁇ l-2 was subcloned into the M13mpl8 and M13mpl9 cloning vectors (Yanisch-Perron et al., 1985, Gene 33:103-119) and both strands were sequenced using the dideoxy chain- termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467). The deduced amino acid sequence of simian TGF- ⁇ l is presented directly above the cDNA sequence.
  • the human TGF- ⁇ l nucleotide sequence is aligned with and presented directly below the simian cDNA sequence; dots indicate homologous nucleotide residues within the sequences. Amino acid differences between the human and simian proteins are indicated in the top line. The mature TGF- ⁇ l sequence is boxed and the signal peptide is overlined.
  • FIG. 2 Nucleotide sequence of human TGF- ⁇ 2-442 cDNA and deduced amino acid sequence.
  • the 2597 BP insert of PC-21 was subcloned into pEMBL (Dante et al., 1983, Nucleic Acids Res. 11:1645-1654) and sequenced on both strands using the dideoxy chain-termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467).
  • the coding sequence is shown and the deduced amino acid sequence is presented directly above.
  • the mature TGF- ⁇ 2 sequence is boxed and the signal peptide is overlined. Potential glycosylation sites are indicated by asterisks.
  • the arrow indicates the putative signal sequence cleavage site.
  • the nucleotide sequence of simian TGF- ⁇ 2-414 cDNA is identical to the human TGF- ⁇ 2-442 cDNA sequence except that (a) nucleotides 346 through 432 (bracketed) are deleted and replaced by the sequence AAT, and (b) several silent nucleotide changes occur elsewhere in the structure (indicated by single letters directly below the changed nucleotide) .
  • the deduced amino acid sequence for simian TGF- ⁇ 2-414 precursor is identical to the human TGF- ⁇ 2-442 precursor amino acid sequence except that Asparagine replaces amino acid residues 116 through 144 in the human TGF- ⁇ 2-442 structure.
  • the nucleotide sequence of a human TGF- ⁇ 2-414 cDNA has been sequenced through the region indicated by broken underlining and was found to be perfectly homologous to the human TGF- ⁇ 2-442 cDNA sequence except that nucleotides 346 through 432 are deleted and replaced by the sequence AAT.
  • FIG. 3 Nucleotide sequence of hybrid TGF- ⁇ l/ ⁇ 2 precursor DNA and deduced amino acid sequence. The coding sequence is shown and the deduced amino acid sequence is presented directly above. The mature TGF- ⁇ 2 sequence is boxed and the precursor signal peptide is overlined. Glycosylation sites are indicated by asterisks. The arrow indicates the putative signal sequence cleavage site.
  • the TGF- ⁇ 2 mature coding sequence depicted is of human origin. The simian TGF- ⁇ 2 mature coding sequence is nearly identical to the human sequence: only 3 silent base changes occur and are indicated by single letters directly below the changed nucleotide.
  • the present invention is directed to methods of modulating blood pressure in an animal using TGF- ⁇ polypeptides, antagonists and/or combinations thereof.
  • the invention is based upon the discovery that parenterally administered mature rTGF- ⁇ l rapidly and significantly lowers blood pressure in cynomologus monkeys.
  • one aspect of the invention relates to the use of TGF- ⁇ s as antihypertensive/hypotensive agents.
  • TGF- ⁇ antagonists capable of inhibiting the antihypertensive/hypotensive effects of TGF- ⁇ .
  • the invention encompasses the use of anti-TGF- ⁇ antibodies, TGF- ⁇ receptors and other compositions capable of inhibiting TGF- ⁇ -induced hypotension.
  • TGF- ⁇ s as antihypertensive/hypotensive agents.
  • Applicants' initial data indicates that rTGF- ⁇ l can rapidly and significantly lower blood pressure in simian test subjects at a dosage which appears to be at or close to the physiologically tolerable limit.
  • parenteral administration of a TGF- ⁇ at such a dose may be acceptable in hypertensive emergencies requiring agressive treatment.
  • Lower does of a TGF- ⁇ may also be effective at reducing blood pressure, and such doses may be appropriate for patients with moderate to severe hypertension. In these patients, less agressive therapy may be desirable where adverse side effects can not be tolerated.
  • the TGF- ⁇ anithypertensive may be administered alone or in combination with other antihypertensive agents in suitable pharmacological carriers via any appropriate route. In hypertensive emergencies, parenteral administration will provide the fastest decrease in BP and is therefore the recommended route of administration in such situations. Additionally, the TGF- ⁇ may be linked to a carrier or targeting molecule and/or incorporated into liposomes, microcapsules, and controlled release preparations prior to administration in vivo.
  • TGF- ⁇ mature and/or precursor forms of TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3, TGF- ⁇ l/ ⁇ 2, etc.
  • TGF- ⁇ used may be obtained from a variety of sources, including but not limited to isolating natural TGF- ⁇ s from appropriate sources, producing TGF- ⁇ by recombinant DNA techniques, or by chemical synthetic methods, etc.
  • Natural TGF- ⁇ l can be isolated from a variety of sources. This potent modulator of cell behavior is synthesized by a variety of normal and transformed cells in culture (Roberts et al., 1981, Proc. Natl. Acad. Sci. USA 78:5339-5343) and has been purified from various sources including placenta (Frolik et al., 1983, Proc. Natl. Acad. Sci. USA 80:3676-3680), kidney (Roberts et al., 1983, Biochemistry 22:5692-5698), urine (Twardzik et al., 1985, J. Cell. Bioche .
  • TGF- ⁇ l Large quantities of TGF- ⁇ l may be obtained by recombinant DNA techniques using eucaryotic host cells transfected with recombinant DNA vectors containing the TGF- ⁇ l coding sequence controlled by expression regulatory elements. Examples of such methods are described in copending application Serial No. 07/353,728 filed August 17, 1989, which application is incorporated by reference herein it its entirety. Briefly, a cDNA clone coding for simian TGF- ⁇ l precursor was obtained from a cDNA library made from an African Green Monkey cell line, BSC-40. The deduced amino acid sequence of the mature simian TGF- ⁇ l shown in FIG. l has 100% homology with that of the mature human TGF- ⁇ l.
  • Expression vectors were constructed which contain the entire coding sequence for the simian TGF- ⁇ l precursor placed under the control of SV40 expression elements. They were used to transfect Chinese Hamster Ovary cells (CHO cells) . The resulting CHO transfectants produce and secrete primarily a high molecular weight complex from which mature bioactive TGF- ⁇ may be liberated by a routine acidification procedure.
