SUSTAINED FORMULATION FOR PREVENTION OR TREATMENT OF AUTOIMMUNE DISEASE CONTAINING LOW-DOSE NALTREXONE AND METHOD USING THE SAME
20220370437 · 2022-11-24
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K9/5031
HUMAN NECESSITIES
A61P21/00
HUMAN NECESSITIES
A61K9/0021
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
A61K9/16
HUMAN NECESSITIES
Abstract
The present disclosure provides a sustained formulation for prevention or treatment of autoimmune disease, comprising microparticles comprising naltrexone or pharmaceutically acceptable salts thereof, and biodegradable polymers, and a method using the same. Accordingly, it may be used to prevent or treat autoimmune diseases for a prolonged period of time with a single administration.
Claims
1. A sustained formulation for prevention or treatment of autoimmune disease, comprising microparticles comprising naltrexone or pharmaceutically acceptable salts thereof, and biodegradable polymers.
2. The formulation of claim 1, wherein the biodegradable polymer comprises one or more selected from the group consisting of polylactide, polylactic acid, polylactide-co-glycolide, polylactic-co-glycolic acid, polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalate, polyhydroxybutyrate, and polyamino acids.
3. The formulation of claim 2, wherein the molar ratio of glycolide to lactide in the polylactide-co-glycolide is 50:50 to 90:10.
4. The formulation of claim 2, wherein the biodegradable polymer comprises one or more of polylactide and polylactide-co-glycolide.
5. The formulation of claim 2, wherein the biodegradable polymer comprises one or more polylactide and one or more of polylactide-co-glycolide.
6. The formulation of claim 5, wherein the weight ratio of polylactide and polylactide-co-glycolide is 1:10 to 10:1.
7. The formulation of claim 5, wherein the weight ratio of polylactide and polylactide-co-glycolide is 1:1 to 2:1.
8. The formulation of claim 2, wherein the biodegradable polymer comprises two or more of polylactide-co-glycolide.
9. The formulation of claim 8, wherein the two or more polylactide-co-glycolides are polylactide-co-glycolides in a weight ratio of 1:10 to 10:1.
10. The formulation of claim 9, wherein the two or more of polylactide-co-glycolides are polylactide-co-glycolides in a weight ratio of 1:1 to 2:1.
11. The formulation of claim 2, wherein an intrinsic viscosity (N) of the polylactide is 0.1 dl/g to 0.5 dl/g.
12. The formulation of claim 2, wherein an intrinsic viscosity of the polylactide-co-glycolide is 0.1 dl/g to 1.5 dl/g.
13. The formulation of claim 4, wherein an intrinsic viscosity of the polylactide is 0.1 dl/g to 0.5 dl/g.
14. The formulation of claim 4, wherein an intrinsic viscosity of the polylactide-co-glycolide is 0.1 dl/g to 1.0 dl/g.
15. The formulation of claim 1, wherein the end of the biodegradable polymer is capped or uncapped.
16. The formulation of claim 1, wherein the residual amount of the solvent in the microsphere is 1000 ppm or less.
17. The formulation of claim 16, wherein the residual amount of the solvent in the microsphere is 800 ppm or less.
18. The formulation of claim 16, wherein the solvent is dichloromethane.
19. The formulation of claim 1, wherein the biodegradable polymer is stirred with 200 to 400 rpm at 10° C. to 20° C. for 30 minutes to 2 hours, with 200 to 400 rpm at 25° C. to 35° C. for 30 minutes to 2 hours, and with 200 to 400 rpm at 35° C. to 45° C. for 30 minutes to 4 hours to remove the solvent.
20. The formulation of claim 1, wherein the microparticle comprises biodegradable polymers and naltrexone or a pharmaceutically acceptable salt thereof in a weight ratio of 1:1 to 10:1.
21. The formulation of claim 1, wherein a median particle size (D50) of the microparticles is 25 μm to 100 μm.
22. The formulation of claim 1, wherein naltrexone or a pharmaceutically acceptable salt thereof is homogenously dispersed in the microparticles.
23. The formulation of claim 1, wherein the formulation is a parenteral formulation.
24. The formulation of claim 1, wherein the formulation is an injection for subcutaneous administration or intramuscular administration.
25. The formulation of claim 1, wherein the formulation comprises 0.1 mg to 1 g of naltrexone or a pharmaceutically acceptable salt thereof per unit dosage form.
26. The formulation of claim 1, wherein the formulation comprises low-dose naltrexone or a pharmaceutically acceptable salt thereof.
27. The formulation of claim 1, wherein the formulation is administered once a day to once a year.
28. The formulation of claim 27, wherein the formulation is administered once every 1 week to 2 months.
29. The formulation of claim 1, wherein the formulation is for administration at a dose of 0.1 mg/kg body weight to 1 g/kg body weight.
30. The formulation of claim 1, wherein the autoimmune diseases are selected from the group consisting of rheumatoid arthritis, multiple sclerosis, hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi disease, vasculitis, adult onset Still's disease, inflammatory myositis, Behcet disease, lgG4-associated disease, Sjogren syndrome, Giant cell arteritis, Temporal arteritis, type 1 diabetes, atopic dermatitis, Crohn's disease, systemic sclerosis, psoriasis, Grave's hyperthyroidism, Hashimoto's disease, Pernicious anemia, Ankylosing spondylitis, Myasthenia, Vitiligo, Guillain-Barre syndrome, Glomerulonephritis, ANCA-associated vasculitis (AAV), Antiphospholipid syndrome, Pemphigus, cancer, Autoimmune hepatitis, Encephalomyelitis, Fibromyalgia, and Psoriatic arthritis.
Description
BRIEF DESCRIPTION OF DRAWINGS
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BEST MODE
[0114] Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present disclosure is not limited to these examples.
Example 1. Preparation of Injectable Composition Containing Low-Dose Sustained-Naltrexone
[0115] 1. Preparation of Naltrexone-Containing Microspheres
[0116] (1) Confirmation of Pharmacokinetic Profile According to Lactide Ratio and Intrinsic Viscosity
[0117] Differences in the release of active ingredients contained in microspheres depending on the type of polymers, the composition of polylactide and polylactide/glycolide copolymer, and the intrinsic viscosity (IV) of the polymer were confirmed by pharmacokinetic profiles.
[0118] For the preparation of the oily solution, naltrexone Base Anhydrous (manufactured by Mallinckrodt, hereinafter the same shall apply) and a polymer was prepared. As a polymer, PDLO2A, PDLG7510, PDLG7504A, PDLG7502A, and a combination thereof were used as shown in Table 1 below.
[0119] [Table 1]
TABLE-US-00001 TABLE 1 Intrinsic Molecular viscosity weight Polymer (dl/g) (kg/mol) Poly (DL-lactide) PDL02A 0.2 17 75/25 DL- PDLG7510 1.0 153 lactide/glycolide PDLG7504A 0.4 44 co-polymer PDLG7502A 0.2 17
[0120] A single polymer (Corbion) or a mixture thereof listed in Table 1 was dissolved in dichloromethane to prepare 18.29% (w/w) of a polymer solution. For the polymer mixture, a mixture of PDLG7510+ PDLG7502A (weight ratio 5:5), PDLO2A+ PDLG7502A (weight ratio 5:5), and PDLO2A+ PDLG7504A (weight ratio 5:5) was used. A drug solution of 29.41% (w/w) was prepared by dissolving naltrexone base in benzyl alcohol. The ratio of drug and polymer was 1:2, and the final oil phase solution was prepared by mixing and stirring the polymer solution and the drug solution.
