MXPA98004640A - Use of prolactin as an antagonist of tgf-b - Google Patents

Abstract

This invention is based on the discovery that prolactin has been found to counteract the effect that TGF-a has on cells. An object of the present invention is to claim a method for treating a patient who may suffer from a disease or disorder that is associated with the presence of TGF-α, by administering an effective amount of a pharmaceutically acceptable composition containing prolactin.

Description

"USE OF PROLACTIN AS AN ANTAGONIST OF TGF-BETA" BACKGROUND OF THE INVENTION The transformation growth factor ß (TGF-β) belongs to a family of polypeptide factors that share certain structural and functional characteristics (Massague et al., Cell, 49: 437 (1987)). Other members include activins, inhibins, the Müllerian inhibitory substance (MIS), bone morphogenic proteins (BMPs) and the decapentaplegic gene product (DPP-C) found in Drosophila. The transforming growth factor a is an unrelated peptide that shares a high degree of homology with the epidermal growth factor (EGF), and binds to the same receptor (Todaro et al., Proc. Nati. Acad. Sci. USA, 77: 5258 (1980)). TGF-β was discovered as a product of the transformed cells of murine sarcoma virus (Delarco et al., Proc. Nati, Acad. Sci. USA, 75: 4001 (1978)). TGF-β is produced by a large number of cell types, including fibroblasts, myocytes, chondrocytes (Ja owlew et al., J. Cell. Physiol., 150: 377 (1992)), astrocytes (da Cunha et al., J. Neuroimmunol, 36: 157 (1992)), and epithelial cells (Steigerwalt et al., Mol.Carcin., 5:32 (1992)). Named in accordance with its ability to stimulate anchoring-independent growth of normal fibroblasts, TGF-β has been isolated from other sources, including a human glioblastoma, where it was identified as the factor-1 (TIF-1) inducer of tumor (Iwata et al., Cancer Res., 45: 2689 (1985)) and bovine bone, from which two cartilage-inducing factors (CIF-A and CIF-B) have been purified (Seyedin et al., Proc. Nati. Acad. Sci. USA, 82: 2267 (1985)). The sequence information has Proc. Nati Acad. Sci. USA, 82: 2267 (1985)). The sequence information has established that the CIF-A is identical to TGF-β1 and that CIF-B represents a second isoform, TGF-β2. TGF-β has the possibility of being identical to the Dicidual Suppressor Factor (DSF) (Lea et al., J. Immunol, 148: 778 (1992)). There are several well-defined isoforms of TGF-β; these disulfide-linked homodimers are designated numerically as TGF-β1 to TGF-β5. A heterodimer, TGF-β2.2, has been identified in porcine platelets. TGF-bl and TGF-ß2 are produced in many cell types, while TGF-ß3 is expressed mainly by cells of mesenchy origin (Roberts and Sporn in Human Cytokines, Blackwell Scientific, page 399 (1992); Lyons and Moses, Eur. J. Biochem, 187-467 (1990), Derynck et al., EMBO J, 7: 3737 (1988), and Massague, Ann. Rev. Cell Biol., 6: 597 (1990)). The amino acid homology between the isoforms varies from 70 percent (ßl versus ß2) to 79 percent (ß2 versus ß3). Each of TGF-ßl, TGF-ß2 and TGF-ß3 that are isoforms exhibits more than 98 percent amino acid sequence homology between species (Massague (1990); and Kondiah et al., J. Biol. Chem., 265: 1089. (1990)). The detection of the TGF-β4 and TGF-β5 isoforms have been limited to chicken embryo chondrocytes and Xenopus embryos, respectively (Roberts and Sporn in Human Cytokines, (1992)). The isoforms of TGF-β are synthesized as larger protein precursors. Proteolytic dissociation at the site of the basic amino acid residues yields the mature monomer consisting of the amino acids of the C-terminal 112-114. A biologically inactive latent complex is formed from the non-covalent association of the mature TGF-β dimer and two non-covalently interacting processes (the protein associated with LAP latency) (Roberts and Sporn in Human Cytokines, (1992); Lyons and Moses ( 1990)). Subsequent to the secretion of the cell source, activation must occur to release the biologically active dimer form. While acidification can release the TGF-β dimer in vitro activation in vivo is still subject to investigation.