  • Natural TGF- ⁇ 2 used in accordance with the invention can be obtained from a variety of sources.
  • a protein isolated from bovine demineralized bone has been identified as being related to TGF- ⁇ (Seyedin et al., 1987, J. Biol. Chem. 262:1946-1949).
  • the protein has also been isolated from porcine platelets (Cheifetz et al., 1987, Cell 48:409-415), a human prostatic adenocarcinoma cell line PC-3 (Ikeda et al., 1987, Biochemistry 26:2406-2410), and a human glioblastoma cell line (Wrann et al., 1987, EMBO 6:1633- 1636) . Partial amino acid sequence of this protein indicated that it was homologous to TGF- ⁇ and has been termed TGF- ⁇ 2.
  • TGF- ⁇ 2 Large quantities of TGF- ⁇ 2 may be obtained by recombinant DNA techniques using eukaryotic host cells transfected with recombinant DNA vectors containing a TGF- ⁇ 2 coding sequence controlled by expression regulatory ele ents. Examples of such methods are described in copending application Serial No. 07/446,020 filed December 5, 1989, which application is incorporated by reference herein in its entirety. Briefly, cDNA clones coding for human TGF- ⁇ 2 precursor were obtained from a cDNA library made from a tamoxifen treated human prostatic adenocarcinoma cell line, PC-3. The cDNA sequence of one such clone is shown in FIG.
  • TGF- ⁇ 2 is synthesized as a 442 amino acid polypeptide precursor from which the mature 112 amino acid TGF- ⁇ 2 subunit is derived by proteolytic cleavage.
  • This TGF- ⁇ 2 precursor termed TGF- ⁇ 2-442, shares a 41% homology with the precursor of TGF- ⁇ l.
  • cDNA clones coding for simian TGF- ⁇ 2 precursor were obtained from a cDNA library made from an African green monkey kidney cell line, BCS-40.
  • TGF- ⁇ 2 is also synthesized as a 414 amino acid polypeptide precursor from which the mature 112 amino acid TGF- ⁇ 2 subunit is derived by proteolytic cleavage.
  • This TGF- ⁇ 2 precursor termed TGF- ⁇ 2-414, has an amino acid sequence of 414 amino acid residues and is identical to the amino acid sequence of TGF- ⁇ 2-442, except that it contains a single Asparagine residue instead of the 29 amino acid sequence from residue numbers 116 to 135 of the human TGF- ⁇ 2-442 sequence.
  • Hybrid precursor TGF- ⁇ molecules may be prepared using recombinant DNA techniques or synthetic methods, as described in co-pending application Serial No. 07/353,728 filed August 17, 1987. Briefly, expression vectors containing the TGF- ⁇ 2 mature coding sequence joined in-phase (i.e. , in the same translational reading frame) to the TGF- ⁇ l signal and precursor sequences (see FIG. 3) were constructed and used to transfect Chinese Hamster Ovary cells (CHO cells) . The resulting CHO transfectants produce and secrete mature, biologically active TGF- ⁇ 2.
  • conservative substitutions would involve the substitution of one or more amino acids within the sequences shown with another amino acid having similar polarity and hydrophobicity/hydro-philicity characteristics resulting in a silent alteration and a functionally equivalent molecule.
  • conservative substitutions include but are not limited to substitutions within the following groups of amino acids: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine; and methionine, norleucine.
  • LATENT TGF- ⁇ COMPLEX TGF- ⁇ l may be isolated from tissues or tissue culture cells in an inactive, biologically latent form which may be activated by chaotropic agents, proteases, or in vivo.
  • CHO cell transfected with the simian TGF- ⁇ l precursor coding sequence secrete a high molecular weight latent complex involving both the mature and "pro" regions of the TGF- ⁇ precursor.
  • the association of the "pro" region of the TGF- ⁇ precursor has also been observed in latent TGF- ⁇ l complex isolated from platlets. Although the mechanism of activation in vivo is unknown, it is possible that the latent complex provides an important level of regulation on TGF- ⁇ l bioactivity.
  • latent TGF- ⁇ complex may be useful as a means of controlling the hypotensive effect induced by the bioactive form of TGF- ⁇ by releasing it at the situs of natural in vivo activation mechanisms.
  • the identification, isolation and characterization of latent TGF- ⁇ l complex from recombinant CHO cells is described more fully in copending application Serial No. 07/353,728 filed August 17, 1989, which application is incorporated herein by reference in its entirety. 5.2. USE OF TGF- ⁇ ANTAGONISTS AS PRESSOR AGENTS
  • rTGF- ⁇ l is capable of rapidly and significantly lowering blood pressure suggests that the TGF- ⁇ s may be involved in the regulation of BP and/or in the genesis of hypotension.
  • antagonists of TGF- ⁇ s may be useful as pressor/hypotensor agents capable of elevating BP. Any compostion which effectively antagonizes the hypotensive effect of a TGF- ⁇ may be used for this purpose, including but not limited to anti-TGF- ⁇ antibodies and TGF- ⁇ receptors.
  • TGF- ⁇ antagonists may be useful in treating medical conditions characterized by a loss of BP where the elevation of BP to normal levels is desirable. Such conditions include, for example, shock associated with blood volume loss, cardiac emergencies, and hypotension in acute renal failure.
  • the TGF- ⁇ antagonists may be administered alone or in combination and/or together with other pressors/hypotensors such as dopamine, epinephrine, aminophylline, etc.
  • Compounds containing effective doses of TGF- ⁇ antagonist formulated in a suitable pharmacological carrier may be administered to patients experiencing hypotension or conditions associated with hypotension via any appropriate route including but not limited to injection, infusion and selective catheterization in order to elevate BP.
  • the TGF- ⁇ antagonist may be linked to a carrier or targeting molecule and/or incorporated into liposomes, microcapsules, and controlled release preparations prior to administration in vivo.
  • ANTI-TGF- ⁇ ANTIBODIES Antibodies capable of inhibiting the hypotensive effect of TGF- ⁇ may be useful as pressor agents.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of TGF- ⁇ s. For the production of antibodies, various host animals can be immunized by injection with a TGF- ⁇ , or a synthetic TGF- ⁇ peptide, including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peotides, oil emulsions, keyhold lympet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • a monoclonal antibody to an epitope of a TGF- ⁇ can be prepared by using any technique which provides for the production of antibody molecules by continous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497) , and the more recent human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. , pp. 77-96) .
  • Antibody fragments which contain the idiotype of the molecule may be generated by known techniques.
