WO2013100657A1 - Pharmaceutical composition comprising thalidomide for treating lupus nephritis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising thalidomide or lenalidomide for coadministration for preventing or treating adrenal disorders. The amount of albuminuria is significantly reduced in a lupus nephritis animal model by using the pharmaceutical composition for preventing and treating adrenal disorders according to the present invention, and thus, the composition of the present invention exhibits an effect for treating and preventing lupus nephritis.

Description

Pharmaceutical composition for treatment of lupus nephritis containing thalidomide

The present invention relates to a pharmaceutical composition for the prevention or treatment of concomitant kidney disease comprising thalidomide or lenalidomide.

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease with a complex clinical symptom called lupus, caused by excessive antibody production of hyper-activated B and T cells. It is an autoimmune disease caused by pathogenic autoantibodies and immunocomplexes.

Lupus nephritis is a serious complication of systemic lupus erythematosus in patients with systemic lupus erythematosus, which not only directly contributes to morbidity and mortality, but also induces kidney disorders (Agrawal et al. , 2006). Although many other immune and non-immune factors contribute to the development of lupus nephritis, the production of autoantibodies against the nucleal and endogenous antigens and the formation of glomerular immune deposits Plays an important role in the development of lupus nephritis (Deocharan et al. 2002; Lefkowith and Gilkeson, 1996). In many studies, anti-DNA antibodies are important diagnostic markers that bind cell surface antigens or directly cross-reactive antigen (s) or inflammatory processes in the kidneys. The ability to indirectly bind glomerular basal membrane components through starting chromatin materials has been reported to be associated with the development of lupus nephritis (Yung and Chan, 2008). Furthermore, cytokines and chemoattractants induced by kidney cells and invasive immune cells exacerbate immunocomplex mediated renal imjury (Aringer and Smolen, 2005; Kulkarni). and Anders, 2008). Lupus therapy is currently focused on immunosuppressants such as corticosteroids, cyclophosphamide, azathioprine, and mycophenolate mofetil (Waldman and Appel, 2006). . However, these drugs carry dangerous side effects that are susceptible to infectious diseases and cancer (Radis et al., 1995). This raises interest in the development of molecules that directly counter inflammatory responses, as well as low-toxic drugs for controlling immune complex formation and deposition.

Thalidomide has been used to treat multiple myeloma with dexamethasone or other cytotoxic drugs prior to organ transplantation or maintenance therapy of autologous tissues. Thalidomide exhibits various anti-myeloma characteristics, such as immune-modulating, anti-angiogenic, anti-inflammatory, anti-acute effects, induction of cell death, and reduced drug resistance in multiple myeloma cells, and cytokines, cell adhesion proteins and angiogenesis It is known to exhibit features that alter the expression of angiogenesis. In addition, thalidomide modulates NF-κB activity in myeloma cells, indicating a decrease in anti-apoptotic protein and an increase in pro-apoptotic modulators. Recently, NF-κB has been known to be a major factor regulating the proliferation of vascular mesenteric cells in lupus mice.

In view of the functional mechanism of thalidomide and therapeutic effects in multiple myeloma, the present inventors anticipated that thalidomide may have a novel therapeutic effect on lupus nephritis, and a combination of thalidomide and corticosteroids in the animal model When administered, it was confirmed that the degree of lupus nephritis was significantly reduced and the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of concomitant kidney disease comprising thalidomide or lenalidomide.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of concomitant kidney disease comprising thalidomide or lenalidomide.

In another embodiment, the present invention provides a dietary supplement for preventing or improving kidney disease, including thalidomide or lenalidomide.

In the present invention, the pharmaceutical composition and dietary supplement may further comprise a corticosteroid-based material, wherein the pharmaceutical composition and dietary supplement are cyclophosphamide, azathioprine and mycophenolate. It may be characterized in that it further comprises any one selected from the group consisting of mofetil. In addition, the pharmaceutical composition and dietary supplement further comprises any one selected from the group consisting of a) corticosteroid-based material and b) cyclophosphamide, azathioprine and mycophenolate mofetil. can do.