Biologically active TGF-β isoforms are all 25 kD dimers, which yield monomers from 11.5 to 12.5 kD under reducing conditions. While the TGF-b precursor contains 3 to 4 N-glycosylation sites, none are present in the monomers that make up the mature TGF-β dimer. There are nine cysteines per mature TGF-β monomer (Roberts and Sporn in Human Citokines (1992)). The Chou-Fasman analysis of TGF-β suggests an extensive leaf-b structure with very little a-helical character (Garnier et al., J. Mol. Biol., 120: 97 (1978)). A number of TGF-β receptors mediate the biological effects of the TGF-β dimer. The five TGF-β receptors have been identified. Type I of angstrom units (53-65 kD), type II (83-110 kD), type III (250-310 kD), type IV (60 kD), and type V (400 kD) receptors (Segarini, Clinical Applications of TGF-b.

Wiley, page 29 (1991); 0'Grady et al., J. Biol. Chem., 266: 8563 (1991); Massague, Cell, 69: 1067 (1992); and Cheifetz et al., J. Biol. Chem., 263: 17225 (1988)). The type of receptors I, II, III and V are co-expressed in most of the cells examined, with the exception of a few tumor lines. The type IV receptor has been identified only in pituitary cells (Cheifetz (1988)). Since the loss of cellular response to TGF-β correlates with the - - loss of type I and / or type II receptors (Laiho et al., J. Biol. Chem. 266: 9108 (1992)), have been studied in the greatest detail. The differential affinities of the TGF-β1, TGF-β2 and TGF-β3 receptors exist among the types of receptors. For example, most type I and II receptors are linked to TGF-β1 and TGF-β3 with higher affinity than TGF-β2 (Massague (1992)). However, there is no direct relationship between binding affinity and biological potency since TGF-β2 is equipotent with respect to TGF-β1 in many biological assays. The potency of the isoform is linked to many factors, including the combination of receptor types present, the number of each type of receptor present and the presence of subgames type I and type II receptors that bind the three isoforms with the same affinity ( Massague (1992), Cheifetz (1988), Laiho (1992), and Cheifetz et al., J. Biol. Chem., 265: 20533 (1990)). Cloning of the type II receptor demonstrated that the cytoplasmic domain contains a functional serine / threonine kinase (Lin et al., Cell, 69: 775 (1992)). Cloning of the type III receptor demonstrated that the betaglycan structure contains a short cytoplasmic domain without a signal motif (Wang et al., Cell, 67: 797 (1991)). The type III receptor can function as a reservoir for surplus TGF-b or as a regulator of the binding capacity of the coordinating group or surface expression of type I or type II receptors (Wang (1991)). The cloning of the other TGF-β receptors is necessary to determine if they share a similar pattern of phosphorylation with the type II receptor. Other observations that provide clarification for the mechanism of TGF-b signals include: 1. induction of changes in Jun B, c-fos, and c-myc expression (Ohtsuki et al., Mol. Cell. Biol., 12: 261 (1992)); 2. prevention of phosphorylation that depends on the cell cycle of the retinoblastoma protein (Ohtsuki (1992)); 3. involvement of guanine nucleotide binding proteins (Diaz-Meco et al., Mol.Cel Biol., 12: 302 (1992)); 4. return of phosphatidyl inositol (Segarini (1991)); Y . translocation of the cytosol protein C kinase C to the cell membrane (Segarini (1991)). TGF-β works both as an inhibitory factor and as a stimulant. TGF-β1 and TGF-β2 exert similar effects with only a few exceptions (Roberts and Sporn in Human Cytokines (1992); Rizzino, Dev. Biol., 130: 411 (1988); Weinberg et al., J. Immunol, 148: 2109 (1992)).

- Stimulatory activities include: 1. fibroblast stimulation and osteoblast proliferation; 2. Improvement of matrix protein synthesis by fibroblasts, osteoblasts, and endothelial cells; 3. induction of cytokine production by monocytes; 4. promotion of fibroblast, monocyte and neutrophil chemotaxis; 5. improvement of the function of the in vivo effect and memory phenotype of the auxiliary T cells of the antigen; and 6. stimulation of IgA secretion from cells B.

