HK1124260B - Methods of treating fibrosing diseases by induction of immune tolerance - Google Patents

Description

Methods of treating fibrotic diseases by inducing immune tolerance

Technical Field

The present invention relates generally to the treatment of fibrotic diseases (fibrosing diseases). In particular, the invention relates to the treatment of fibrotic diseases by inducing immune tolerance.

Background

Acquired fibrotic disease in humans has several common features. Tissue fibrosis is preceded by injury and/or inflammation of normal tissue. Infiltration of tissues by T cells and monocytes is seen early in the development of fibrosis.

Systemic sclerosis (SSc, scleroderma) is a common Systemic fibrotic disease associated with increased accumulation of collagen types I, III, IV, VI, VII, XVI, XVIII. Cellular and/or humoral immunity to types I, III and IV has been described in patients with SSc. The disease is primarily characterized by thickening of the skin and tight association with underlying structures. The internal organs commonly involved are the gastrointestinal tract, lungs, kidneys and heart.

The T-lymphatic system regulates the function of fibroblasts and monocytes/macrophages as well as a variety of other target cells by synthesizing different types of cytokines. For fibrosis, T cells produce fibrous factors such as IL-4, TGF-. beta.1 and. beta.2, which directly stimulate collagen synthesis by cultured fibroblasts. T cells activate macrophages by synthesizing Interferon (IFN) γ, which synthesize a variety of fibroblast cytokines including platelet-derived growth factors, TGF- β 1 and β 2, which stimulate fibroblasts to synthesize collagen.

Summary of The Invention

The present invention provides methods for treating fibrotic diseases by orally administering tissue proteins derived from tissue undergoing fibrosis.

Fibrotic diseases that may be treated according to the invention include, but are not limited to, scleroderma (SSc), skin fibrosis, cirrhosis, renal fibrosis, pulmonary fibrosis, cardiac fibrosis, gastrointestinal fibrosis and vascular fibrosis.

In one embodiment, the invention is used to treat a patient suffering from a fibrotic disease for at least 3 years, preferably for at least 5 years.

In another embodiment, fibrotic diseases are treated by oral administration of collagen from tissue undergoing fibrosis. Depending on the tissue type, different types of collagen may be used for this treatment. Collagen may be prepared from tissue that is undergoing fibrosis in a human patient, or from the corresponding tissue of an animal such as a bird or mammal. Alternatively, chemically synthesized or recombinantly produced collagen may be used. Fragments of collagen or mixtures thereof may also be used in the present invention.

In a preferred embodiment, the collagen or collagen fragment is provided to the patient by oral administration at about 500 μ g/day for about 12 months.

Brief Description of Drawings

FIG. 1 is a bar graph showing the changes in MRSS at different time points in different subsets of SSc patients.

Figure 2 shows the percentage of patients with SSc versus the percentage of MRSS improvement at month 12.

Figure 3 shows the percentage of SSc patients improved relative to MRSS at month 15.

FIG. 4 shows the cleavage of α 1(I) and α 2(I) with CNBr.

Detailed Description

The present invention demonstrates for the first time that orally administered type I Collagen (CI) induces CI tolerance and alleviates the clinical manifestations of the disease in patients with systemic sclerosis (SSc).

SSc is a common systemic fibrotic disease associated with increased accumulation of extracellular matrix proteins such as collagen. Without being bound by any particular theory, it is believed that oral administration of tissue proteins (e.g., collagen) present in a tissue site undergoing fibrosis where T cells are activated by various stimuli may down-regulate T cells. Thus, the effects of T cells secreting fibroblast cytokines and cytokines that stimulate monocytes/macrophages are inhibited, which would otherwise stimulate fibroblast cells at the tissue site to produce extracellular matrix proteins such as collagen.

Accordingly, the present invention provides a method of treating fibrotic disease by orally administering tissue proteins derived from tissue undergoing fibrosis.