In the present invention, the corticosteroid-based material may be selected from the group consisting of prednisolone, methyl prednisolone and dexamethasone.

In the present invention, the kidney disease may be selected from the group consisting of lupus nephritis, glomerulonephritis, kidney stones, renal tuberculosis and renal tumor.

In the present invention, the pharmaceutical composition and the dietary supplement may be characterized by reducing the amount of proteinuria, the pharmaceutical composition is a suspension, powder, powder, granules, tablets, sustained-release preparation, injection, ointment or capsule It may be characterized in that the form, the health functional food may be in the form of capsules, tablets, granules, powders or drinks.

In addition, the pharmaceutical composition may be characterized as targeting a human.

The composition of the present invention may be prepared as a pharmaceutical composition, a neutraceutical composition or a food composition.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. The dosage of the pharmaceutical composition of the present invention may be 0.001-100 mg / kg body weight, 0.01-80 mg / kg body weight, 0.1-60 mg / kg body weight per day on an adult basis. In addition, depending on the judgment of the doctor or pharmacist may be divided administration once a day to several times.

The pharmaceutical and health food composition of the present invention may be formulated by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. It may be prepared in a dosage form or incorporated into a multi-dose container.

According to an embodiment of the invention, the formulation of the composition of the invention is a solution, suspension, syrup, emulsion, liposome, extract, powder, powder, granule, tablet, capsule, and may further comprise a dispersing or stabilizing agent. have.

The use of a pharmaceutical composition for the prevention or treatment of concomitant kidney disease comprising thalidomide or lenalidomide according to the present invention is effective in treating and preventing lupus nephritis by significantly reducing the amount of proteinuria in a lupus nephritis animal model. There is.

Figure 1 shows the change in the amount of proteinuria according to the treatment group of thalidomide.

Figure 2 shows the extent of histological damage according to the treatment group of thalidomide.

Figure 3 shows the difference in immune complex deposition according to treatment group of thalidomide.

Figure 4 shows the serum concentration changes of anti-ds DNA and IgG according to the treatment group of thalidomide.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Animal Models and Test Groups

All animal models used in the present invention were used according to the regulations on animal use and handling of Yonsei University. 43 female NZB / WF1 mice, 18 weeks old, were purchased and raised in specific sterile breeding facilities under sterile conditions. According to the first experiment, 25-30% of mice not treated with thalidomide died during the experiment, and eight mice in the untreated group and each treated group (group 1: untreated, group 2: 1.7 mg / kg thalidomide treated) , Group 3: 5 mg / kg thalidomide treatment, group 4: 10 mg / kg thalidomide treatment, group 5: 1.7 mg / kg thalidomide and PL treatment, group 6: 5 mg / kg thalidomide and PL treatment, group 7: 10 mg 5 mice in / kg thalidomide and PL treatment, group 8: MMF and PL treatment). Treatment of thalidomide, MMF, and PL started at week 24, with three doses of thalidomide, MMF, and PL per day until 31 weeks.

Measurement of proteinuria

Proteinuria was measured twice a week during the experiment in spot urine collected from each mouse using an albumin reagent strip (URiSCA; Yeongdong Pharm., Korea). Proteinuria is expressed semi-quantitatively: 0 = none or trace, 1+ = 100 mg / dL or less, 2+ = 300 mg / dL or less, 3+ = 2,000 mg / dL or less, and 4+ = 2,000 mg / dL More than.

Histopathological examination

Kidney specimens were obtained from 31 week old mice, fixed in buffered formalin and stored in gaseous nitrogen. Formalin-coagulated kidney specimens were placed in paraffin and then sectioned 4 micrometers thick. It was then stained with periodic-acid Schiff (PAS) according to conventional methods. Renal histological modification scores in mice, including glomerular injury, vascular injury, and vascular injury, were performed semi-quantitatively on a four-point scale by two pathologists, with a mean score of 0 = no change (no change, 1 = mild change, 2 = moderate changes, 3 = significantly change. At least 30 glomeruli per mouse were used for the experiment.