  • such fragments include but are not limited to the F(ab')_ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the Ffab')., fragment, and the two Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • TGF- ⁇ RECEPTORS Exogenous TGF- ⁇ receptor molecules may be useful pressor agents inasmuch as they are capable of binding circulating TGF- ⁇ and/or out-competing endogenous receptors which may initiate the hypotensive effect of TGF- ⁇ .
  • TGF- ⁇ receptors may be prepared by the methods described in copending application Serial No. 269,524 filed November 11, 1988, which application is incorporated by reference herein in its entirety.
  • rTGF- ⁇ l has a profound reducing effect on blood pressure.
  • CYNOMOLGUS MONKEYS One adult male and two adult female cynomoigus monkeys were monitored for blood pressure changes resulting from daily TGF- ⁇ l treatment. Monkeys were fed commerically available chow daily and were provided water ad libitum. Blood pressures were measured via chronic arterial catheters surgically implanted at least 5 days prior to the initiation of the study. One of the female monkeys (39-181) underwent general anesthesia and a chronic venous catheter was implanted in the right illiac vein. Approximately one month later, this catheter was removed, and the monkey was asepically implanted with chronic arterial and venous catheters in the illiac artery and vein.
  • test article dosing solution (0.0213 mg/ml) was prepared by mixing 0.66 ml of rTGF- ⁇ l stock solution (0.46 mg/ml in 5mM HCl) with 9.34 ml of 0.1% monkey serum albumin in PBS solution. The pH of the dosing solution was recorded after formulation and prior to administration each day. Dose volumes were calculated based on the most recent non- tethered body weights and rounded to the nearest 0.1 ml.
  • Recombinant mature TGF- ⁇ l was isolated and purified from the supernatants of cultured Chinese Hamster Ovary cells transfected with the complete simian TGF- ⁇ l precursor coding sequence as described in co-pending application Serial No. 07/353,728 filed August 17, 1989, which application is incorporated by reference herein in its entirety.
  • BLOOD PRESSURE MEASUREMENTS Blood pressure measurements were recorded from monkeys 29-825, 39-181, and B344 via chronic arterial catheters. Blood pressure measurements were recorded for at least one minute prior to and after completion of the administration of the test article. Blood pressure measurements were also recorded as amended or at the discretion of the study director if such measurements were clinically relevant.

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Abstract

The use of TGF-β and TGF-β antagonists to modulate blood pressure is described. In a specific embodiment described by way of example herein, recombinant mature TGF-β1 isolated and purified from transfected Chinese Hamster Ovary cells induced rapid, significant and sustained decreases in arterial blood pressure of cynomolgus monkeys receiving daily injections of the rTGF-β1. The TGF-β used to lower blood pressure may be obtained from native sources or may be produced by recombinant DNA or chemical synthetic techniques.

Description

METHODS OF MODULATING BLOOD PRESSURE USING TGF-β AND ANTAGONISTS THEREOF
1. INTRODUCTION The present invention is directed to the use of transforming growth factor-beta (TGF-9) and TGF- antagonists to modulate blood pressure (BP) . The method of the invention is demonstrated by way of example in which mature recombinant TGF-βl (rTGF-j8) is used to rapidly lower blood pressure in adult cynomoigus monkeys. However, the scope of the invention is not limited to the use of rTGF- l but rather encompasses the use of mature and precursor forms of all members of the TGF-9 family effective at modulating blood pressure, including natural and recombinant mature TGF-/31, TGF-02, TGF- 3, TGF-04, etc., as well as TGF-y8 hybrids, analogs and latent TGF-^ complexes. Similarly, the invention includes the use of any and all compositions effective at antagonizing TGF- activity, including but not limited to anti-TGF-^ antibodies and TGF-y8 receptors.
2. BACKGROUND OF THE INVENTION
2.1. TRANSFORMING GROWTH FACTOR-BETA TGF-β is a member of a recently described family of polypeptides that regulate cellular differentiation and proliferation. Other members of this family include Mullerian inhibitory substance (Cate et al., 1986, Cell 45:685-698), the inhibins (Mason et al., 1985, Nature 318:659-663) and a protein predicted from a transcript of the decapentaplegic gene complex of Drosophila (Padgett et al., 1987, Nature 325: 81-84.
Four types of TGF-β have been identified and designated TGF- l, TGF-^2, TGF-βl .2 , and TGF-β3 . The first described type, TGF-91, consists of two identical disulfide linked subunits having molecular weights of 13,000 (Assoian et al., 1983, J. Biol. Chem. 258:7155-7160; Frolik et al., 1983, Proc. Natl. Acad. Sci. USA 80:3676-3680; Frolik et al., 1984, J. Biol. Chem. 260:10995-11000). It has been purified from several tissue sources including placenta (Frolik et al., 1983, Nature 325:81-84), blood platelets (Childs et al., 1982, Proc. Natl. Acad. Sci. USA 79:5312- 5316; Assoian et al., 1983, J. Biol. Chem. 258:7155-7160) kidney (Roberts et al., 1983, Biochemistry 22:5692-5698), and demineralized bone (Seyedin et al., 1985, Proc. Natl. Acad. Sci. USA 82:119-123). cDNA clones coding for human (Derynσk et al., 1985, Nature 316:701-705), mouse (Derynck et al., 1986, J. Biol. Chem. 261:4377-4379) and simian (Sharpies et al., 1987, DNA 6:239-244) TGF-βl have been isolated. DNA sequence analysis of these clones indicates that TGF-βl is synthesized as a large precursor polypeptide, the carboxy terminus of which is cleaved to yield the mature TGF-β monomer. Strong sequence homology has been found throughout the TGF-βl precursor protein from all of the above sources.
In the presence of 10% serum and epidermal growth factor, TGF-βl promotes the anchorage independent growth of normal rat kidney fibroblasts (Roberts et al., 1981, Proc. Natl. Acad. Sci. USA 78:5339-5343; Roberts et al., 1982, Nature 295:417-419; Twardzik et al., 1985, J. Cell. Biochem. 28:289-297) ; in the presence of 10% serum alone, it is able to induce colony formation of AKR-2B fibroblasts (Tucker et al., 1983, Cancer Res. 43:1518-1586). TGF-βl has also been shown to cause fetal rat muscle mesenchymal cells to differentiate and produce cartilage specific macromolecules (Seyedin et al., 1986, J. Biol. Chem. 261:5693-5695).
