Field of the Invention

This invention is directed to methods of treatment of inflammatory disorders by reducing the level of activation of phagocytes. More particularly, this invention relates to treatment of these disorders with transforming growth factor-beta (TGF-s).

Background of the Invention

Phagocytic cells (neutrophils, eosinophils, basophils, monocytes/ acrophages) have among their functions the phagocytosis and destruction of microorganisms, malignant cells and certain foreign particles. The phagocytes, when activated by appropriate stimuli, release toxic agents which assist in the destruction and removal of the microorganisms, malignant cells and foreign particles. Some of these cytotoxic agents include reactive oxygen intermediates, such as superoxide anion, hydrogen peroxide and hydroxyl radicals, and proteolytic and other digestive enzymes. Many of these tissue degradative enzymes are activated by oxidation or have enhanced activity in an environment where reactive oxygen intermediates are present. Further, some protease inhibitors, such as human α-1-proteinase inhibitor, are inactivated by oxidative pathways, see Johnson et al.. The oxidative inactivation of human β-1-proteinase inhibitor: further evidence for methionine at the reactive center. J. Biol. Chem., 254:4022-4026 (1979), and see Ossanna et al., Oxidative regulation of neutrophil elastase-alpha-1-proteinase inhibitor interactions. J. Clin. Invest., 77:1939-1951.

Phagocytic cell activation is essential for host survival from infection and contributes to surveillance and containment of tumorigenic cells. However, when activated, phagocytic cells in their defensive _^ role have the potential to also destroy a great deal of surrounding normal host tissue.

Both microbes and tumor cells, therefore, may be under selective pressure to inhibit or reverse the activation of phagocytes. This reasoning ^" led to the demonstration of macrophage deactivating factors from both microbes and tumor cells, see Confer et al.. Science 2T7, 948-950 (1982); Ding et al., J. Immunol. 1 , 1971-1977(1987); Szuro-Sudol et al., J. Exp. Med. 156, 945-961(1982); Szuro-Sudol et al., J. Immunol., 131, 384-387(1983); and ^" Tsunawaki et al., J. Exp. Med. 104_, 1319-1331(1986). In some circumstances the host itself probably requires the ability to deactivate phagocytes. Macrophages are essential to the healing of wounds and repair of damaged tissues. Yet the cytotoxic products of the activated macrophages can damage endothelium, fibroblasts, smooth muscle, and parenchymal cells, as described in Cross et al., Ann. Int. Med. I<λ7, 526-545(1987). Thus, after an inflammatory site has been sterilized, the impact of phagocyte activation on the host can shift from benefit to detriment.

There are many inflammatory disorders which contribute to significant morbidity and mortality in humans and other mammals. Although the absence of inflammation leads to a compromised host, excessive inflammation leads to inflammatory disorders. The accumulation and subsequent activation of phagocytic cells are central events in the pathogenesis of virtually all forms of inflammation, see Gallin et al., Inflammation, Basic Principles and Clinical Correlates, Raven Press, New York, 1988, hereby incorporated by reference.

It is an object of the subject invention to provide a method of treating disorders involving excessive phagocyte activation.

It is a further object of the present invention -to provide improved methods of suppressing acute or chronic inflammatory responses.

Other objects, features and characteristics of the present invention will become apparent upon consideration of the following description and the appended claims.

Summary of the Invention

The subject invention relates to a method of treating an inflammatory disorder involving excessive phagocyte activation by administering .to a patient a therapeutically effective amount of TG ^' F-3. The invention also relates to a pharmaceutical composition suitable for administration to a patient having an inflammatory disorder comprising a therapeutically effective amount of: TGF-s and at least one additional immunosuppressant.

Brief Description of the Drawinσs

Figure 1 shows suppression of macrophage H ₂ 0 ₂ releasing capacity following two days incubation in TGF-sl and TGF-S2, but not any of the nine other polypeptide growth factors tested.

Figure 2 shows time course for suppression of H ₂ 0 ₂ releasing capacity by (A) TGF-sl or (3) TGF-32.