[0121] As an aqueous solution, a 0.5% (w/v) of polyvinyl alcohol (PVA) solution was prepared.
[0122] A 100 μm of microchannel was assembled into a microsphere manufacturing module, and an oil phase solution and an aqueous phase solution were connected to an oil phase line and an aqueous phase line, respectively. A pressure of 400 mbar for the oil phase solution and 2500 mbar for aqueous phase solution was applied, and the oil phase solution and the aqueous phase solution were allowed to flow at a temperature of 17′C. Microspheres were to be prepared at the point where the flows of the oil phase solution and the aqueous phase solution met. After the preparation of microspheres was completed, the product was stirred at 300 rpm at 15° C. for 1 hour, 300 rpm at 30° C. for 2 hours, and 300 rpm at 40° C. for 3 hours to remove the solvent. Microspheres having a diameter between 25 μm and 63 μm were obtained by filtration through a sieve of 25 μm and 63 μm. The obtained microspheres were lyophilized then stored until use.
[0123] The prepared microspheres were dispersed in a diluent to prepare an injection formulation. The microspheres of the injection formulation were administered to the beagle dog once by subcutaneous injection at a dose of 190 mg/2 mL (based on naltrexone) (microspheres, 570 mg). The concentration (ng/mL) of naltrexone in the blood over the administration time (hour) was measured, and the results were shown in
[0124] In general, the degradation rate of PDLO2A-based microspheres is about 6 months to about 9 months, and the degradation rate of PDLG7502A-based microspheres is about 2 months to about 3 months. This means that the degradation rate of microspheres is decreased and the microspheres are maintained for a longer period in poly lactide compared to poly lactide-glycolide copolymer.
[0125] According to the results of
[0126] In consideration of the naltrexone release pattern, initial release, and maintenance period of microsphere according to the results of
[0127] (2) Change in Dichloromethane Residual Amount According to Stirring Conditions
[0128] Changes in the residual amount of dichloromethane (DCM) according to the stirring conditions during the preparation of microspheres were confirmed.
[0129] PDLG7510+ PDLG7502A (weight ratio 5:5) and PDLO2A+ PDLG7502A (weight ratio 5:5)-based microspheres were prepared by a method described in Example 1.1(2). The residual amount of dichloromethane according to the stirring conditions was shown in Table 2 below.
TABLE-US-00002 TABLE 2 PDLG7510 + PDL02A + Conditions PDLG7502A (5:5) PDLG7502A (5:5) Size of a particle 46.92 109.5 50.25 51.38 (μl)(X50) Amount in an 5000 5000 5000 5000 aqueous (Reservoir, mL) 15° C./rpm 1 H/150 1 H/300 1 H/300 1 H/300 30° C./rpm 2 H/300 3 H/600 2 H/300 2 H/300 40° C./rpm 3 H/600 — 1 H/300 3 H/300 Residual amount 644.6 530,027.1 7,596.3 619.0 (ppm) of DCM
[0130] As shown in Table 2, if PDLO2A+ PDLG7502A (5:5)-based microspheres were stirred at 300 rpm at 15° C. for 1 hour, at 300 rpm at 30° C. for 2 hours, and at 300 rpm at 40° C. for 3 hours, the residual amount of dichloromethane was the lowest.
[0131] 2. Preparation of Microsphere-Based Low-Dose Naltrezone Injection Composition and Confirmation of its Particle Size Distribution.
2-1. Preparation of Microsphere-Based Low-Dose Naltrexone Injection Composition Preparation Example 1
[0132] Microspheres containing low dose naltrexone were prepared according to the method described in Example 1.1.
[0133] Specifically, for the preparation of the oil phase solution, a polymer solution of 18.29% (w/w) was prepared by dissolving PDLO2A and PDLG7502A (5:5) mixture in dichloromethane. A drug solution of 29.41% (w/w) was prepared by dissolving naltrexone base in benzyl alcohol. The final oil phase solution was prepared by mixing and stirring the polymer solution and the drug solution, so that the ratio of drug and polymer was 1:2.
[0134] As an aqueous phase solution, a 0.5% (w/v) of polyvinyl alcohol (PVA) solution was prepared.
[0135] A 100 μm microchannel was assembled into a microsphere manufacturing module, and an oil phase solution and an aqueous phase solution were connected to an oily line and an aqueous line, respectively. A pressure of 400 mbar for the oil phase solution and 2500 mbar for aquious phase solution was applied, and the oil phase solution and the aqueous phase solution were allowed to flow at a temperature of 17° C. Microspheres were allowed to be prepared at the point where the flow of the oil phase solution and the flow of the aqueous phase solution met. After the preparation of microspheres was completed, the product was stirred at 300 rpm at 15° C. for 1 hour, 300 rpm at 30° C. for 2 hours, and 300 rpm at 40° C. for 3 hours to remove the solvent. Microspheres having a diameter between 25 μm and 63 μm were obtained by filtration through a sieve of 25 μm and 63 μm. The obtained microspheres were lyophilized and stored until use.
[0136] The prepared microspheres were confirmed with a scanning electron microscope (SEM), and the images were shown in
2-2. Preparation of Microsphere-Based Low-Dose Naltrexone Injection Composition (Preparation Example 2)
[0137] Microspheres containing low dose naltrexone were prepared according to the method described in Example 1.1.
[0138] Specifically, for the preparation of an oil phase solution, PDLG7504 was dissolved in ethyl acetate and benzyl alcohol, and a drug solution was prepared by dissolving naltrexone base. The final oil phase solution was prepared by mixing and stirring the polymer solution and the drug solution, so that the ratio of drug and polymer was 1:2.
[0139] As an aqueous phase solution, a 1.0% (w/v) of polyvinyl alcohol (PVA) solution was prepared.
[0140] A 100 μm of microchannel was assembled into a microsphere manufacturing module, and an oil phase solution and an aqueous phase solution were connected to an oily line and an aqueous line, respectively. A pressure of 400 mbar for the oil phase solution and 2500 mbar for aquious phase solution was applied, and the oil phase solution and the aqueous phase solution were allowed to flow at a temperature of 7° C. Microspheres were allowed to be prepared at the point where the flows of the oil phase solution and the aqueous phase solution met. After the preparation of the microspheres was completed, the solvent was removed by extracting the solvent from an aqueous ethanol solution at 10° C. for 10 hours. Microspheres having a diameter between 25 μm and 63 μm were obtained by filtration through a sieve of 25 μm and 63 μm. The obtained microspheres were lyophilized and stored until use.
2-3. Preparation of Microsphere-Based Low-Dose Naltrexone Injection Composition (Preparation Example 3)
[0141] Microspheres containing low dose naltrexone were prepared according to the method described in Example 1.1.