In contrast to its effect on cell proliferation, TGF-βl purified from human platelets has been shown to inhibit the growth of certain cells in culture (Tucker et al., 1984, Science 226:705-707). TGF-βl has also been shown to inhibit the growth of several human cancer cell lines (Roberts et al., 1985, Proc. Natl. Acad. Sci. USA 82:119- 123) . This inhibitory/stimulatory effect of TGF-βl may depend on several factors including cell type and the physiological state of the cells (for review see Sporn et al., 1986, Science 233:532-534).
TGF-β2, like TGF-βl, is a polypeptide of molecular weight 26,000 composed of two identical 13,000-dalton subunits which are disulfide like (Chiefetz et al., 1987, Cell 48:409-415; Ikeda et al., 1987, Biochemistry 26:2406- 2410) and has been isolated from bovine demineralized bone (Seydin et al., 1987, J. Biol. Chem. 262:1946-1949), porcine platelets Chiefetz et al., 1987, 48:409-415), a human prostatic adenocarcinoma cell line, PC-3 (Ikeda et al., 1987, biochemistry 26:2406-2410), and a human gliablastoma cell line (Wrann et al., 1987, EMBO 6:1633-1636). cDNA clones coding for human and simian TGF-β2 have been isolated (Madisen et al., 1988, DNA 7:1-8; Webb et al., 1988, DNA 7:493-497). The mature TGF-β2 monomer is cleaved from one of two larger precursor polypeptides, the mRNAs of which may arise via differential splicing (Webb et al., 1988, DNA 7:493-497) .
TGF-βl and TGF-β2 share 71% amino acid sequence identity in their mature regions, and 41% identity in their precursor structures. TGF-β3, the amino acid sequence of which has very recently been deduced from cDNA clones, appears to contains C-terminal 112 amino acid sequence with about 80% homology to the mature monomers of TGF-βl and TGF-β2 (Dijke et al., 1988, Proc. Natl. Acad. Sci. USA 85:4715-4719). TGF-βl.2 is a heterodimeric form comprising a βl and β2 subunit linked by disulfide bonds (Chiefetz et al., 1987, Cell 48:409-415). 3. SUMMARY OF THE INVENTION The present invention is directed to methods of modulating blood pressure using TGF-β polypeptides, TGF-β antagonists, and/or combinations thereof. The invention may be subdivided into two categories solely for the purpose of description.
First, the invention relates to the use of TGF-βs as antihypertensive agents capable of rapidly and significantly lowering blood pressure. This aspect of the invention encompasses the use of any and all TGF-β polypeptides having a hypotensive activity, including mature and precursor forms of TGF-βl, TGF-β2, TGF-β3, hybrid TGF- βs, latent TGF-β complexes, TGF-β analogs, etc. In a specific embodiment of the invention, described more fully by way of example herein (Section 6., infra), simian recombinant TGF-βl is administered parenterally to induce rapid significant, and sustained decreases of arterial blood pressure in cynomoigus monkeys. In a related embodiment, TGF-βs may be used to rapidly lower blood pressure to normal levels in patients facing acute hypertension and emergency conditions associated with extreme hypertension.
Second, the invention relates to the use of TGF-β antagonists to elevate blood pressure through the inhibition of hypotension induced by TGF-β and/or related factors. Any composition which antagonizes TGF-β activity may be useful in this regard, including for example, anti-TGF-β antibodies and TGF-β receptors. Additionally, methods which lower and/or maintain the level of circulating TGF-β in an individual may result in a similar pressor effect. For example, anti-TGF-β antisense RNA molecules may inhibit synthesis and release of bioactive TGF-βs, thereby preventing excessive hypotensive signal generation and resulting hypotension. 4. DESCRIPTION OF THE FIGURES FIG. 1. Nucleotide sequence of simian TGF-βl cDNA and deduced amino acid sequence. The 1600 bp insert of pTGF-βl-2 was subcloned into the M13mpl8 and M13mpl9 cloning vectors (Yanisch-Perron et al., 1985, Gene 33:103-119) and both strands were sequenced using the dideoxy chain- termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467). The deduced amino acid sequence of simian TGF-βl is presented directly above the cDNA sequence. The human TGF-βl nucleotide sequence is aligned with and presented directly below the simian cDNA sequence; dots indicate homologous nucleotide residues within the sequences. Amino acid differences between the human and simian proteins are indicated in the top line. The mature TGF-βl sequence is boxed and the signal peptide is overlined.
FIG. 2. Nucleotide sequence of human TGF-β2-442 cDNA and deduced amino acid sequence. The 2597 BP insert of PC-21 was subcloned into pEMBL (Dante et al., 1983, Nucleic Acids Res. 11:1645-1654) and sequenced on both strands using the dideoxy chain-termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467). The coding sequence is shown and the deduced amino acid sequence is presented directly above. The mature TGF-β2 sequence is boxed and the signal peptide is overlined. Potential glycosylation sites are indicated by asterisks. The arrow indicates the putative signal sequence cleavage site. The nucleotide sequence of simian TGF-β2-414 cDNA is identical to the human TGF-β2-442 cDNA sequence except that (a) nucleotides 346 through 432 (bracketed) are deleted and replaced by the sequence AAT, and (b) several silent nucleotide changes occur elsewhere in the structure (indicated by single letters directly below the changed nucleotide) . The deduced amino acid sequence for simian TGF-β2-414 precursor is identical to the human TGF-β2-442 precursor amino acid sequence except that Asparagine replaces amino acid residues 116 through 144 in the human TGF-β2-442 structure. The nucleotide sequence of a human TGF-β2-414 cDNA has been sequenced through the region indicated by broken underlining and was found to be perfectly homologous to the human TGF-β2-442 cDNA sequence except that nucleotides 346 through 432 are deleted and replaced by the sequence AAT.
FIG. 3. Nucleotide sequence of hybrid TGF-βl/β2 precursor DNA and deduced amino acid sequence. The coding sequence is shown and the deduced amino acid sequence is presented directly above. The mature TGF-β2 sequence is boxed and the precursor signal peptide is overlined. Glycosylation sites are indicated by asterisks. The arrow indicates the putative signal sequence cleavage site. The TGF-β2 mature coding sequence depicted is of human origin. The simian TGF-β2 mature coding sequence is nearly identical to the human sequence: only 3 silent base changes occur and are indicated by single letters directly below the changed nucleotide.
5. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to methods of modulating blood pressure in an animal using TGF-β polypeptides, antagonists and/or combinations thereof. The invention is based upon the discovery that parenterally administered mature rTGF-βl rapidly and significantly lowers blood pressure in cynomologus monkeys. Thus, one aspect of the invention relates to the use of TGF-βs as antihypertensive/hypotensive agents. Precisely the opposite effect, i.e., raising and/or maintaining blood pressure, may be achieved by TGF-β antagonists capable of inhibiting the antihypertensive/hypotensive effects of TGF-β. In this regard, the invention encompasses the use of anti-TGF-β antibodies, TGF-β receptors and other compositions capable of inhibiting TGF-β-induced hypotension.
5.1. USE OF TGF-βs AS ANTIHYPERTENSIVE AGENTS
One aspect of the invention relates to the use of TGF-βs as antihypertensive/hypotensive agents. Applicants' initial data indicates that rTGF-βl can rapidly and significantly lower blood pressure in simian test subjects at a dosage which appears to be at or close to the physiologically tolerable limit. In this regard, parenteral administration of a TGF-β at such a dose may be acceptable in hypertensive emergencies requiring agressive treatment. Lower does of a TGF-β may also be effective at reducing blood pressure, and such doses may be appropriate for patients with moderate to severe hypertension. In these patients, less agressive therapy may be desirable where adverse side effects can not be tolerated.
Human patients with diastolic blood pressure greater than 130 mm Hg and complications such as hypertensive encephalopathy, progressive renal failure, acute pulmonary edema, cerebral accident, papilledema, or multiple fresh retinal hemorhages are generally treated agressively with a parenteral antihypertensive agent such as, for example, nitroprusside and diazoxide. Treatment of hypertension characterized by such acute complications generally aims to lower BP to about 100 mm Hg within 30 to 60 minutes, since rapid decrease is a key determinant of survival in patients facing these emergencies.
The TGF-β anithypertensive may be administered alone or in combination with other antihypertensive agents in suitable pharmacological carriers via any appropriate route. In hypertensive emergencies, parenteral administration will provide the fastest decrease in BP and is therefore the recommended route of administration in such situations. Additionally, the TGF-β may be linked to a carrier or targeting molecule and/or incorporated into liposomes, microcapsules, and controlled release preparations prior to administration in vivo.
5.1.1. SOURCES OF TGF-β In accordance with the invention, mature and/or precursor forms of TGF-βl, TGF-β2, TGF-β3, TGF-βl/β2, etc., may be used to lower blood pressure. The TGF-β used may be obtained from a variety of sources, including but not limited to isolating natural TGF-βs from appropriate sources, producing TGF-β by recombinant DNA techniques, or by chemical synthetic methods, etc.
5.1.1.1. TGF-βl
Natural TGF-βl can be isolated from a variety of sources. This potent modulator of cell behavior is synthesized by a variety of normal and transformed cells in culture (Roberts et al., 1981, Proc. Natl. Acad. Sci. USA 78:5339-5343) and has been purified from various sources including placenta (Frolik et al., 1983, Proc. Natl. Acad. Sci. USA 80:3676-3680), kidney (Roberts et al., 1983, Biochemistry 22:5692-5698), urine (Twardzik et al., 1985, J. Cell. Bioche . 28:289-297) and blood platelets (Childs et al., 1982, Proc. Natl. Acad. Sci. USA 79:5312-5316). Additionally, the human (Derynck et al., 1985, Nature 316:701-705), mouse (Derynck et al., 1986, J. Biol. Chem. 261:4377-4379), and simian (Sharpies et al., 1987, DNA 6:239-244) TGF-βl have been described.
Large quantities of TGF-βl may be obtained by recombinant DNA techniques using eucaryotic host cells transfected with recombinant DNA vectors containing the TGF-βl coding sequence controlled by expression regulatory elements. Examples of such methods are described in copending application Serial No. 07/353,728 filed August 17, 1989, which application is incorporated by reference herein it its entirety. Briefly, a cDNA clone coding for simian TGF-βl precursor was obtained from a cDNA library made from an African Green Monkey cell line, BSC-40. The deduced amino acid sequence of the mature simian TGF-βl shown in FIG. l has 100% homology with that of the mature human TGF- βl. Expression vectors were constructed which contain the entire coding sequence for the simian TGF-βl precursor placed under the control of SV40 expression elements. They were used to transfect Chinese Hamster Ovary cells (CHO cells) . The resulting CHO transfectants produce and secrete primarily a high molecular weight complex from which mature bioactive TGF-β may be liberated by a routine acidification procedure.
5.1.1.2. TGF-β2
Natural TGF-β2 used in accordance with the invention can be obtained from a variety of sources. A protein isolated from bovine demineralized bone has been identified as being related to TGF-β (Seyedin et al., 1987, J. Biol. Chem. 262:1946-1949). The protein has also been isolated from porcine platelets (Cheifetz et al., 1987, Cell 48:409-415), a human prostatic adenocarcinoma cell line PC-3 (Ikeda et al., 1987, Biochemistry 26:2406-2410), and a human glioblastoma cell line (Wrann et al., 1987, EMBO 6:1633- 1636) . Partial amino acid sequence of this protein indicated that it was homologous to TGF-β and has been termed TGF-β2.
Large quantities of TGF-β2 may be obtained by recombinant DNA techniques using eukaryotic host cells transfected with recombinant DNA vectors containing a TGF-β2 coding sequence controlled by expression regulatory ele ents. Examples of such methods are described in copending application Serial No. 07/446,020 filed December 5, 1989, which application is incorporated by reference herein in its entirety. Briefly, cDNA clones coding for human TGF-β2 precursor were obtained from a cDNA library made from a tamoxifen treated human prostatic adenocarcinoma cell line, PC-3. The cDNA sequence of one such clone is shown in FIG. 2 and predicts that TGF-β2 is synthesized as a 442 amino acid polypeptide precursor from which the mature 112 amino acid TGF-β2 subunit is derived by proteolytic cleavage. This TGF-β2 precursor, termed TGF-β2-442, shares a 41% homology with the precursor of TGF-βl. In another embodiment, cDNA clones coding for simian TGF-β2 precursor were obtained from a cDNA library made from an African green monkey kidney cell line, BCS-40. The cDNA sequence of one such clone predicts that TGF-β2 is also synthesized as a 414 amino acid polypeptide precursor from which the mature 112 amino acid TGF-β2 subunit is derived by proteolytic cleavage. This TGF-β2 precursor, termed TGF-β2-414, has an amino acid sequence of 414 amino acid residues and is identical to the amino acid sequence of TGF-β2-442, except that it contains a single Asparagine residue instead of the 29 amino acid sequence from residue numbers 116 to 135 of the human TGF-β2-442 sequence.