Immunofluorescence staining of kidney

All mice were placed in OCT compositions and frozen at minus 20 ° C. Samples were sectioned 4 micrometers thick and fixed in 4% para-formaldehyde for 15 minutes and washed three times with cold PBS (phosphate buffer saline). Nonspecific binding is blocked for 30 minutes by 1% standard Xinyang serum in phosphate buffer saline tween (PBST) and the sample is goat anti-mouse IgG (1: 100, Sigma-aldrich, St. Luis, MO) and rabbit anti Incubated with mouse C3 (1: 100; Abcam, Cambridge, Mass.) for 4 hours at room temperature and then washed three times with PBS. The secondary antibody was incubated with alexa flour 488 donkey anti-goat IgG and alexa flour 568 donkey anti-rabbit IgG (both 1: 100, Molecular probe, Invitrogen, Carlsbad, Calif.) For 1 hour at room temperature and washed three times with PBS. After installation with a sealant (Vector laboratories, Southfield, MI), it was observed with a laser microscope (LSM 710 confocal microscope, Carl Zeiss AG, Germany).

Measurement of serum concentration of anti-ds DNA and IgG

Blood samples were obtained from experimental animals and serum was isolated and stored at minus 80 ° C. Mouse anti-ds DNA IgG concentration was measured by sandwich ELISA according to the manufacturer's instructions (Alpha diagnostic international, San Antonio, TX). In addition, IgG1, IgG2a, IgG2b and IgG3 were analyzed with Milliplex MAP mouse immunoglobulin isotype kit (Millipore, Billerica, Mass.).

Statistical analysis

All statistical analyzes were performed using the SPSS package of Windows version 15 (SPSS Inc., Chicago, IL). Representative values are average values of values obtained from each mouse of each group, and all values of the treated group were compared with those of the untreated group. All results are expressed as mean ± standard deviation. Kaplan-Meier plot of survival was analyzed by log-rank test. Statistical comparisons of proteinuria, histological damage, and serum serum levels of the two groups were assessed by the Mann-Whitney U test. When comparing the values between the untreated groups and each treatment group, an asterisk was marked for the mean value of each treatment group which was statistically significant (P-value <0.05).

Example 1

Proteinuria Measurement in NZB / WF1 Mice

To measure the effects of the combined treatment of thalidomide, PL and thalidomide, and to compare the effects with the effects of general induction therapy, the amount of proteinuria in lupus nephritis mice was measured and the survival rate was analyzed. When experimented with 24 week old mice, most mice showed proteinuria levels ranging from 2 to 2.2 on average. In untreated thalidomide mice and mice treated with 1.7 mg / kg of thalidomide, proteinuria increased gradually with time, and at 31 weeks, untreated mice reached 3.1 and mice treated with thalidomide at 1.7 mg / kg reached 2.6. Mice treated with 10 mg / kg of thalidomide and mice treated with 1.7 mg / kg of thalidomide and PL slightly reduced proteinuria at 31 weeks compared to the amount of proteinuria at the starting point, whereas the amount of proteinuria in mice treated with 5 mg / kg of thalidomide This hardly decreased. In addition, the amount of proteinuria was significantly reduced at week 31 as compared to untreated mice in mice treated with 5 mg / kg of thalidomide and PL, mice treated with 10 mg / kg of thalidomide and PL, and mice treated with MMF and PL. (Reduced 67%, 99% and 62%, respectively). In particular, mice treated with 10 mg / kg of thalidomide and PL showed a greater therapeutic effect than mice treated with 5 mg / kg of thalidomide and PL and mice treated with MMF and PL (FIG. 1A). Two mice in the untreated group and one mouse in the group treated with 1.7 mg / kg of thalidomide died during the experiment, while in the other group all mice survived during the experiment (FIG. 1B). From this, it can be seen that the group treated with 5 or 10 mg / kg of thalidomide and PL together than the group treated with thalidomide alone showed superior therapeutic effect in lupus nephritis mice. Moreover, the combination of thalidomide and PL in clinically used doses in humans is expected to have a superior therapeutic effect than conventional induction therapy.