[0142] Specifically, for the preparation of an oily solution, PDLG7504 was dissolved in dichloromethane, and a drug solution was prepared by dissolving naltrexone base. The final oil phase solution was prepared by mixing and stirring the polymer solution and the drug solution, so that the ratio of drug and polymer was 1:2.
[0143] As an aqueous solution, a 0.5% (w/v) of polyvinyl alcohol (PVA) solution was prepared.
[0144] A 100 μm of microchannel was assembled into a microsphere manufacturing module, and an oil phase solution and an aqueous phase solution were connected to an oily line and an aqueous line, respectively. A pressure of 600 mbar for the oil phase solution and 3000 mbar for aquious phase solution was applied, and the oil phase solution and the aqueous phase solution allowed to flow at a temperature of 10° C. Microspheres allowed to be prepared at the point where the flows of the oil phase solution and the aqueous phase solution met. The product was stirred at 300 rpm at 10° C. for 1 hour, at 300 rpm at 30° C. for 2 hours, and at 300 rpm at 40′C for 3 hours to remove the solvent. Microspheres having a diameter between 25 μm and 63 μm were obtained by filtration through a sieve of 25 μm and 63 μm. The obtained microspheres were lyophilized and then stored until use.
2-4. Preparation of Microsphere-Based Low-Dose Naltrexone Injection Composition (Preparation Example 4)
[0145] Microspheres containing low dose naltrexone were prepared according to the method described in Example 1.1.
[0146] Specifically, for the preparation of an oily solution, PDLG7504 was dissolved in dichloromethane and diethylether, and a drug solution was prepared by dissolving naltrexone base. The final oil phase solution was prepared by mixing and stirring the polymer solution and the drug solution, so that the ratio of drug and polymer was 1:2.
[0147] As an aqueous solution, a 0.5°% (w/v) of polyvinyl alcohol (PVA) solution was prepared.
[0148] A 100 μm of microchannel was assembled into a microsphere manufacturing module, and an oil phase solution and an aqueous phase solution were connected to an oily line and an aqueous line, respectively. A pressure of 600 mbar for the oil phase solution and 3000 mbar for the aqueous phase solution was applied, and the oil phase solution and the aqueous phase solution were allowed to flow at a temperature of 10′C. Microspheres were allowed to be prepared at the point where the flows of the oil phase solution and the aqueous phase solution met. The product was stirred at 300 rpm at 10° C. for 1 hour, at 300 rpm at 30° C. for 2 hours, and at 300 rpm at 40′C for 3 hours to remove the solvent. Microspheres having a diameter between 25 μm and 63 μm were obtained by filtration through a sieve of 25 μm and 63 μm. The obtained microspheres were lyophilized and stored until use.
[0149] The prepared microspheres were confirmed with a scanning electron microscope (SEM), and the images were shown in
[0150] 3. Pharmacokinetic Properties of Naltrexone-Containing Microspheres
[0151] The microspheres prepared as described in Example 1.2-1 (Preparation Example 1) were dispersed in a diluent to prepare an injection formulation. The microspheres of the injection formulation were administered to the beagle dog once by subcutaneous injection at a dose of 190 mg/2 mL (naltrexone base) (microspheres, 570 mg). The concentration (ng/mL) of naltrexone in the blood over the administration time (hour) was measured, and the results were shown in FIG. 3A.
[0152] The microspheres of the injection formulation were administered once by intramuscular injection at a dose of 300 mg/4 mL (based on naltrexone) (microspheres, 900 mg). Blood of beagle dogs was obtained regularly until 31 days after administration. The concentration (ng/mL) of naltrexone in the blood according to the administration time (hour) was measured, and the results were shown in
[0153] As shown in
[0154] Further, the microspheres prepared as described in Example 1.2-4 (Preparation Example 4) were dispersed in a diluent to prepare an injection formulation. The microspheres of the injection formulation were administered to the beagle dog once by intramuscular injection at a dose of 300 mg/head (based on naltrexone) (microspheres, 900 mg). The concentration (ng/mL) of naltrexone in the blood over the administration time (hour) was measured, and the results were shown in
[0155] As shown in
Example 2. Efficacy Evaluation of Naltrexone-Containing Microspheres for Rheumatoid Arthritis (First)
[0156] 1. In Vivo Testing Method of Naltrexone-Containing Microspheres for Rheumatoid Arthritis
[0157] In vivo efficacy was evaluated to determine whether naltrexone-containing microspheres, i.e., the naltrexone drug delivery systems, have therapeutic efficacy for rheumatoid arthritis.
[0158] As a model of murine collagen-induced arthritis (CIA), 6 to 10 week-old male mice of DBA/1 J strain was prepared. Mice were kept and tested in a specific pathogen free (SPF) laboratory under an environment of a temperature of 21° C. to 23° C. and a relative humidity of 40% to 45%. The number of experimental animals per cage was kept under 6, and a cage was exchanged twice a week and feed was supplied.
[0159] A 2 mg/mL collagen solution was prepared by dissolving bovine type 2 collagen in 10 mM acetic acid. After emulsifying by mixing 2 mg/mL of Complete Freund's adjuvant and collagen solution in a 1:1 (v/v) ratio, it was intradermally injected into the tail of mouse at a dose of 100 μl/animal (first immunization, day 0). After emulsifying by mixing incomplete Freund's adjuvant (WA) and collagen solution in a 1:1 (v/v) ratio on the 21.sup.th day, it was intradermally injected into the tail of mouse at a dose of 100 μl/animal (the secondary immunization, on the 21.sup.th day). After that, mice were randomly divided into groups and assigned to experimental groups.
[0160] As a material to be tested, naltrexone DDS prepared as described in Preparation Example 1 was prepared. Since the ratio of polymer to active ingredient in naltrexone DDS is about 1:2, 9 mg of microspheres contains about 3 mg of naltrexone as an active ingredient. As a negative control, DDS containing a carrier was used instead of naltrexone, and as a positive control, methotrexate (MTX, MTX injection, 50 mg/2 mL, JW Pharmaceutical) and naltrexone (Revia Tablet) were used. The refrigerated test substance was standed at room temperature 1 hour before administration and dissolved in 0.15 mL of water for injection. 0.15 mL of the test substance was filled using a syringe with a 21 G needle. The needle was changed to 23 G within 2 minutes after filling and the mice were injected.
[0161] Administration information for each administration group is as described in Table 3 below.
TABLE-US-00003 TABLE 3 Admin- Regimen istration and dose of group Route administration Admin- (Each Admin- of (Baseline of istration group, istration admin- the active volume n = 6) drug istration ingredient) (μl) 1 Carrier- Sub- one dose on the 150 (Negative containing cutaneous 23.sup.th day, N/A control) DDS injection 2(Positive metho- intra- Twice/week, 1 100 control) trexate peritoneal mg/kg body injection weight/each time 3 (Positive Naltrexone Oral Once/day, 10 200 control) mg/kg body weight/each time 4 Naltrexone Sub- Administration 150 (Test DDS cutaneously once on the 23.sup.th group) day, 3 mg/mouse
[0162] Based on the blind evaluation data for each evaluation index, statistical analysis between the negative control group and the test group or between the two test groups was performed using SPSS. Student's t-test was used for comparison between the two groups. To compare differences between treatment groups at multiple time points, repeated measures ANOVA with Turkey's post-hoc test was used. The significance level was defined as a p-value of 0.05 or less.