Clones from the BSC-40 cDNA library which encode a simian TGF-β2-442 precursor as well as clones from the human PC-3 cDNA library which encode a human TGF-β2-414 precursor have also been identified. The human and simian TGF-β2-442 precursors appear to be perfectly homologous at the amino acid level, as do the human and simian TGF-β2-414 precursors. 5.1.1.3. HYBRID MATURE AND PRECURSOR TGF-βs Hybrid mature TGF-β molecules may be prepared using recombinant DNA techniques or synthetic methods. Examples of such methods are also described in copending applications Serial No. 284,972, filed December 15, 1988, which application is incorporated by reference herein in its entirety.
Hybrid precursor TGF-β molecules may be prepared using recombinant DNA techniques or synthetic methods, as described in co-pending application Serial No. 07/353,728 filed August 17, 1987. Briefly, expression vectors containing the TGF-β2 mature coding sequence joined in-phase (i.e. , in the same translational reading frame) to the TGF- βl signal and precursor sequences (see FIG. 3) were constructed and used to transfect Chinese Hamster Ovary cells (CHO cells) . The resulting CHO transfectants produce and secrete mature, biologically active TGF-β2.
5.1.1.4. MODIFIED TGF-β Variations in the amino acid sequences shown herein for the different TGF-β molecules, as well as variations in the steric configuration, the type of covalent bonds which link the amino acid residues, and/or addition of groups to the amino- or carboxy-terminal residues are within the scope of the invention. For example, the TGF-β molecules used in accordance with the invention may include altered sequences such as conservative alterations which result in a silent change thus producing a functionally equivalent molecule. Thus, the amino acid sequences shown in FIGS. 1-3 may be altered by various changes such as insertions, deletions and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use. As used herein, conservative substitutions would involve the substitution of one or more amino acids within the sequences shown with another amino acid having similar polarity and hydrophobicity/hydro-philicity characteristics resulting in a silent alteration and a functionally equivalent molecule. Such conservative substitutions include but are not limited to substitutions within the following groups of amino acids: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine; and methionine, norleucine.
5.1.1.5. LATENT TGF-β COMPLEX TGF-βl may be isolated from tissues or tissue culture cells in an inactive, biologically latent form which may be activated by chaotropic agents, proteases, or in vivo. Similarly, CHO cell transfected with the simian TGF-βl precursor coding sequence secrete a high molecular weight latent complex involving both the mature and "pro" regions of the TGF-β precursor. The association of the "pro" region of the TGF-β precursor has also been observed in latent TGF-βl complex isolated from platlets. Although the mechanism of activation in vivo is unknown, it is possible that the latent complex provides an important level of regulation on TGF-βl bioactivity. In accordance with the method of the invention, latent TGF-β complex may be useful as a means of controlling the hypotensive effect induced by the bioactive form of TGF-β by releasing it at the situs of natural in vivo activation mechanisms. The identification, isolation and characterization of latent TGF-βl complex from recombinant CHO cells is described more fully in copending application Serial No. 07/353,728 filed August 17, 1989, which application is incorporated herein by reference in its entirety. 5.2. USE OF TGF-β ANTAGONISTS AS PRESSOR AGENTS
Applicants' discovery that rTGF-βl is capable of rapidly and significantly lowering blood pressure suggests that the TGF-βs may be involved in the regulation of BP and/or in the genesis of hypotension. In this regard, through their ability to impede the hypotensive effect of TGF-β, antagonists of TGF-βs may be useful as pressor/hypotensor agents capable of elevating BP. Any compostion which effectively antagonizes the hypotensive effect of a TGF-β may be used for this purpose, including but not limited to anti-TGF-β antibodies and TGF-β receptors.
For example, TGF-β antagonists may be useful in treating medical conditions characterized by a loss of BP where the elevation of BP to normal levels is desirable. Such conditions include, for example, shock associated with blood volume loss, cardiac emergencies, and hypotension in acute renal failure. The TGF-β antagonists may be administered alone or in combination and/or together with other pressors/hypotensors such as dopamine, epinephrine, aminophylline, etc. Compounds containing effective doses of TGF-β antagonist formulated in a suitable pharmacological carrier may be administered to patients experiencing hypotension or conditions associated with hypotension via any appropriate route including but not limited to injection, infusion and selective catheterization in order to elevate BP. In addition, the TGF-β antagonist may be linked to a carrier or targeting molecule and/or incorporated into liposomes, microcapsules, and controlled release preparations prior to administration in vivo. 5.2.1. ANTI-TGF-β ANTIBODIES Antibodies capable of inhibiting the hypotensive effect of TGF-β may be useful as pressor agents. Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of TGF-βs. For the production of antibodies, various host animals can be immunized by injection with a TGF-β, or a synthetic TGF-β peptide, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peotides, oil emulsions, keyhold lympet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
A monoclonal antibody to an epitope of a TGF-β can be prepared by using any technique which provides for the production of antibody molecules by continous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497) , and the more recent human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. , pp. 77-96) .
Antibody fragments which contain the idiotype of the molecule may be generated by known techniques. For example, such fragments include but are not limited to the F(ab')_ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the Ffab')., fragment, and the two Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
The generation of anti-TGF-β antibodies is described in copending application Serial No. 7/353,728 filed August 17, 1989, and in copending application Serial No. 07/446,020 filed December 5, 1989.
5.2.2. TGF-β RECEPTORS Exogenous TGF-β receptor molecules may be useful pressor agents inasmuch as they are capable of binding circulating TGF-β and/or out-competing endogenous receptors which may initiate the hypotensive effect of TGF-β. TGF-β receptors may be prepared by the methods described in copending application Serial No. 269,524 filed November 11, 1988, which application is incorporated by reference herein in its entirety.
6. EXAMPLE: EFFECT OF PARENTERALLY ADMINISTERED rTGF-βl ON BLOOD PRESSURE IN CYNOMOLGUS MONKEYS
Described below is part of a study designed to evaluate the pliarmacotoxic effects of rTGF-βl following daily intravenous infusions to cynomoigus monkeys (Macaca fascicularis) . The results described herein indicate that rTGF-βl has a profound reducing effect on blood pressure.