Example 2

Confirmation of histological damage of NZB / WF1 mouse kidney

In order to measure the histological deformation of kidneys in lupus nephritis mice, three categories of injury scores were measured: glomerular, tubular and vascular damage. As a result, increased glomerular cell proliferation was observed in untreated mice, whereas mice treated with 5 mg / kg of thalidomide and PL, mice treated with 10 mg / kg of thalidomide and PL, and mice treated with MMF and PL Relatively little cell proliferation, general structure, was observed (FIG. 2A). Histological evaluation revealed that glomerular damage was 62% and 85% in mice treated with 5 mg / kg of thalidomide and PL, mice treated with 10 mg / kg of thalidomide and PL, and mice treated with MMF and PL, respectively. Significantly decreased to% and 81%, and tube damage was reduced to 67%, 72% and 72%, respectively. In the degree of glomerular damage, mice treated with thalidomide and PL at 10 mg / kg showed a lower degree of injury than mice treated with MMF and PL. In addition, mice treated with 1.7% mg / kg of thalidomide and PL showed reduced glomerular damage and vascular damage, and mice treated with 10 mg / kg of thalidomide showed a similar degree to the group treated with thalidomide and PL. The degree of reduced tube damage was shown (FIG. 2B). However, the degree of vascular damage did not show a significant difference in all experimental groups.

Example 3

Deposition of Immune Complex in the Kidney

To determine the amount of immune complex deposits, kidneys from NZB / WF1 mice were stained with antibodies specific for C3 (green) and IgG (red) and analyzed intensively by immunofluorescence. In the untreated group, immune complexes were present in capillary rings and glomerular epilepsy, and a large accumulation of C3 and IgG was observed in the glomeruli (in the case of the presence of C3 and IgG as immune complexes, they appear in yellow and orange color). Similar fluorescence intensity remained in the kidney region in mice treated with 1.7 and 5 mg / kg of thalidomide, whereas fluorescence intensity disappeared in mice treated with 10 mg / kg of thalidomide. In mice treated with 5 mg / kg of thalidomide and PL, mice treated with 10 mg / kg of thalidomide and PL, and mice treated with MMF and PL, compared to untreated mice or mice treated with thalidomide alone, Accumulation was significantly reduced (FIG. 3). From this, it can be seen that when thalidomide and PL are treated together, the effect of treating lupus nephritis is reduced by reducing glomerular accumulation of the immune complex.

Example 4

Measurement of serum concentration of anti-ds DNA and IgG

To determine the effect of the combination treatment of thalidomide and PL on the function of B cells involved in the production of lupus nephritis-specific anti-ds DNA and total IgG, serum concentrations of anti-ds DNA and IgG were measured in all mice. It was. Compared to untreated mice, serum concentrations of anti-ds DNA were increased in mice treated with 5 mg / kg of thalidomide and PL, mice treated with 10 mg / kg of thalidomide and PL, and mice treated with MMF and PL, respectively. Significantly decreased to%, 30% and 24% (FIG. 4A). In addition, the IgG1 concentration was not significantly changed in all experimental groups, but in the serum concentrations of IgG2a, IgG2b and IgG3, mice treated with 5 mg / kg of thalidomide and PL and 10 mg / kg of thalidomide and PL were treated. Significant decrease was observed in mice and mice treated with MMF and PL (FIG. 4B). From this, treatment with 5 mg / kg or 10 mg / kg of thalidomide and PL inhibits the production of anti-ds DNA and the production of B and plasma cells, thus reducing kidney accumulation of immune complexes and relieving lupus nephritis It can be seen that the results are shown.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The use of a pharmaceutical composition for the prevention or treatment of concomitant kidney disease comprising thalidomide or lenalidomide according to the present invention is useful for treating and preventing lupus nephritis by significantly reducing the amount of proteinuria in a lupus nephritis animal model. Do.