[0163] 2. Clinical Evaluation of Arthritis Activity
[0164] After administration of the test substance to the collagen-induced arthritis mouse model, the occurrence and level of inflammation were regularly observed from the date of group separation to the end of the experiment (the 41.sup.th day). Inflammation level was given a score of 0 to 4 for each paw according to the criteria in Table 4 below, and the sum was used as a clinical arthritis index (CAI).
TABLE-US-00004 TABLE 4 Score Symptom 0 Asymptomatic 1 Inflammation on one toe 2 Inflammation on two toes 3 Inflammation on three or more toes and sole 4 Inflammation on all toes and sole
[0165] When the clinical index of each paw was 2 score or higher, it was determined that arthritis occurred, and the incidence was defined as 100% when arthritis occurred in four toes.
[0166] Toe images of normal mouse to which the test substance was not administered, and each administration group was shown in
TABLE-US-00005 TABLE 5 CAI CAI-AUC p-value p-value vs vs Group Mean SEM Vehicle Mean SEM Vehicle Vehicle DDS 10.75 2.73 — 120.3 27.9 — Methotrexate (1 mg/kg) 5.92 2.15 0.0057 46.6 13.8 0.0002 Naltrexone (10 mg/kg) 8.25 4.06 0.0572 59.0 32.5 0.0057 Naltrexone DDS 6.25 2.46 0.0064 46.8 16.5 0.0002 (3 mg/mouse)
[0167] As shown in
[0168] The frequency (%) of arthritis over the time (days) after administration of the test substance was shown in
TABLE-US-00006 TABLE 6 Incidence (%) Incidence-AUC Group Mean SEM p-value Mean SEM p-value Vehicle DDS 70.83 24.58 — 714.6 28.3 — Methotrexate (1 mg/kg) 20.83 18.82 0.0120 185.4 169.8 0.0013 Naltrexone (10 mg/kg) 41.67 34.16 0.0813 320.8 377.5 0.0561 Naltrexone DDS 25.00 22.36 0.0159 204.2 229.8 0.0036 (3 mg/mouse)
[0169] As shown in
[0170] Therefore, it was confirmed that naltrexone DDS showed superior therapeutic efficacy compared to naltrexone for oral administration, and showed an effect similar to that of methotrexate, which is a standard treatment used in clinical practice.
[0171] 3. Histological Estimation
[0172] Mice were sacrificed on the 41.sup.th day, which is the end of the experiment, and the tissues of the hind paws were stained with hematoxylin/eosin. Hematoxylin/eosin staining was used to evaluate the activity of inflammation in the arthritic tissue, and toluidine blue staining was performed to confirm the histological therapeutic effect on cartilage destruction.
[0173] (1) Hematoxylin/Eosin Staining
[0174] The hind paw tissue of the mouse was stained with hematoxylin/eosin, and 4 sites (100 magnification) for each tissue were photographed. Two or more investigators evaluated the items of synovial hyperplasia, pannus formation, cartilage destruction, and bone erosion for hematoxylin/eosin staining. The scoring criteria for each item were described in Table 7, and the average score of 4 sites was calculated as the score of each tissue.
TABLE-US-00007 TABLE 7 Synovial Pannus Cartilage Bone Score hyperplasia formation destruction erosion 0 None None None None 1 Very few Pannus Catilage Bone inflammatory cells formation surface is surface is not are found in the is not not smooth. smooth synovium Mild distinct Focal erosion hyperplastic of cartilage synovium surface region 2 Inflammatory Pannus is Catilage Cell invasion cellular pro- weakly surface in bone is liferation is well invaded in takes a Found. Marked marked leading the bone corrugated loss of bone to thichkened shape surface synovium integrity 3 Extensive Pannus is More than Bone proliferation of strongly 50% of conformation inflammatory cell invaded in cartilage of is almost along with severely the bone either joint disrupted thickening of counterpart is synovium destroyed
[0175] The image of the tissue stained with hematoxylin/eosin was shown in
TABLE-US-00008 TABLE 8 Naltrexone MTX Naltrexone DDS Histochromatographic Vehicle (1 (10 (3 mg/ parameters DDS mg/kg) mg/kg) mouse) Synovial Mean 3.00 0.91 1.27 0.98 hyperplasia SEM 0.00 0.28 0.74 0.29 p-value vs — <0.0001 0.0045 <0.0001 Vehicle Pannus Mean 2.89 0.14 0.58 0.30 formation SEM 0.10 0.21 0.77 0.49 p-value vs — <0.0001 0.0014 0.0001 Vehicle Cartilage Mean 2.61 0.50 0.98 0.66 destruction SEM 0.34 0.18 0.72 0.31 p-value vs — <0.0001 0.0052 <0.0001 Vehicle Bone Mean 2.89 0.08 0.58 0.28 erosion SEM 0.10 0.11 0.77 0.49 p-value vs — <0.0001 0.0014 0.0001 Vehicle
[0176] As shown in
[0177] (2) Toluidine Blue Staining
[0178] The hind paw tissue of the mouse was stained with toluidine blue, and 4 sites (100 magnification) for each tissue were photographed. Two or more investigators evaluated the items of matrix staining, surface regularity, and cartilage thickness. The scoring criteria for each item was described in Table 9, and the average score of 4 sites was calculated as the score of each
TABLE-US-00009 TABLE 9 Score Matrix staining Surface regularity Cartilage thickness 0 Normal Smooth; 75% to 100% >2/3 depth relative to av. GC* depth 1 Slightly Moderate; 50% to 75% 1/2 to 2/3 depth reduced relative to av. GC 2 Markedly Irregular; <50% <1/2 depth relative reduced to av. GC 3 Not staining Severely irregular
[0179] The image of the tissue stained with toluidine blue was shown in
TABLE-US-00010 TABLE 10 Histological MTX Naltrexone Naltrexone Scoring Vehicle (1 (10 DDS (3 parameters DDS mg/kg) mg/kg) mg/mouse) Matrix Mean 2.97 0.61 0.92 0.59 staining SEM 0.09 0.39 0.70 0.65 p-value vs — <0.0001 0.0015 0.0003 Vehicle Surface Mean 2.78 0.34 0.80 0.44 regularity SEM 0.13 0.13 0.74 0.38 p-value vs — <0.0001 0.0024 <0.0001 Vehicle Cartilage Mean 1.78 0.11 0.39 0.16 thickness SEM 0.11 0.12 0.51 0.20 p-value vs — <0.0001 0.0024 <0.0001 V ehicle
[0180] As shown in
[0181] Taken together with the histological estimation results, the naltrexone DDS administration group showed a level of inhibitory effect similar to the methotrexate-administered group as the positive control in the evaluation of the activity of inflammation of the arthritis tissues and the severity of joint damage, and showed a more consistent inhibitory effect compared with the oral administration group of naltrexone. Therefore, naltrexone DDS was histologically confirmed to have an arthritis treatment effect.
[0182] 4. Safety Assessment In Vivo
[0183] The in vivo safety of the test substance was evaluated by measuring the body weight of mouse in each administration group of Example 2.1.