Figure 3 shows prevention of TGF-sl-induced deactivation by coincubation in macrophage activating factors.

Figure 4 shows preservation of phagocytic function after deactivation of macrophage by TGF-sl or TGF-S2.

Detailed Description of the Invention

A new application of transforming growth factor-beta (TGF-s) had been discovered. Surprisingly it has been found that TGF-s is a potent phagocyte deactivator. Among eleven polypeptide growth factors studied that regulate angiogenesis, fibrogenesis, and other aspects of tissue repair, two proteins with 71% homology proved to be macrophage deactivators: transforming growth factor-sl (TGF-sl) and TGF-S2. The term TGF-s as used herein, refers to TGF-sl, TGF-S2, and/or TGF-sl.2 produced, for example from natural source extraction and purification, or from recombinant cell culture systems. The term likewise covers biologically active human TGF-s equivalents; e.g., differing in one or more amino acid(s) in the overall sequence. Further, the term as used in this application is intended to cover substitution, deletion and insertion amino acid variants of TGF-s, or post translational modifications. Advantageously, TGF-s is mature TGF-s from recombinant cell culture, see for example European Patent Application 200341, hereby incorporated by reference.

Growth factors were tested over a broad concentration range for their ability to suppress the capacity of activated cultured mouse peritoneal macrophages to release H ₂ 0 ₂ . The results are presented in detail below. The H ₂ 0 ₂ releasing capacity is a close biochemical correlate of macrophage activation, due to the prominent involvement of reactive oxygen intermediates in the antimicrobial function of macrophages, see Nathan et al.. Trans. Roy. Soc. Trop. Med. Hyg. TT_, 620-630 (1983).

Prominent sources of natural TGF-s include degranulating platelets, endothelial cells, fibroblasts, keratinocytes, tumor cells, and T cells responding to antigen. Thus, wounds, tumors, and T cells may all be able to restrain or reverse macrophage activation through the action of TGF-s. Eukaryotic cells can deactivate macrophages by other routes as well, see Szuro-Sudol et al., J. Exp. Med. supra; Szuro-Sudol et al., J. Immunol., supra; Tsunawaki et al., J. Exp. Med., supra. However, the previously described macrophage activating factor from tumors (Szuro-Sudol et al., J. Exp. Med. supra; Szuro-Sudol et al., J. Immunol., supra; Tsunawaki et al., J. Exp. Med., supra. ) , is immunoche ically and functionally distinct ^' from TGF-s. Macrophages themselves can secrete TGF-s upon exposure to bacterial lipopolysaccharide. This suggests the possibility of a negative feedback that may counter the potential for positive feedback implicit in the ability of the macrophages to produce some of its own activating factors, such as TNF-α and CSF-GM.

The actions of TGF-sl on phagocytes are both pro-inflammatory and anti-inflammatory. TGF-sl induces chemotaxis (EC ₅₀ , 0.004 pM) (EC ₅₀ = the concentration giving 50% of the maximum effect), release of fibroblast growth factors and accumulation of IL-1 mRNA (EC ₅₀ , -40 pM), (see Wahl et al., PNAS, 4, 5788-5792(1987)), and release of angiogenic factors. The virtually complete suppression of macrophage respiratory burst capacity by TGF-sl (EC ₅₀ , 0.6 pM) and TGF-S2 (EC ₅₀ , 4.8 pM) is described herein. These effects may reflect a coordinated response in wound healing, in which macrophages are recruited to scavenge debris and foster the growth of fibroblasts and endothelial cells, while being suppressed in their capacity for a respiratory burst that could be inimical to these cells.

The ability of TGF-sl and TGF-S2 to ablate phagocyte respiratory burst indicates that these agents

have a role in the treatment of inflammatory disorders involving excessive phagocyte activation.

One embodiment of this invention is a method of treating disorders involving excessive phagocyte activation --by administering to a patient a therapeutically effective amount of TGF-s. Acute or chronic inflammatory responses are suppressed by reducing the degree of phagocytic cell activation, particularly by deactivating respiratory burst function. Deactivating respiratory burst function can reduce tissue degradation caused by many digestive enzymes.