Fibrotic diseases that can be treated by the methods of the invention include, but are not limited to, SSc, skin fibrosis, liver cirrhosis, kidney fibrosis, lung fibrosis, cardiac fibrosis (e.g., as found in congestive heart failure), gastrointestinal fibrosis, and vascular fibrosis as found in atherosclerosis. Regardless of its etiology, the methods of the invention can treat these fibrotic diseases

In a particular embodiment, the method of the invention is used to treat a patient suffering from a fibrotic disease for at least 3 years, preferably for at least 5 years.

In accordance with the present invention, fibrotic diseases can be treated by oral administration of collagen from tissue undergoing fibrosis. For example, SSc is known to be associated with excessive accumulation of type I collagen, and thus type I collagen or fragments thereof can be orally administered to patients with SSc. Liver cirrhosis and pulmonary fibrosis, as well as interstitial collagen disease, are associated with the accumulation of collagen types I, III and V, respectively. Thus, type I, III and V collagen, respectively, may be orally administered to patients suffering from liver cirrhosis, pulmonary fibrosis and interstitial collagen diseases. Small synthetic peptides from collagen may also induce tolerance when administered nasally, e.g., by nasal drops or nasal spray, or inhaled as an aerosol.

Collagen may be prepared and extracted from tissue that is undergoing fibrosis in a human patient or from the corresponding tissue of an animal, such as a bird (e.g., poultry) or a mammal (e.g., cattle or swine). Alternatively, chemically synthesized or recombinantly produced collagen may be used. In addition, collagen fragments or mixtures thereof may also be used in the present invention. For example, peptides derived from CNBr cleavage of type I collagen may be used in the treatment of patients with SSc.

Collagen or fragments thereof may be provided by oral administration to a patient at the following doses: about 200-. The treatment may continue for at least 6 months, preferably 12 months or longer, or until the clinical manifestation of the disease is reduced or alleviated.

The present invention is further illustrated by the following examples.

Example 1

To determine whether oral administration of 500 μ g/day of bovine type I Collagen (CI) ameliorated the clinical manifestations of systemic sclerosis (SSc), a multicenter double-blind placebo-controlled study was performed.

Patients for inclusion in the study were screened based on the following criteria:

-male or female at least 18 years old;

clinical diagnosis with diffuse (diffuse) SSc (according to ACR criteria in 1980) for 3 years or less (early), or 4-10 years (late);

stable skin lesions were shown by medical history or physical examination within 6 months prior to addition to the study; and

stable Modified Rodnan Skin Score (MRSS) 1 month prior to addition to the study: the selection was stable MRSS > 16, the following MRSS stable at random assignment:

MRSS at screening	MRSS allowed at random distribution (Baseline period)
		16	up to 20
17-20	16-24
		21-25	±4
26-30	±5
		≥31	±7

168 patients meeting the above criteria were graded and randomized to receive either daily placebo [2ml0.1M acetic acid (HAc) ] or 500 μ g bovine CI for 12 months. MRSS was determined as the first clinical outcome variable during baseline and after 4, 8, 12 and 15 months. Scleroderma Health Assessment Questionnaire (SHAQ), short form 36 questionnaire, physician's comprehensive assessment, patient's comprehensive assessment, blood pressure, weight and serum creatinine as second clinical outcome indicators were determined at baseline and after 4, 8, 12 and 15 months. FVC and DLCO were measured as a second clinical outcome parameter in patients no earlier than 5 weeks and at 12 months prior to baseline. Requiring pre-screening visits to patients receiving any exclusive medication/treatment.

Fig. 1 summarizes the MRSS changes at month 4 (blue), 8 (red), 12 (green), and 15 (orange) from baseline, divided into four subgroups. Each bar indicates the distribution of MRSS changes in each group at each time point; the upper edge is 75%; the lower edge is 25%; the line inside the column indicates that the MRSS changed the median value. Values outside this are indicated by the lines drawn.