[0184] For the safety evaluation of the test substance, the body weight of mouse was measured daily from before the start of administration (The 21t day) to just before the end of administration (The 41t day), and the body weight (%) of the mouse for each administration group over the time (day) was shown in
[0185] As shown in
Example 3. Efficacy Evaluation of Naltrexone-Containing Microspheres for Rheumatoid Arthritis (Secondary)
[0186] 1. In Vivo Testing Method of Naltrexone-Containing Microspheres for Rheumatoid Arthritis
[0187] Following the in vivo efficacy evaluation of naltrexone-containing microspheres for rheumatoid arthritis in Example 2, an additional efficacy experiment was conducted by giving a difference between the dose and the combination administration of methotrexate and the control group.
[0188] A model of murine collagen-induced arthritis prepared in the same manner as in Example 2 (6-10 week-old male mouse of DBA/1J strain; the first immunization on day 0 and secondary immunization on day 21) was used.
[0189] As a material to be tested, naltrexone DDS (“Q-DDS”) was prepared as described in Preparation Example 1. As a negative control, vehicle DDS was used instead of naltrexone, and as a positive control, methotrexate (MTX, MTX injection, 50 mg/2 mL, JW Pharmaceutical) and Tofacitinib (Xeljanz® Tablet, 5 mg, Pfizer Korea) were used.
[0190] Administration information for each administration group is as described in Table 11 below.
TABLE-US-00011 TABLE 11 Admin- Regimen and istration dose of group Route administration Admin- (Each Admin- of (based on istration group, istration admin- the active volume n = 6) drug istration ingredient) (μl) 1 Vehicle Subcutaneous 1 dose on the 23.sup.th 140 (Negative DDS injection day, N/A control) 2 Metho- Intraperitoneal Twice/week, 1 100 (Positive trexate injection mg/kg body control) (MTX) weight/each time 3 Tofacitinib Oral Once/day, 10 200 (Positive (Xeljanz ®) mg/kg body control) weight/each time 4 Q-DDS Subcutaneous Administration 50 (Test injection once on group) the 23.sup.th day, 1 mg/mouse 5 Q-DDS Subcutaneous Administration 160 (Test injection once on group) the 23.sup.th day, 3 mg/mouse 6 Q-DDS Subcutaneous Administration 260 (Test injection once on group) the 23.sup.th day, 5 mg/mouse 7 Q-DDS Subcutaneous Administration 500 (Test injection once on group) the 23.sup.th day, 10 mg/mouse 8 Q-DDS Subcutaneous Administration 1000 (Test injection once on group) the 23.sup.th day, 20 mg/mouse 9 MTX + Intraperitoneal + MTX: 160 (Test Q-DDS subcutaneous Twice/week, group) injection 1 mg/kg body weight/each time Q-DDS: Administration once on the 23.sup.th day, 3 mg/mouse
[0191] Based on the blind evaluation data for each evaluation index, statistical analysis between the negative control group and the test group or between the two test groups was performed using SPSS. For comparison between the two groups, Student's t-test or Mann Whitney U test was used. To compare differences between treatment groups at multiple time points, repeated measures ANOVA with Turkey's post-hoc test was used. The significance level was defined as a p-value of 0.05 or less.
[0192] 2. Clinical Evaluation of Arthritis Activity
[0193] After administration of the test substance to the collagen-induced arthritis mouse model, the occurrence and level of inflammation were regularly observed from the date of group separation to the end date of the experiment (the 41.sup.th day). Inflammation level was given a score of 0 to 4 for each paw according to the criteria in Table 4 above, and the sum was used as a clinical arthritis index (CAI).
[0194] When the clinical index of each paw was 2 score or higher, it was determined that arthritis occurred, and the incidence was defined as 100% when arthritis occurred in four toes.
[0195] Toe Images for each administration group were shown in
TABLE-US-00012 TABLE 12 CAI (D41) CAI-AUC P value vs P value vs Group Mean SEM Vehicle Mean SEM Vehicle Vehicle DDS 11.75 1.20 — 150.65 12.03 — Methotrexate (1 mg/kg) 6.35 1.05 <0.0001 71.20 8.19 <0.0001 Xeljanz ® (10 mg/kg) 6.25 0.75 <0.0001 74.50 9.63 <0.0001 Q-DDS (1 mg/mouse) 7.30 1.28 <0.0001 91.13 16.43 <0.0001 Q-DDS (3 mg/mouse) 7.60 1.69 <0.0001 88.05 14.86 <0.0001 Q-DDS (5 mg/mouse) 6.30 1.32 <0.0001 73.30 10.77 <0.0001 Q-DDS (10 mg/mouse) 5.25 0.69 <0.0001 66.08 8.09 <0.0001 Q-DDS (20 mg/mouse) 6.85 1.30 <0.0001 78.63 11.93 <0.0001 Methotrexate (1 mg/kg) + 5.95 1.30 <0.0001 63.60 10.75 <0.0001 Q-DDS (3 mg/mouse)
[0196] As shown in
[0197] The incidence (%) of arthritis over the time (days) after administration of the test substance was shown in
TABLE-US-00013 TABLE 13 Incidence (%) Incidence-AUC P value vs P value vs Group Mean SEM Vehicle Mean SEM Vehicle Vehicle DDS 70.0 9.9 — 835.0 89.2 — Methotrexate (1 mg/kg) 30.0 11.5 <0.0001 170.0 77.1 <0.0001 Xeljanz ® (10 mg/kg) 27.5 9.2 <0.0001 263.8 122.7 <0.0001 Q-DDS (1 mg/mouse) 35.0 12.1 0.0002 337.5 185.4 0.0012 Q-DDS (3 mg/mouse) 35.6 14.7 0.0001 307.5 103.3 <0.0001 Q-DDS (5 mg/mouse) 22.5 12.4 <0.0001 196.3 121.3 <0.0001 Q-DDS (10 mg/mouse) 12.5 6.6 <0.0001 131.3 71.9 <0.0001 Q-DDS (20 mg/mouse) 22.5 12.4 <0.0001 216.3 112.1 <0.0001 Methotrexate (1 mg/kg) + 25.0 11.8 <0.0001 167.5 90.9 <0.0001 Q-DDS (3 mg/mouse)
[0198] As shown in
[0199] In addition, since the group administered with methotrexate and naltrexone DDS 3 mg/mouse (combination test group) had a lower incidence of arthritis than the group administered with methotrexate alone or naltrexone DDS 3 mg/mouse, a synergistic effect could be confirmed.
[0200] 3. Histological Estimation
[0201] Mice were sacrificed on the 41.sup.th day, which is the end of the experiment, and the tissues of the hind paws were stained with hematoxylin/eosin. Hematoxylin/eosin staining was used to evaluate the activity of inflammation in the arthritic tissue, and toluidine blue staining was performed to confirm the histological therapeutic effect on cartilage destruction.
[0202] (1) Hematoxylin/Eosin Staining
[0203] Hematoxylin/eosin staining was performed and evaluated in the same manner as in Example 2.3. (1) above, and the score for items of synovial hyperplasia, pannus formation, cartilage destruction, and bone erosion was calculated by scoring each tissue as the average of the score of 4 sites based on Table 7 above.