Inflammatory disorders involving excessive phagocyte activation include the following. Each of the following articles is hereby incorporated by reference.

Adult Respiratory Distress Syndrome: (ARDS)

See Gallin et al., supra, pgs. 815-824 Chapter 45; Cochrane et al., Pathogenesis of the adult respiratory distress syndrome. J. Clin. Invest., 71:754- 761 (1983); Balwin et al., Oxidant activity in expired breath of patients with adult respiratory distress syndrome. Lancet, 1:11-13 (1986); Johnson et al.. Acute and progressive lung injury after contact with phorbol myristate acetate. Am. J. Pathol., 107:29-35 (1982); and Johnson et al.. Mediator of I_A induced lung injury in the rat. Role of macrophages and reactive oxygen products. Lab. Invest., 54:499-506 (1986).

Rheumatoid Arthritis and Other Arthritic Diseases Including Crystal Induced Inflammation

See Gallin et al., supra, Chapters 41-42, pgs. 751-783.

Asthma

See Calhoun et al.. Increased superoxide release from alveolar macrophages in symptomatic asthma. Am. Rev. Respir. Dis., 135:224A (abstract) (1987); and Serhan et al., Lipoxins: Novel series of biologically active compounds formed from arachidonic acid in human leukocytes, PNAS 81:5335-5339 (1984).

Emphysema

See Mittman et al.. Second International Symposium on Pulmonary Emphysema and Proteolysis. Academic Press, New York (1986); and Theron et al., Investigation of the protective effects of the antioxidants ascorbate, cysteine and dapsone on the phagocyte-mediated oxidative inactivation of human alpha^protease inhibitor iτι vitro. Am. Rev. Respir. Dis., 132:1049-1054 (1985); and Werb et al.. Degradation of connective tissue matrices by macrophages. I.

Proteolysis of elastin, glycoproteins and collagen by proteinases isolated from macrophages. J. Exp. Med., 152:1340-1357 (1980).

Acute Glo erular Nephritis

See Johnson et al., A new mechanism for glomerular injury: the myelo-peroxidase-hydrogen peroxide-halide system. J. Clin. Invest., 79:1379-1387 (1987).

Inflammatory Bowel Disease or Bowel-Associated Dermatosis

See Jorizzo et al., Bowel-associated dermatosis-arthritis syndrome: Immune complex-mediated

vessel damage and increased neutrophil migration. Arch. Intern. Med., 144:738-740 (1984).

Neutrophilic Dermatoses Especially Psoriasiform Dermatoses ^* ~

See Gallin et al., supra, Chapter 43, pgs. 785-802; Cram et al.. Psoriasis: Current advances in etiology and treatment. J. Am. Acad. Dermatol., 4:1-14 (1981); and Ragaz et al., Evolution, maturation, and regression of lesions of psoriasis. Am. J. Dermatopathol., 1:119-214 (1979).

Disorders Characterized by Fibrosis Including Pulmonary Fibrosis

See Johnson et al.. In vivo damage of rat lungs by oxygen metabolites. J. Clin. Invest. 67:983-993 (1981).

Sarcoidosis

See Robinson et al.. Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis. J. Clin. Invest., 75:1488-1495 (1985).

The TGF-s is administered as a pharmaceutical composition comprising therapeutically effective amounts of TGF-s (i.e. amounts that provide a therapeutic effect by reducing the level of activation of phagocytes) together with suitable diluents, adjuvants and/or carriers useful in immunosuppressive therapy. The TGF-s compositions are administered by continuous infusion, sustained release formulation, or injection at empirically determined frequencies. Where the disorder

permits, site-specific delivery is made. TGF-s sustained release formulations can include biodegradable microcapsular particles or i plantable articles. TGF-S is optionally delivered as an aerosol to the activated phagocytic- ^' cells in inflammatory disorders of the lung and airways.