Inhibitory activities include: 1. Inhibition of lymphocyte growth, endothelial cells, hepatocytes, ceratinocytes and certain tumor cell lines; 2. inhibition of IgG and IgM secretion from B cells; 3. inhibition of respiratory rupture capacity in monocytes; 4. suppression of hematopoietic progenitors that depend on IL-3; 5. megacariotopoiesis inhibition; 6. inhibition of steroidogenesis in adrenocortical cells and Leidig; and 7. inhibition of adipocyte and myocyte differentiation. The expression of TGF-β isoforms in a manner that depends on time during development and the ability to induce a mesodermal marker in the developmental Xenopus (Rosa et al., Science, 239: 783 (1988)) may demonstrate a role for TGF-β in embryogenesis. Released at the site of a wound by degranulation of platelets, it is reported that TGF-β causes infiltration of other effector cells, synthesis of the protein matrix and secretion of other factors that combine with TGF-β to mediate angiogenesis , and fibrosis associated with wound healing. Similarly, TGF-ß is believed to stimulate congrogenesis and mediate bone fracture healing. TGF-β has been associated with different forms of cancer (Macias et al., Anticancer Res., 7: 1271 (1987); Shirai et al., Japan J Cancer Res., 83: 676 (1992); Reed et al., Am. J Pathology, 145: 97 (1994)) and liver and lung disease that frequently develops as a complication, of chemotherapy and bone marrow transplantation procedures used to treat cancer patients (Kong et al., Ann Surg 222: 155 (1995); and Murase et al., Bone Marrow Transplantation, 15: 173 (1994); - Anscher et al., N Eng J Med, 328: 1592 (1993). Increased secretion of TGF-β by peripheral blood mononuclear cells (PBMC) from HIV infected donors has also been reported (Kekow et al., J Clin Invest 87: 1010 (1991) .Therefore, any TGF antagonist. -β can be useful to treat these forms of cancer, as well as it can be useful to counteract any of the numerous effects caused by TGF-β.

SUMMARY OF THE INVENTION This invention is based on the discovery that prolactin has been found to counteract the effect that TGF-β has on cells. It is an object of the present invention to claim a method for treating a patient who may be suffering from a disease or disorder that is associated with the presence of TGF-β by administering an effective amount of a pharmaceutically acceptable composition containing prolactin. Another object of the present invention is to claim a method for preventing inhibition of cell growth by TGF-β by administering an effective dose of a composition containing prolactin. Orro object of the present invention is a method for improving the growth of the cell by administering an effective amount in a composition containing prolactin.

BRIEF DESCRIPTION OF THE DRAWINGS The Figure illustrates the effect of TGF-β on Clone of hybridoma 5C6. Figure lb illustrates the effect of prolactin on the Clone 5C6 hybridoma. Figure 1c illustrates the ability of prolactin to overcome the effects of TGF-β on the hybridoma Clone 5C6. Figure 2 is a table containing the results of Example 2. Figure 3 illustrates the ability of prolactin to overcome the effects of TGF-β in stimulated human PBL.

DETAILED DESCRIPTION OF THE INVENTION This invention is based on the discovery that prolactin has been found to counteract the effect of TGF-β on the cells. It was found that the inhibitory effect of TGF-β counteracted by prolactin in both mouse hybridoma cells and human peripheral blood mononuclear cells.

Definitions As used herein, the term "prolactin" refers to a polypeptide obtained from tissue cultures or by recombinant techniques and other techniques known to those skilled in the art, which exhibit in the spectrum of activities that characterize this protein. . The word includes not only human prolactin (hPRL), but also another macular prolactin such as, e.g., mouse, rat, rabbit, primate, pig and bovine prolactin. Reference is made herein to the recombinant PRL (r-PRL). The term "recombinant prolactin", designated as r-PRL, preferably human prolactin, refers to prolactin having comparable biological activity to native prolactin prepared by recombinant DNA techniques known to those skilled in the art. In general, the gene coding for prolactin is derived from its native plasmid and inserted into a cloning vector to be cloned and then inserted into the expression vector, which is used to transform a host organism. The host organism expresses the foreign gene to produce prolactin under expression conditions. As used herein, the term "patient" has its conventional meaning, ie, one who suffers from a disease or disorder and is under treatment for it (Stedmans Medical Dictionary, (1987)). As used herein, the term "disease" has its conventional meaning, that is, morbid: ill, disease; an interruption, cessation or disorder of the functions of the body, systems or organs (Stedmans Medical Dictionary, (1987)). As used herein, the term "disorder" has its conventional meaning, ie, the presence of an alteration of function, structure or both (Stedmans Medical Dictionary (1987)).