[0204] Images for tissues stained with hematoxylin/eosin were shown in
TABLE-US-00014 TABLE 14 p-value Histological Scorning vs vs vs Value Mean SEM Vehicle MTX Xeljanz ® Synovial Negative control 3.00 0.00 — <0.0001 0.0012 hyperplasia (Vehicle DDS) MTX 1.38 0.29 <0.0001 — 0.6489 Xeljanz ® 1.56 0.39 0.0012 0.6489 — Q-DDS 1.98 0.54 0.0441 0.2564 0.4634 (1 mg/mouse) Q-DDS 2.00 0.43 0.0183 0.1737 0.3825 (3 mg/mouse) Q-DDS 0.98 0.27 <0.0001 0.2485 0.1658 (5 mg/mouse) Q-DDS 0.63 0.10 <0.0001 0.0127 0.0175 (10 mg/mouse) Q-DDS 1.58 0.56 0.0108 0.6942 0.9720 (20 mg/mouse) MTX + Q-DDS 0.79 0.18 <0.0001 0.0599 0.0529 (3 mg/mouse) Pannus Negative control 3.00 0.00 — <0.0001 0.0004 formation (Vehicle DDS) MTX 0.92 0.36 <0.0001 — 0.6608 Xeljanz ® 1.13 0.44 0.0004 0.6608 — Q-DDS 1.58 0.67 0.0273 0.3075 0.5004 (1 mg/mouse) Q-DDS 1.58 0.59 0.0147 0.2627 0.4629 (3 mg/mouse) Q-DDS 0.21 0.14 <0.0001 0.0489 0.0358 (5 mg/mouse) Q-DDS 0.06 0.05 <0.0001 0.0165 0.0150 (10 mg/mouse) Q-DDS 1.17 0.68 0.0079 0.6985 0.9529 (20 mg/mouse) MTX + Q-DDS 0.06 0.08 <0.0001 0.0175 0.0156 (3 mg/mouse) Cartilage Negative control 2.98 0.03 — <0.0001 0.0004 destruction (Vehicle DDS) MTX 1.02 0.30 <0.0001 — 0.3907 Xeljanz ® 1.38 0.38 0.0004 0.3907 Q-DDS 1.71 0.56 0.0199 0.2157 0.5592 (1 mg/mouse) Q-DDS 1.75 0.45 0.0071 0.1294 0.4519 (3 mg/mouse) Q-DDS 0.65 0.20 <0.0001 0.2356 0.0627 (5 mg/mouse) Q-DDS 0.42 0.06 <0.0001 0.0380 0.0117 (10 mg/mouse) Q-DDS 1.38 0.51 0.0033 0.4839 0.9975 (20 mg/mouse) MTX + Q-DDS 0.46 0.14 <0.0001 0.0654 0.0187 (3 mg/mouse) Bone Negative control 3.00 0.00 — <0.0001 0.0004 erosion (Vehicle DDS) MTX 0.92 0.36 <0.0001 — 0.6608 Xeljanz ® 1.13 0.44 0.0004 0.6608 — Q-DDS 1.58 0.678 0.0273 0.3075 0.5004 (1 mg/mouse) Q-DDS 1.58 0.59 0.0147 0.2627 0.4629 (3 mg/mouse) Q-DDS 0.19 0.15 <0.0001 0.0445 0.0330 (5 mg/mouse) Q-DDS 0.04 0.05 <0.0001 0.0145 0.135 (10 mg/mouse) Q-DDS 1.17 0.68 0.079 0.6985 0.9529 (20 mg/mouse) MTX + Q-DDS 0.06 0.08 <0.0001 0.0175 0.0156 (3 mg/mouse)
[0205] As shown in
[0206] (2) Toluidine Blue Staining
[0207] In the same manner as in Example 2.3. (2), the hind paw tissue of the mouse was stained with toluidine blue, and items such as photographing and matrix staining, surface regularity, and cartilage thickness were evaluated. The scoring criteria for each item were described in Table 9, and the average score of 4 sites was calculated as the score of each tissue.
[0208] Images for tissues stained with toluidine blue were shown in
TABLE-US-00015 TABLE 15 p-value Histochromatographic vs vs vs parameters Mean SEM Vehicle MTX Xeljanz ® Matrix Negative control 3.00 0.00 — <0.0001 <0.0001 staining (Vehicle DDS) MTX 1.15 0.16 <0.0001 — 0.2676 Xeljanz ® 1.42 0.23 <0.0001 0.2676 — Q-DDS 1.92 0.36 0.0040 0.0366 0.1814 (1 mg/mouse) Q-DDS 1.96 0.45 0.0177 0.0639 0.2198 (3 mg/mouse) Q-DDS 0.92 0.18 <0.0001 0.2767 0.0659 (5 mg/mouse) Q-DDS 0.63 0.06 <0.0001 0.0037 0.0023 (10 mg/mouse) Q-DDS 1.46 0.60 0.0104 0.5517 0.9384 (20 mg/mouse) MTX + Q-DDS 0.83 0.09 <0.0001 0.0612 0.0163 (3 mg/mouse) Surface Negative control 3.00 0.00 — <0.0001 <0.0001 regularity (Vehicle DDS) MTX 1.02 6.19 <0.0001 — 0.6652 Xeljanz ® 1.13 0.22 <0.0001 0.6652 Q-DDS 1.75 0.34 0.0011 0.0442 0.0868 (1 mg/mouse) Q-DDS 1.83 0.49 0.0159 0.0886 0.1383 (3 mg/mouse) Q-DDS 0.56 0.24 <0.0001 0.0946 0.0587 (5 mg/mouse) Q-DDS 0.35 0.10 <0.0001 0.0032 0.0027 (10 mg/mouse) Q-DDS 1.15 0.68 0.0074 0.8321 0.9721 (20 mg/mouse) MTX + Q-DDS 0.46 0.14 <0.0001 0.0147 0.0101 (3 mg/mouse) Cartilage Negative control 1.96 0.05 — <0.0001 <0.0001 thickness (Vehicle DDS) MTX 0.38 0.15 <0.0001 — 0.3341 Xeljanz ® 0.56 6.17 <0.0001 0.3341 — Q-DDS 1.04 0.24 0.0009 0.0150 0.0719 (1 mg/mouse) Q-DDS 1.10 0.39 0.0245 0.0589 0.1521 (3 mg/mouse) Q-DDS 0.21 0.12 <0.0001 0.3035 0.0625 (5 mg/mouse) Q-DDS 0.10 0.06 <0.0001 0.0652 0.0115 (10 mg/mouse) Q-DDS 0.67 0.47 0.0075 0.4855 0.8040 (20 mg/mouse) MTX + Q-DDS 0.19 0.09 <0.0001 0.2094 0.0375 (3 mg/mouse)
[0209] As shown in
[0210] Taken together with the histological estimation results, naltrexone DDS 5 mg/mouse administration group and 10 mg/mouse administration group, and combination administration group not only had an anti-inflammatory effect compared to the negative control group in the evaluation of the activity of inflammation of arthritic tissues and the severity of joint damage but also showed a more effective arthritis inhibitory effect than the positive control group, such as the standard antirheumatic drugs methotrexate or Xeljanz® administration group. Therefore, it was confirmed that naltrexone DDS is histologically effective for treating arthritis, and when administered in an appropriate amount, it has a superior effect to existing standard antirheumatic agents.