Claims (21)

1. A pharmaceutical composition for preventing or treating kidney disease for concomitant administration comprising thalidomide or lenalidomide.
2. The method of claim 1,

   The pharmaceutical composition is a pharmaceutical composition for preventing or treating kidney disease, characterized in that it further comprises a corticosteroid-based material.
3. The method of claim 1,

   The pharmaceutical composition is a pharmaceutical composition for preventing or treating kidney disease, further comprising any one selected from the group consisting of cyclophosphamide, azathioprine and mycophenolate mofetil.
4. The method of claim 1,

   The pharmaceutical composition further comprises any one selected from the group consisting of a) corticosteroid-based substances and b) cyclophosphamide, azathioprine, and mycophenolate mofetil. Pharmaceutical composition for.
5. The method according to claim 2 or 4,

   The corticosteroid-based material is a pharmaceutical composition for preventing or treating kidney disease, characterized in that selected from the group consisting of prednisolone, methyl prednisolone and dexamethasone.
6. The method of claim 1,

   The kidney disease is a pharmaceutical composition for preventing or treating kidney disease, characterized in that selected from the group consisting of lupus nephritis, glomerulonephritis, kidney stones, renal tuberculosis and renal tumor.
7. The method of claim 6,

   The kidney disease is a pharmaceutical composition for preventing or treating kidney disease, characterized in that the lupus nephritis.
8. The method of claim 1,

   The pharmaceutical composition is a pharmaceutical composition for preventing or treating kidney disease, characterized in that the human.
9. The method of claim 1,

   The pharmaceutical composition is a suspension, powder, powder, granules, tablets, sustained release formulation, injection, ointment or capsule in the form of a pharmaceutical composition for preventing or treating kidney disease.
10. The method of claim 1,

    The pharmaceutical composition is a pharmaceutical composition for preventing or treating kidney disease, characterized in that to reduce the amount of proteinuria.
11. Health functional food for preventing or improving kidney disease, including thalidomide or lenalidomide.
12. The method of claim 11,

    The health functional food is a health functional food for preventing or improving kidney disease, characterized in that it further comprises a corticosteroid-based material.
13. The method of claim 11,

    The health functional food is a health functional food for preventing or improving kidney disease, characterized in that it further comprises any one selected from the group consisting of cyclophosphamide, azathioprine and mycophenolate mofetil.
14. The method of claim 11,

    The dietary supplement further comprises any one selected from the group consisting of a) corticosteroid-based substances and b) cyclophosphamide, azathioprine and mycophenolate mofetil. Health functional food for improvement.
15. The method according to claim 12 or 14,

    The corticosteroid-based material is a functional food for preventing or improving kidney disease, characterized in that selected from the group consisting of prednisolone, methyl prednisolone and dexamethasone.
16. The method of claim 11,

    The kidney disease is a health functional food for preventing or improving kidney disease, characterized in that selected from the group consisting of lupus nephritis, glomerulonephritis, kidney stones, renal tuberculosis and renal tumor.
17. The method of claim 16,

    The kidney disease is a functional health food for preventing or improving kidney disease, characterized in that the lupus nephritis.
18. The method of claim 11,

    The health functional food is a health functional food for preventing or improving kidney disease, characterized in that the capsule, tablets, granules, powder or beverage form.
19. The method of claim 11,

    The health functional food is a functional health food for preventing or improving kidney disease, characterized in that to reduce the amount of proteinuria.
20. A method of preventing or treating kidney disease by administering thalidomide or lenalidomide to a mammal.
21. Use of thalidomide or lenalidomide to prevent or treat kidney disease.

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