General Method Formulations or compositions containing prolactin to counteract the effects of TGF-β are most conveniently administered by injection, even when other methods of administration are possible. Normal formulations are either liquid or solid injectables that can be admitted into appropriate liquids such as suspensions or solutions for injection. Suitable excipients are for example, water, saline dextrose, glycerol, ethanol and so on. Non-toxic auxiliary substances such as wetting agents, stabilizers or emulsifiers can also be added. The sustained and sustained release formulations are of a considerable variety and could be used in the method of the present invention as will be understood by those skilled in the art. In addition, because we have shown that prolactin can counteract the effects of TGF-β it is believed that exogenous administration of the prolactin gene would result in the expression of prolactin in vivo that could be obtained to function in order to counteract TGF-β whether administered through a conventional means or through inoculation of the gene. The gene sequence for prolactin is disclosed in (U.S. Patent No. 4,725,549). This could be produced by inserting prolactin cDNA into a DNA delivery vehicle (e.g., plasmid vectors, liposomes, viral vectors). This could be achieved as described by Pellegrini I, and others, Molec. Endocrinolgy, 6, 1023 (1992), Maniatis T, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989) and Felger P. et al., Proc. Nati Acad. Sci., 84, 7413, (1991).

EXAMPLIFICATION Example 1: Dose Response of TGF-J3 and PRL The Mouse Hybridoma Cell Lines (including 5C6) were maintained in the Hybridoma Growth Medium (HGM) consisting of 25 percent DMEM (Gibco), media conditioned with 40 percent NSO, 10 percent NCTC 109 (Gibco), 15 percent of FCS (Gibco), 0.1 percent ITS (Sigma 1-884), 0.05 mM 2-mercaptoethanol, 2 mM L-glutamine (Gibco), 10 percent ORIGIN (Igen, Inc.). The cells were fed the day before use to ensure the growth of the stub phase. On the day of the assay, cells were delivered to 15 milliliter conical tubes and centrifuged at 1000 revolutions per minute for 8 minutes. The medium was aspirated from the cells and washed twice with 5 milliliters of a test medium consisting of 88 percent RPMI-1640 (Gibco), 10 percent FCS (Gibco), 2 mM L-Glutamine, 0.05 mM 2-mercaptoethanol, 100 U / milliliter of Pen / Strep (Gibco). The cells were resuspended in 2 milliliters of the test medium from RPMI-1640, were found and diluted with assay media of 2 x 10 ^ cells per milliliter. A volume of 100 microliters of cells from a 96-well sterile tissue culture plate was delivered to the wells. The plate was placed in the incubator at 37 ° C until it was used.

A dilution series of recombinant human prolactin (Genzyme Corporation, batch 02A, 800 micrograms per milliliter) and TGF-b (Genzyme Corporation, batch 94F003, 96 micrograms per milliliter) were prepared in assay media. Twenty-five microliters of each reagent was added to the appropriate wells. In all cases, TGF-β was added first. Cells were incubated at 37 ° C / 5 percent CO2 for 72 hours and the amount of proliferation was measured by incorporation of tritiated thymidine. Tritiated thymidine (0.5 icroCi) was added during the last 18 hours. The radioactivity associated with the cell was measured by scintillation counting (1205 Betaplate LSC, Wallac) after harvesting the cells in fiberglass filters using a TOMEC 96 Harvester. The sample results in Figures la-c. The Figure provides a graph demonstrating the dose response for TGF-β in the proliferation of the hybridoma cell. TGF-β can significantly inhibit the growth of these cells in a dose-dependent manner. Figure lb demonstrates the dose response of r-hPRL in this cell line. The molecule clearly stimulates the proliferation of these cells in a manner that depends on the dose.

Figure 1c demonstrates the effect of incubating various concentrations of r-hPRL with 0.4 microgram per milliliter of TGF-β, a concentration of TGF-β which was shown to inhibit proliferation by > 50 percent in these cells. R-hPRL antagonized the suppressive effect of TGF-β on these cells in a dose-related manner.