[0211] 4. Safety Assessment In Vivo
[0212] The in vivo safety of the test substance was evaluated by measuring the body weight of mouse in each administration group of Example 3.1.
[0213] For the safety evaluation of the test substance, the body weight of mouse was measured daily from before the start of administration (The 21.sup.th day) to just before the end of administration (The 41.sup.th day), and the body weight (%) of the mouse for each administration group over the time (day) was shown in
TABLE-US-00016 TABLE 16 D23 body D41 body Change of body weight Change of body weight weight (%, D23 body weight 100%) weight Mean SEM Mean SEM Mean SEM Negative control 22.6 0.3 20.5 1.0 90.8 3.7 (Vehicle DDS) MTX 22.7 6.5 23.8 1.2 104.7 3.5 Xeljanz ® 21.7 0.5 21.6 0.8 99.6 2.5 Q-DDS 21.4 0.6 20.7 0.9 96.9 3.6 (1 mg/mouse) Q-DDS 22.2 0.9 21.7 0.6 98.2 4.0 (3 mg/mouse) Q-DDS 22.8 0.6 22.5 1.3 98.5 4.3 (5 mg/mouse) Q-DDS 22.6 0.5 22.5 0.8 99.6 2.0 (10 mg/mouse) Q-DDS 22.7 0.7 23.4 1.1 103.2 4.3 (20 mg/mouse) MTX + Q-DDS 22.2 0.5 22.3 1.0 100.3 2.8 (3 mg/mouse)
[0214] As shown in
[0215] 5. Imaging Evaluation of Arthritis Using Micro-CT
[0216] After treatment with the test substance in Example 3.1, and fixing the paw tissue representing the results of each group, micro-CT scanners (Quantum FX, Perkin Elmer, MA) were used to photograph it. The radiographs were 3D rendered using the Quantum FX μCT imaging system (Perkin Elmer, MA), and then the radiological score was evaluated, and the bone volume ratio (Bone volume [BV]/Tissue volume [Tissue volume; TV]) and bone surface density (Bone surface area (BS)/Bone volume (BV)) were also measured. Two or more investigators performed blind evaluation for joint destruction, and the indicators in Table 17 below were used as the scoring criteria. The average value of the researchers' blind evaluation data was used as the score, and the average score of the paw tissue was calculated as the score of each group.
TABLE-US-00017 TABLE 17 score Joint destruction 0 No damage 1 Minor bone destruction observed in one enlightened spot 2 Moderate change, 2-4 spots in one area 3 Marded change, 2-4 spots in more area 4 Severe erosion afflicting the joint 5 Complete destruction of the joints
[0217] Representative images for each group photographed with micro-CT were shown in FIG. 11A, and in particular, images magnifying joint portions were shown in
TABLE-US-00018 TABLE 18 p-value Radiological analysis vs vs vs value Mean SEM Vehicle MTX Xeljanz ® Bone Negative control 13.50 1.06 — 0.0002 0.0097 volume/ group tissue (Vehicle DDS) volume MTX 31.25 0.62 0.0002 — 0.0624 (%) Xeljanz ® 25.27 1.92 0.0097 0.0624 — Q-DDS 28.46 2.92 0.0141 0.4652 0.4730 (1 mg/mouse) Q-DDS 23.57 4.18 0.1131 0.1906 0.7653 (3 mg/mouse) Q-DDS 32.24 0.27 0.0001 0.2726 0.0358 (5 mg/mouse) Q-DDS 35.82 1.09 0.0002 0.0341 0.0144 (10 mg/mouse) Q-DDS 26.81 6.10 0.1363 0.5654 0.8444 (20 mg/mouse) MTX + Q-DDS 31.93 0.16 0.0001 0.4119 0.0403 (3 mg/mouse) Bone Negative control 9.00 0.23 — 0.0004 0.0011 surface group area/bone (Vehicle DDS) volume MTX 6.12 0.06 0.0004 — 0.0703 (mm.sup.−1) Xeljanz ® 6.52 0.13 0.0011 0.0703 — Q-DDS 6.23 0.14 0.0008 0.5389 0.2600 (1 mg/mouse) Q-DDS 6.84 0.47 0.0233 0.2578 0.5947 (3 mg/mouse) Q-DDS 6.09 0.06 0.0004 0.8030 0.0587 (5 mg/mouse) Q-DDS 6.12 0.04 0.0004 0.9423 0.0639 (10 mg/mouse) Q-DDS 6.75 0.45 0.0180 0.2981 0.6934 (20 mg/mouse) MTX + Q-DDS 6.14 0.03 0.0004 0.7847 0.0673 (3 mg/mouse) Radiological Negative control 4.67 0.41 — 0.0002 0.0010 score group (Vehicle DDS) MTX 1.67 0.49 0.0002 — 0.5078 Xeljanz ® 2.08 0.56 0.0010 0.5078 — Q-DDS 2.33 0.66 0.0044 0.3466 0.7314 (1 mg/mouse) Q-DDS 2.33 0.72 0.0062 0.3705 0.7433 (3 mg/mouse) Q-DDS 1.42 0.33 <0.0001 0.6171 0.2368 (5 mg/mouse) Q-DD 1.08 0.40 <0.0001 0.2862 0.1046 (10 mg/mouse) Q-DDS 1.83 0.61 0.0008 0.7989 0.7176 (20 mg/mouse) MTX + Q-DDS 1.08 0.33 <0.0001 0.2563 0.0884 (3 mg/mouse)
[0218] As noted in
[0219] As a result of measurement of bone volume ratio, the naltrexone DDS 10 mg/mouse administration group showed a higher value than the negative control group (Vehicle DDS vs Q-DDS 10 mg/mouse, 13.50%±1.06% vs 35.82%±1.09%, p<0.001) and showed increased values compared to the positive control group (MTX vs Xeljanz(10 vs Q-DDS 10 mg/mouse, 31.25%±0.62% vs 25.27%±1.92% vs 35.82%±1.09%, p<0.05). As a result of measurement of bone surface density, it was lower in all treatment groups compared to the negative control group, and in particular, in the naltrexone DDS 5 mg/mouse administration group, 10 mg/mouse administration group, and the combination administration group, the MTX administration group, it was similar to the normal joint at a level similar to that of the MTX administration group among the positive control groups (Vehicle DDS vs Q-DDS 5 mg/mouse vs Q-DDS 10 mg/mouse vs MTX+Q-DDS, 9.00 mm.sup.−1±0.23 mm.sup.−1 vs 6.09 mm.sup.−1±0.06 mm.sup.−1 vs 6.12 mm.sup.−1±0.04 mm.sup.−1 vs 6.14 mm.sup.−1±0.03 mm.sup.−1, p<0.001), and showed lower level compared to Xeljanz® administration group. As a result of radiographic score for joint destruction, when compared with the negative control group, the radiographic score was decreased in the naltrexone DDS administration group including the positive control group, and showed a decrease in radiological score compared to the positive control group, in particular, in the naltrexone DDS administration group, 5 mg/mouse administration group and 10 mg/mouse administration group and a combination administration group. In the end, it was confirmed that naltrexone DDS not only treats the inflammation of arthritis, but also inhibits the progression of joint destruction as a result.