In another embodiment of the subject invention, one or more additional immunosuppressants such as an additional phagocyte deactivator and/or an anti-inflammatory agent, are administered with TGF-s. Examples of anti-inflammatory substances are nonsteroidal anti-inflammatory drugs, salicylate, penicilla ine, gold salts, and antagonists of: TNFα, s, IL-1, and λ-interferon. Several variables will be taken into account by the ordinary artisan in determining the concentration of TGF-S in the therapeutic compositions and the dosages to be administered. Therapeutic variables include the half-life of the TGF-s preparation, administration route, and the clinical condition of the patient.

The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.

EXPERIMENTAL

Suppression of Hydrogen Peroxide Releasing Capacity

Activated macrophages were collected 4 d after intraperitoneal injection of sodium caseinate and plated at 1.2-1.3x10 ^s per well in 96-well trays in Eagle's minimum essential medium (α-variant) with 10% horse serum (complete medium). Nonadherent cells were washed off at 2 h and test media added to triplicate wells for the times indicated before the media were flicked out and the plates washed in saline. H ₂ 0 ₂ release was then

measured in the absence of cytokines over 90 in in response to 167 nM phorbol myristate acetate (PMA) by the horseradish peroxidase catalyzed oxidation of fluorescent scopoletin, and related to the protein content in ^{^} the same wells as described in de la Harpe et al., J. Immunolog. Meth. 7 , 323-325(1985).

Two days incubation in 0.04-100 ng/ml of TGF-sl suppressed macrophage H ₂ 0 ₂ release by 86 ± 11% (means ± SEM, n = 83 in 25 experiments) compared to macrophages incubated in medium alone; the latter released 243 ± 65 nmol H ₂ 0 ₂ /mg cell protein per 90 min (n = 33 in 25 experiments). Results were similar with TGF-S2, although -8-fold higher concentrations were required. The concentrations causing 50% inhibition of H ₂ 0 ₂ releasing capacity (EC5 _Q ) were 0.6 pM for TGF-sl and 4.8 pM for TGF-S2 (Fig. 1), suggesting a physiologic role for this action of the cytokines. In contrast, the following growth factors suppressed macrophage H ₂ 0 ₂ releasing capacity by -21% to 30%, when tested over a > 10,000-fold dose range up to 100 ng/ml (Fig. 1): natural mouse nerve growth factor (NGF) , natural murine interleukin (IL)-3, recombinant human IL-ls, natural bovine fibroblast growth factor (FGF)-a and -b, natural porcine platelet derived growth factor (PDGF), recombinant mouse colony stimulating factor (CSF) for granulocytes and macrophages (CSF-GM), recombinant human CSF for granulocytes (CSF-G), and natural mouse epidermal growth factor (EGF) (which shares receptors with TGF-o) . Caseinate-elicited mouse peritoneal macrophages were incubated in the indicated concentrations of cytokines before being washed and challenged with 167 nM PMA to trigger secretion of H ₂ 0 ₂ . One of 3 similar experiments is shown in Figure 1 (2 for EGF). For clarity, only means are presented in Figure 1; SΞM for the triplicates averaged 7.5% of the means, n =

natural; r = recombinant; suffixes designate species of origin. All reagents were pure except IL-3 which was partially purified. TGF-sl was prepared from human platelets as described in Assoian et al., J. Biol. Chem. 2_5_8_ 7155-7160(1983) , except that the urea was removed by desalting on C18 HPLC. TGF-s2 was purchased from R&D Systems, Minneapolis MN, see Marquandt et al., J. Biol. Chem. !26_2, 12127-12131 (1987).

Time Course for Suppression

Deactivation induced by TGF-sl or TGF-82 was not evident over the first 9 h of incubation, but became half-maximal by 21-23 h at concentrations of 1-10 ng/ml, and by -48 h for 0.1 ng/ml (Fig. 2). After incubation for the indicated periods in 0.001-10 ng/ml TGF-s, the macrophages were washed and challenged with 167 nM PMA to measure H ₂ 0 ₂ release (nmol/mg protein/90min) . Values are expressed as a percent of the results for controls incubated for the same time periods without TGF-s. The control values were nearly constant over 3 days. The mean ± SE for the means of 8 control triplicates was 372 ± 13 in (A) and 362 ± 19 in (B). One of two similar experiments is shown in Figure 2. These results show that TGF-s did not deactivate macrophages by triggering their respiratory burst, since exhaustion of respiratory burst capacity by triggering agents is evident immediately after the burst ceases (-1.5-3.5 h). Moreover, TGF-sl at 1-100 ng/ml did not elicit H ₂ 0 ₂ release from macrophages when added directly to the scopoletin assay.