The results showed no statistical difference in mean change at month 12 for the CI treated group and the placebo group. Similar conclusions are seen for other clinical and laboratory parameters (see tables 1 and 2). However, at month 15, MRSS in CI-treated advanced patients (the "advanced collagen" group) was 7.9, a very significant difference compared to 2.9 in advanced patients in the placebo group (the "advanced placebo" group). As shown in fig. 1, at month 15, the median values in the orange columns were significantly lower in the advanced collagen treatment group than in the other orange columns, and were actually the lowest of all columns. This indicates the greatest improvement in MRSS in the late patient subgroup of CI treatment. P-value for the difference in the mean MRSS values in each group of treatment-receiving advanced patients was 0.0063; while all other experiments were not evident for the level of 0.05. Note that the variable MRSS itself is not normally distributed, but that the changes in MRSS at months 12 and 15 compared to baseline are normally distributed. Thus, p values were obtained from the t-test. Nonparametric tests were also used to determine the MRSS change between the treatment and placebo groups, i.e. rank-sum tests, with similar resulting p-values.

In the case where changes in MRSS produced bifurcations and the percentage of patients with cutaneous MRSS changes was determined, two graphs were obtained (fig. 2 and 3). Each graph shows the percentage of patients in each of the four subgroups who were administered a different degree of improvement in MRSS. For example, in fig. 2, almost 50% of patients in the advanced collagen group had a 20% reduction in MRSS at month 12. Whereas only about 19% of the early collagen group experienced similar improvement. Both figures clearly show that advanced patients benefit most from collagen treatment compared to the other subgroups. In the collagen group, chi-square test demonstrated a significantly higher proportion of at least 25% improvement in MRSS in advanced patients compared to early patients at month 15.

In summary, the above study shows that oral administration of 500 μ g/day CI for 12 months significantly reduces MRSS in patients with a disease course of 4-10 years or more at study month 15, indicating that oral collagen treatment has a delayed effect on skin fibrosis. In this study, oral CI had no significant effect on PFT or HAQ and no side effects caused by CI treatment. The delayed effect of oral collagen treatment is associated with the need for some time to "gently settle" the fibroblasts once the T cells are stimulated to be neutralized. These results also indicate that T cells provide the major source of fibroblast signals only in advanced patients.

Example 2

This example describes an experiment to determine whether oral CI treatment at 500 μ g/day induces CI tolerance in patients enrolled in the study described in example 1.

Serum and PBMCs were obtained from patients before treatment and after 12 months of oral bovine CI treatment, or patients who dropped from 3 months or more to 11 months or less (drop-out). PBMCs were collected with or without bovine α 1(I) chain, bovine α 2(I) chain, native bovine CI, or α 1(I) or α 2(I) CB (CNBr) peptide. CB peptides were isolated by cleavage of bovine or human α 1(I) and α 2(I) with CNBr (shown in fig. 4 and table 3) and purification by ion exchange chromatography. Purified α 1(I) and α 2(I) CB peptides and unseparated α 1(I) and α 2(I) CB peptides were used to culture PBMCs of SSc patients at baseline and at month 12 prior to CI or placebo administration. At months 0 and 12, PBMC supernatants were analyzed by ELISA for IFN γ and IL-10. Chain-stimulated PBMC produce either a decrease in IFN γ or an increase in IL-10 following oral administration of CI as a first immunological outcome variable. The results are summarized in tables 4-9.

As shown in tables 5-6, a significant reduction in PBMC IFN γ production was observed for α 1(I) CB peptide mixtures as well as α 1(I) CB7 in the population of patients at the sum early disease stage treated with oral CI for 12 months. Furthermore, a significant increase in IL-10 production by PBMCs was observed for human α 2(I) as well as α 1(I) CB7 in the population of patients at the early disease stage of the sum (tables 7-8). These results indicate that oral bovine CI is effective for treating patients with diffuse SSc for 4 years or longer by modulating TH1/TH2 production. Upregulation of antigen-specific IL-10 production suggests that CI tolerance is induced in LD patients.