Example 2: Effect of TGF-J3 and PRL on Murine Hybridoma Clones The mouse hybridoma cell lines were maintained in a Hybridoma Growth Medium (HGM) as described above. On the day of the assay, the cells were delivered in 15 milliliter conical tubes and centrifuged at 1000 revolutions per minute for 8 minutes. The medium was aspirated from the cells and washed twice with 5 milliliters of the assay medium consisting of 88 percent RPMI-1640 (Gibco), 10 percent FCS (Gibco), 2 mM L-Glutamine, 0.05 mM of 2-mercaptoethanol, 100 Units / milliliter of Pen / Strep (Gibco). The cells were resuspended in 2 milliliters of the RPMI-1640 assay medium, found and diluted with the test media to 2 x 10 cells per milliliter. A volume of 100 microliters of cells from a 96-well sterile tissue culture plate was delivered to the wells and placed in an incubator at 37 ° C until used. A 10-fold dilution series of TGF-β (Genzyme Corporation, batch 94F003, 96 micrograms / milliliter) will be prepared in the assay media and twenty-five microliters added to the appropriate wells. Subsequently, 1 microgram of PRL was added to the wells and the cells were incubated at 37 ° C / 5 percent CO2 for 72 hours. Tritiated thymidine (0.5 icroCi) was added during the last 18 hours. The amount of proliferation measured by tritiated thymidine incorporation was measured by scintillation counting (1205 Betaplate LSC, Wallac) after stitching the cells in a glass fiber filter using a TOMEC 96 harvester. The results are shown in the Table (Figure 2). ). Hybridomas varied in their sensitivity to TGF-β, but in all cases a decrease in cell proliferation was observed in the presence of TGF-β. The ability of prolactin to overcome the suppressive effects of TGF-β depended in part on the concentration of TGF-β. It was observed that prolactin exceeded suppressive TGF-β with all hybridomas 8/8 incubated with 0.1 microgram of TGF-β, with 7/8 hybridomas incubated with - 1 microgram of TGF-β, and with 5/8 hybridomas incubated with 10 micrograms of TGF-β.

Example 3: Effect of TGF-β and PRL on Human Peripheral Blood Mononuclear Cells Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Plaque gradient centrifugation (Pharmacia). Cells were washed twice in PBS and resuspended in RPMI-1640 medium (Gibco) containing 1 percent FCS (Gibco), 2mM glutamine (Gibco), 15mM HEPES (Gibco) and 100 units / milliliter of penicillin (Gibco) and 100 micrograms per milliliter of streptomycin (Gibco). The cells were adjusted to a final cell density of 2 10 ^ cells per milliliter and plated 0.1 milliliter in wells of microevaluation tissue culture plates. Cells were incubated overnight in a humane incubator at 37 ° C, 5 percent CO2 • The next day, 20 micrograms per milliliter of PHA was added to the wells to stimulate human peripheral blood lymphocytes (PBL). Immediately, TGF-β was added to the cultures at concentrations of 0, 0.5 and 10 ng per milliliter of TGF-β, followed by 0, 10 and 20 micrograms per milliliter of PRL.

The cultures were incubated for 96 hours and boosted during the last 8 hours with tritiated thymidine / well of 0.5microCi. Cells were harvested on a glass fiber filter using a TomTec Mach II cell harvester and incorporated by counting using a Betaplate Wallac 1205 Counter. The results are shown in Figure 3. Prolactin antagonized the suppression of TGF-β from Human PBL simulated with PHA.

Claims (9)

1. CLAIMS: 1. The method for treating a patient suffering from a disease or disorder associated with TGF-β by administering an effective amount of a pharmaceutically acceptable composition containing prolactin.
2. 2. A method for preventing inhibition of cell growth by TGF-β by administering an effective dose of a composition containing prolactin.
3. 3. A method for improving the growth of the cell by administering an effective amount of a composition containing prolactin.
4. 4. The methods of claims 1, 2 and 3, wherein the prolactin is human prolactin.
5. 5. The methods of claims 1, 2 and 3, wherein the prolactin is recombinant prolactin.
6. 6. The method of claim 1, 2 and 3, wherein the cells are lymphoid cells.
7. The method of claim 1, 2 and 3, wherein the cells are endothelial cells.
8. The method of claim 1, 2 and 3, wherein the cells are epithelial cells.
9. 9. The method for treating a patient suffering from a disease or disorder associated with TGF-β by administering an effective amount of a pharmaceutically acceptable composition containing prolactin DNA.