Coincubation with Macrophage Activating Factors

To determine whether the state of macrophage activation reflects the balance between activating and

deactivating factors, macrophages were incubated in TGF-sl alone, macrophage activating factors alone, or combinations of these cytokines (Fig. 3). Activated macrophages were exposed to the following cytokines alone or in ^" the indicated combinations: TGF-sl, and one of three pure recombinant proteins (A) murine interferon-λ (rIFNλ-mo, specific activity 5x10^ ϋ/mg), (B) tumor necrosis factor-o-hu, (TNFa, specific activity 3.6xl0 ⁷ ϋ/mg) and (C) TNF-8-hu (lymphotoxin, specific activity 1.2xl0 ⁸ ϋ/mg). After 2 days, the cells were washed and challenged with PMA. TGF-sl concentrations were 0 (o), 0.1 (Δ), or 1 ([]) ng/ml. Concentrations of the other cytokines are indicated on the abscissa. One of four similar experiments is shown. in Figure 3. Suppression of H ₂ 0 ₂ releasing capacity caused by TGF-sl could be overcome by interferon-λ, tumor necrosis factor (TNF)-α, or TNF-s, at concentrations similar to those required to activate resident peritoneal macrophages, see Ding et al., supra.

Phaσocvtic Function Af ter Deactivation of Macroϋhaαes

Suppress ion of H ₂ 0 releasing capacity by TGF-s did not reflect toxicity to the macrophages . Thus , > 95% of macrophage excluded trypan blue after 2 d incubation in > 1 ng/ml TGF-s . Macrophages incubated for 2 d in 1 or 10 ng/ml TGF-s retained a capacity for extens ive phagocytosis : treated macrophages took up -20-25 starch granules per cell ( each granule 2.6 in diameter ) , marginally less than control cells , as assessed by a method optimized to detect dif ferences in maximal phagocytic capacity ( Fig . 4 ) , see Nathan et al . . Cell Immunol . 2_9 , 295-311 ( 1977 ) . 8.5xl0 ⁵ macrophages were plated on 13 -mm glass coverslips and nonadherent cells removed 2 h later . Test media were added for 2 days as

indicated: complete medium alone (open bar), or complete medium containing TGF-sl (solid bars) or TGF-S2 (hatched bars) at the concentrations indicated in ng/ml. After 2 days, macrophages were washed and incubated in complete medium for 2 h with 2 mg/coverslip of

^ ^■4 C-acetylated starch granules isolated from seeds of Amaranthus caudatus. These conditions optimize the quantification of maximal phagocytic capacity by macrophages, see Nathan et al., Cell. Immunol,, supra. The coverslips were washed and the raonolayers solubilized to determine the mg particles phagocytized per mg cell protein. Means ± SE of triplicates are shown in Figure 4. However, TGFs did cause macrophages to become less tightly adherent to the plastic culture surface. This was reflected by a dose-dependent decrease in the amount of cell protein remaining after vigorous washing (IC ₅₀ -0.8 pM for TGF-sl, -5.2 pM for TGF-S2) (IC _5Q = the concentration giving 50% of the maximum inhibitory effect). Overall, after treatment for 2 days with 0.04-100 ng/ml of TGF-sl, ^' the adherent cell protein in vigorously washed monolayers averaged 59 ± 20% of control (n=83 in 25 experiments).

While the invention has been described in what is considered to be its preferred embodiments, it is not to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and equivalents included within the spirit and scope of the appended claims, which scope is to be accorded the broadest interpretation so as to encompass all such modifications and equivalents.