6.1. PROTOCOL 6.1.1. CYNOMOLGUS MONKEYS One adult male and two adult female cynomoigus monkeys were monitored for blood pressure changes resulting from daily TGF-βl treatment. Monkeys were fed commerically available chow daily and were provided water ad libitum. Blood pressures were measured via chronic arterial catheters surgically implanted at least 5 days prior to the initiation of the study. One of the female monkeys (39-181) underwent general anesthesia and a chronic venous catheter was implanted in the right illiac vein. Approximately one month later, this catheter was removed, and the monkey was asepically implanted with chronic arterial and venous catheters in the illiac artery and vein. The other female monkey (29-825) and the male monkey (B-344) were also implanted with chronic venous and arterial catheters in the illiac artery and vein. The catheters were exteriorized via a tether apparatus as a venous access to facillitate the administration of the test article or vehicle control.
6.1.2. TEST ARTICLE FORMULATION The formulation of the test and/or control articles was performed daily prior to administration. Each 10 ml of test article dosing solution (0.0213 mg/ml) was prepared by mixing 0.66 ml of rTGF-βl stock solution (0.46 mg/ml in 5mM HCl) with 9.34 ml of 0.1% monkey serum albumin in PBS solution. The pH of the dosing solution was recorded after formulation and prior to administration each day. Dose volumes were calculated based on the most recent non- tethered body weights and rounded to the nearest 0.1 ml.
Recombinant mature TGF-βl was isolated and purified from the supernatants of cultured Chinese Hamster Ovary cells transfected with the complete simian TGF-βl precursor coding sequence as described in co-pending application Serial No. 07/353,728 filed August 17, 1989, which application is incorporated by reference herein in its entirety.
6.1.3. TREATMENT Monkey 39-181 received 1% monkey albumin in PBS (vehicle control) at a volume of 8 ml/kg daily for five consecutive days. Monkey 69-168 was treated for five consecutive days at a dose of 0.17 mg/kg and at a concentration of 0.0213 mg/ml. rTGE-βl was administered to Monkeys 29-825, 39-181 and B344 at a dose of 0.51 mg/kg and at a concentration of 0.0639 mg/ml. Monkey 29-825 was treated for three consecutive days, monkey 39-181 for three consecutive days, and monkey B-344 for one day. The body weights used to calculate the dosages of either the test article or control were the most recent body weights obtained without the encumbrance of the tether apparatus. rTGF-βl or vehicle control was administered intravenously through chronic venous catheters at a rate of 1.60 ml of dosing solution/minute via an infusion pump (Harvard Apparatus) , and calibrated according to the standard operating procedures of the test facility. Prior to the administration of the test article and control, catheter patency was maintained via periodic flushing of the catheter with 0.9% sterile saline. The volume, time, and date of administration of rTGF-βl and control were recorded.
6.1.4. BLOOD PRESSURE MEASUREMENTS Blood pressure measurements were recorded from monkeys 29-825, 39-181, and B344 via chronic arterial catheters. Blood pressure measurements were recorded for at least one minute prior to and after completion of the administration of the test article. Blood pressure measurements were also recorded as amended or at the discretion of the study director if such measurements were clinically relevant.
6.2. CLINICAL OBSERVATIONS The monkeys were observed daily over 29 days for clinical abnormalities, food and water intake, body temperature, respiration rate, blood pressure, and other parameters. Additionally, blood samples were collected from each monkey to provide samples for hematology, serum chemistry and immunological analyses.
6.2.1. EFFECT OF rTGF-βl ON BP Two of the three monkeys receiving rTGF-βl injections experienced immediate, significant and progressive decreases in arterial blood pressure. The third experimental monkey also experienced BP loss, but these results are somewhat more difficult to interpret in view of the extreme hypotension existing in this animal prior to treatment. No significant BP fluctuations were observed in the control monkey. The individual BP observations for the three experimental monkeys are tabulated in TABLE I.
Figure imgf000021_0001
39-181 F 0.51 44
48 50 60 32 46 40 32 12 18 32
29-825 F 0.51 108
80 48 76 76 20 52 50
B-344 M 0.51 104
60 36 20 32 22 20
Figure imgf000021_0002
36 Monkeys 29-825 and B-344 experienced an immediate (l hour post-administration) BP reduction of 26% and 42%, respectively. Four hours after treatment, BP had dropped by 55% in monkey 29-825 and by 65% in monkey B-344. These initial drops in BP were sustained in the subsequent treatment days, resulting in hypotension and shock. Monkey 39-181 did not respond to initial TGF-βl treatment (day 1) with a reduction in BP, possibly because of its pre-existing hypotensive condition. Interestingly, a slight elevation in BP was observed on day 2 prior to treament, and a sustained decrease in BP was observed thereafter.
In addition to the dramatic decrease in BP and the accompanying shock/hypotension observed in all three treated animals, other observed effects directly attributable to TGF-βl included hemotopoietiσ changes (decrease in erythrocytes, lymphocytes and platlets) and immunological compromise (decrease in lymphocyte responsiveness to itogen) . Additionally, all the treated monkeys had no or minimal appetite, and all were inactive during the treatment period. Monkey 29-825 was recumbent on day 4, required fluid therapy on days 4 and 5, and was euthanized on day 5 because of its moribund condition. Monkey 39-181 had darkened blood on days 3 and 4, developed septicemia on day 8, and was euthanized because of its deteriorating condition on day 8. Monkey B-344 appeared normal from days 6 to 29.
The present invention is not to be limited in scope by the cell lines, TGF-β molecules and assays exemplified which are intended as but single illustrations of one aspect of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method of treating hypertension comprising administering a TGF-β to an individual at a dose effective at lowering blood pressure.
2. The method of claim 1 wherein the TGF-β comprises a mature TGF-βl.
3. The method of claim 1 wherein the TGF-β comprises a mature TGF-β2.
4. The method of claim 1 wherein the TGF-β comprises a mature TGF-βl/β2 hybrid.
5. The method of claim 1 wherein the TGF-β comprises a TGF-βl precursor.
6. The method of claim 1 wherein the TGF-β comprises a TGF-β2 precursor.
7. The method of claim 1 wherein the TGF-β comprises a hybrid TGF-βl/TGF-β2 precursor.
8. The method of claim 1 wherein the TGF-β comprises a latent TGF-βl complex.
9. The method of claim 1 wherein the TGF-β comprises a latent TGF-β2 complex.
10. A method of lowering blood pressure in a mammal comprising administering TGF-β to the mammal in an amount and for a time period effective at inducing the desired hypotensive effect.