For the total SSc population, there is an inverse relationship between the disease stage and the IL-10 production caused by: α 1(I) CB3(p ═ -0.0059.N ═ 153); α 1(I) CB7(p ═ 0.0335, N ═ 150); human α 1(I) (p ═ -0.0166, N ═ 152); α 2(I) CB mixtures (p ═ -0.0032.N ═ 154).

In early patients, there is an inverse relationship between the production of IFN γ by MRSS and α 2(I) CB2 (p ═ 0.026, N ═ 94).

For the total SSc population, SF-36 and α 1(I) CB4 produce IFN γ in inverse proportion (p ═ 0.0448, N ═ 143). For advanced patients, SF-36 and α 1(I) CB4 are inversely proportional to PHA-induced IFN γ production (p-0.0364, N-57; p-0.028, N-58, respectively).

For the total diffuse SSc population, IL-10 production by FVC was directly proportional to PBMC cultured with α 1(I) CB4 and human α 2(I) (p ═ 0.0122, N ═ I52; p ═ 0.0072, N ═ 94, respectively).

For early stage patients, FVC is proportional to IL-10 induced by human α 2(I) (p ═ 0.0062, N ═ 94).

For early patients, FEB1 and the α 2(I) CB4 and α 1(I) CB mixture resulted in an inverse ratio of IL-10 production (p ═ 0.0067, N ═ 92; p ═ 0.0041, N ═ 94, respectively). For the total diffuse SSc population, FEV1 and α 1(I) CB mixtures resulted in an inverse ratio of IL-10 (p 0.0241, N154).

In early patients, DLCO was proportional to IFN γ production by α 1(I) CB7(p ═ 0.0367, N ═ 90). In advanced patients, DLCO is directly proportional to IFN γ production by α 2(I) CB2 (p 0.0383, N59).

In summary, immune response experiments performed by culturing PBMCs of patients with CI and CI-derived peptides showed that, overall, increased IFN γ and IL-10 production by cultured PBMCs was seen in patients with early stage disease (< 4 years disease course). IFN γ production by the antigen candida albicans was absent in early and late stage patients, suggesting an impaired Th1 response to the common environmental antigen. Native bovine CI elicited a significant increase in IFN γ and IL-10 production in both early and late stage patients. Specific CI CB peptides that are unable to elicit IFN γ or IL-10 production in advanced stage patients include α 1(I) CB2, 4, 5, and 7, and α 2(I) CB2, 3, and 3-5. The most consistent IFN γ and IL-10 responses in early and late stage patients were found in α 1(I) CB8, α 1(I) CB6, α 2(I) CB4, suggesting that these portions of α 1(I) and α 2(I) contain epitopes that elicit T cell responses in the disease duration of most disseminated SSc patients. PBMC IFN γ or IL-10 responses specific for CI and CI-derived peptides suggest that there may be a subset of patients in which a particular cytokine responding to a particular CI epitope may affect disease expression.

Claims (5)

1. Use of a substance comprising one or more collagen fragments selected from the group consisting of α 1(I) CB1, CB2, CB3, CB4, CB5, CB6, CB7, CB8 and α 2(I) CB1, CB2, CB3, CB4 and CB5 for the manufacture of a medicament for the treatment of scleroderma (SSc).

2. The use of claim 1, wherein the one or more collagen fragments are selected from the group consisting of: α 1(I) CB2, CB4, CB5 and CB7, and α 2(I) CB2, and CB 3-5.

3. The use of claim 1, wherein the one or more collagen fragments are type I collagen fragments.

4. Use according to claim 3, wherein the dose of the substance is 500 μ g.

5. The use of claim 3 wherein the type I collagen fragment is an α 1(I) CB peptide mixture.