11. The method of claim 10 wherein the TGF-β comprises a mature TGF-βl.
12. The method of claim 10 wherein the TGF-β comprises a mature TGF-β2.
13. The method of claim 10 wherein the TGF-β comprises a mature TGF-βl/β2 hybrid.
14. The method of claim 10 wherein the TGF-β comprises a TGF-βl precursor.
15. The method of claim 10 where in the TGF-β comprises a TGF-β2 precursor.
16. The method of claim 10 where in the TGF-β comprises a hybrid TGF-βl/TGF-β2 precursor.
17. The method of claim 10 where in the TGF-β comprises a latent TGF-βl complex.
18. The method of claim 10 where in the TGF-β comprises a latent TGF-β2 complex.
19. A method of treating hypotension comprising administering to an individual a TGF-β antagonist capable of inhibiting the hypotensive activity induced by TGF-β at a dose effective at inducing an elevation in blood pressure.
20. The method of claim 19 wherein the TGF-β antagonist is an anti-TGF-β antibody.
21. The method of claim 19 wherein the TGF-β antagonist is a TGF-β receptor.
22. The method of claim 19 wherein the TGF-β antagonist is TGF-βl antagonist.
23. The method of claim 22 wherein the TGF-βl antagonist is an an i-TGF-βl antibody.
24. The method of claim 22 wherein the TGF-β antagonist is a TGF-βl receptor.
25. The method of claim 19 wherein the TGF-β antagonist is a TGF-β2 antagonist.
26. The method of claim 25 wherein the TGF-β2 antagonist is a TGF-β2 antibody.
27. The method of claim 25 wherein the TGF-β2 antagonist is a TGF-β2 receptor.
28. A method of elevating blood pressure comprising administering to a mammal a TGF-β antagonist in an amount and for a time period effective at inducing the desired blood pressure increase.
29. The method of claim 28 wherein the TGF-β antagonist is an anti-TGF-β antibody.
30. The method of claim 28 wherein the TGF-β antagonist is a TGF-β receptor.
31. The method of 28 wherein the TGF-β antagonist is a TGF-βl antagonist.
32. The method of claim 31 wherein the TGF-βl antagonist is an anti-TGF-βl antibody.
33. The method of claim 31 wherein the TGF-βl antagonist is a TGF-βl receptor.
34. The method of claim 28 wherein the TGF-β antagonist is a TGF-β2 antagonist.
35. The method of claim 34 wherein the TGF-β2 antagonist is a TGF-β2 antibody.
36. The method of claim 34 wherein the TGF-β2 antagonist is a TGF-β2 receptor.
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Cited By (24)

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US5772998A (en) * 1988-12-22 1998-06-30 Celtrix Pharmaceuticals, Inc. Monoclonal antibodies to transforming growth factor-beta and methods of use
US6090383A (en) * 1988-12-22 2000-07-18 Dasch; James R. Monoclonal antibodies to transforming growth factor-β and methods of use
US5783185A (en) * 1988-12-22 1998-07-21 Celtrix Pharmaceuticals, Inc. Monoclonal antibodies to transforming growth factor-beta and methods of use
US6419928B1 (en) 1988-12-22 2002-07-16 Genzyme Corporation Monoclonal antibodies to transforming growth factor-beta and methods of use
EP0624165A4 (en) * 1992-01-27 1996-01-24 Ludwig Inst Cancer Res Large, latent complexes of tgf-beta2 and tgf-beta3.
EP0624165A1 (en) * 1992-01-27 1994-11-17 Ludwig Institute For Cancer Research Large, latent complexes of tgf-beta2 and tgf-beta3
US5693607A (en) * 1992-10-29 1997-12-02 Segarini; Patricia R. Uses of TGF-β receptor fragment as a therapeutic agent
EP0669833A4 (en) * 1992-10-29 1996-07-31 Celtrix Pharma USES OF TGF--g(b) RECEPTOR FRAGMENT AS A THERAPEUTIC AGENT.
EP0669833A1 (en) * 1992-10-29 1995-09-06 Celtrix Pharmaceuticals, Inc. USES OF TGF-$g(b) RECEPTOR FRAGMENT AS A THERAPEUTIC AGENT
EP0684828A4 (en) * 1993-02-19 1996-08-07 Celtrix Pharma EXPANSION OF STEM CELLS IN LONG TERM BONE MARROW CULTURES BY NEUTRALIZATION OF TGF--g(b).
EP0684828A1 (en) * 1993-02-19 1995-12-06 Celtrix Pharmaceuticals, Inc. EXPANSION OF STEM CELLS IN LONG TERM BONE MARROW CULTURES BY NEUTRALIZATION OF TGF-$g(b)
EP1741780A3 (en) * 1995-06-02 2007-03-28 Gilead Sciences, Inc. High affinity oligonucleotide ligands to growth factors
EP1741780A2 (en) * 1995-06-02 2007-01-10 Gilead Sciences, Inc. High affinity oligonucleotide ligands to growth factors
US6383733B1 (en) 1996-04-05 2002-05-07 Boehringer Ingelheim International Gmbh Methods of screening for pharmacologically active compounds for the treatment of tumour diseases
WO1997037678A1 (en) * 1996-04-05 1997-10-16 Boehringer Ingelheim International Gmbh Drug for the treatment of tumours
WO2001066140A1 (en) * 2000-03-09 2001-09-13 Genzyme Corporation Use of tgf-beta antagonists to treat or to prevent loss of renal function
US9974759B2 (en) 2013-05-31 2018-05-22 Indiana University Research And Technology Corporation Beta 2 adrenoceptor antagonists for treating orthostatic hypotension
US9809637B2 (en) 2013-08-22 2017-11-07 Accleron Pharma Inc. Transforming growth factor beta receptor II fusion polypeptides
US10316076B2 (en) 2013-08-22 2019-06-11 Acceleron Pharma Inc. Transforming growth factor-beta receptor type II fusion polypeptides
US10981973B2 (en) 2013-08-22 2021-04-20 Acceleron Pharma Inc. Methods of treating a sclerotic disorder by administering a transforming growth factor beta receptor type II fusion polypeptide
US11008377B2 (en) 2013-08-22 2021-05-18 Acceleron Pharma Inc. Methods of treating a fibrotic disorder by administering transforming growth factor beta receptor II fusion polypeptides
US9884900B2 (en) 2015-08-04 2018-02-06 Acceleron Pharma Inc. Methods for treating Janus kinase-associated disorders by administering soluble transforming growth factor beta type II receptor
US11203624B2 (en) 2015-08-04 2021-12-21 Acceleron Pharma Inc. Method for treating myelofibrosis comprising administering a transforming growth factor beta type II receptor antagonist
US11021527B2 (en) 2017-05-04 2021-06-01 Acceleron Pharma Inc. Transforming growth factor beta receptor type II fusion polypeptides

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