[0220] 6. Multiplex Protein Immunoassay on Arthritic Tissue and Serum
[0221] In order to evaluate the inflammatory activity and confirm the mode of action of the test substance through quantitative analysis of inflammatory substances in mouse arthritis tissue and blood, inflammatory mediators were investigated at the protein level. In order to overcome the limitation of the amount of sample that can be extracted for protein analysis, a multi-analyte-ELISA (multi-analyte-ELISA) method based on Luminex technology was used to analyze the largest amount of protein in a small sample.
[0222] (1) Biomarker Evaluation Through Analysis of Protein Concentration of Inflammation Mediators in Arthritic Tissues
[0223] The mouse joint tissue obtained after treatment with the test substance in the method of Example 3.1 was pulverized through ceramic beads, and all proteins were extracted using a lysis buffer (Cat. 1713040111, Bio-Rad). Using Pro-reagent kit V, the same amount of protein (2.7 mg/mL) for each administration group was diluted ¼ and reacted with the target-binding beads in a culture dish for 30 minutes. After washing with a wash buffer, it was reacted with the target-measurement antibody for 30 minutes, and after washing with a wash buffer again, it was reacted with streptavidin-PE for 10 minutes. Thereafter, the target protein is quantitatively measured for each administration group using the Bio-Plex® 200 system (Bio-Rad). The target-binding beads were composed of a set that can be combined as needed from Bio-Plex Mouse cytokine 4-plex (IL-1β, IL-6, IL-17A, TNF-α; Cat. 171G5002M), Bio-Plex Pro Mouse Cytokine 1-plex (MCP1; Cat. 171G5019M), Bio-Plex Pro Mouse Cytokine II 1-plex (MIP2; Cat 171G6006M), and Bio-Plex Pro Mouse Cytokine 1-plea (IL-6; Cat. 171 G5007M) (all products are from Bio Rad) and were used for measurement.
[0224] For the measurement result, paw tissue weight of the mouse was corrected for the quantified target protein. For each target joint, the hind paw was mainly used, and in order to overcome the limitation of the amount of protein extracted from the joint tissue, up to 4 inflammatory mediators were measured in one well.
[0225] On the other hand, as the target protein to be measured, a representative inflammation-promoting cytokine group A (IL-1β, IL-6, IL-17, and TNF-α) among inflammatory mediators, and a chemokine group B (MCP-1 and MIP-2) among inflammatory mediators was selected to carry out the experiment. The measurement results were summarized in
TABLE-US-00019 TABLE 19 p-value ELISA values in Arthritis vs vs vs Paw (pg/mL) Mean SEM Vehicle MTX Xeljanz ® IL-1β Negative control 26.77 12.21 — 0.0276 0.1321 (Vehicle DDS) MTX 1.05 0.31 0.0276 — 0.0069 Xeljanz ® 12.68 4.38 0.1321 0.0069 — Q-DDS 3.44 0.79 0.0122 0.0034 0.0067 (1 mg/mouse) Q-DDS 15.98 2.27 0.2112 <0.0001 0.3297 (3 mg/mouse) Q-DDS 5.52 0.94 0.0201 0.0001 0.0289 (5 mg/mouse) Q-DDS 0.83 0.25 0.0064 0.4583 0.0009 (10 mg/mouse) Q-DDS 7.64 4.29 0.0473 0.0867 0.2340 (20 mg/mouse) MTX + Q-DDS 3.28 0.31 0.0034 <0.0001 0.0014 (3 mg/mouse) IL-6 Negative control 250.8 187.0 — 0.1341 0.2264 (Vehicle DDS) MTX 1.8 0.7 0.1341 — 0.0359 Xeljanz ® 88.6 44.8 0.2264 0.0359 — Q-DDS 14.0 4.4 0.0750 0.0057 0.0246 (1 mg/mouse) Q-DDS 35.3 10.3 0.1023 0.0017 0.1019 (3 mg/mouse) Q-DDS 14.1 2.0 0.0749 <0.0001 0.0242 (5 mg/mouse) Q-DDS 1.5 0.5 0.0623 0.6586 0.0102 (10 mg/mouse) Q-DDS 15.5 10.1 0.0771 0.1247 0.0298 (20 mg/mouse) MTX + Q-DDS 6.1 2.4 0.0326 0.0505 0.0046 (3 mg/mouse) IL-17 Negative control 8.70 1.11 — <0.0001 0.0022 (Vehicle DDS) MTX 2.269 0.29 <0.0001 — 0.0179 Xeljanz ® 4.57 0.99 0.0022 0.0179 — Q-DDS 3.06 0.35 <0.0001 0.0459 0.0465 (1 mg/mouse) Q-DDS 7.11 0.99 0.1690 0.0001 0.0150 (3 mg/mouse) Q-DDS 3.21 0.45 <0.0001 0.0419 0.0877 (5 mg/mouse) Q-DDS 1.78 0.50 <0.0001 0.2862 0.0018 (10 mg/mouse) Q-DDS 4.87 2.29 0.0824 0.1964 0.8487 (20 mg/mouse) MTX + Q-DDS 3.40 0.71 <0.0001 0.0881 0.1346 (3 mg/mouse) TNF-α Negative control 45.61 7.21 — 0.0001 0.0129 (Vehicle DDS) MTX 6.54 1.60 0.0001 — 0.0077 Xeljanz ® 24.52 6.78 0.0129 0.0077 — Q-DDS 10.14 2.15 <0.0001 0.1037 0.0080 (1 mg/mouse) Q-DDS 42.60 6.97 0.6925 <0.0001 0.0131 (3 mg/mouse) Q-DDS 10.42 1.28 <0.0001 0.0213 0.0075 (5 mg/mouse) Q-DDS 3.54 1.10 <0.0001 0.0504 0.0003 (10 mg/mouse) Q-DDS 45.37 24.23 0.9915 0.0748 0.2310 (20 mg/mouse) MTX + Q-DDS 29.01 3.11 0.0032 <0.0001 0.3208 (3 mg/mouse)
TABLE-US-00020 TABLE 20 p-value ELISA values in Arthritis vs vs vs Paw (pg/mL) Mean SEM Vehicle Vehicle Vehicle MCP-1 Negative control 3371.1 2096.6 — 0.0831 0.4044 (Vehicle DDS) MTX 76.1 11.5 0.0831 — 0.0294 Xeljanz ® 207.9 954.6 0.4044 0.0294 — Q-DDS 281.8 22.6 0.0421 <0.0001 0.0154 (1 mg/mouse) Q-DDS 1062.6 120.1 0.1168 <0.0001 0.1599 (3 mg/mouse) Q-DDS 366.9 57.0 0.0473 <0.0001 0.0204 (5 mg/mouse) Q-DDS 69.8 9.4 0.0316 0.5709 0.0077 (10 mg/mouse) Q-DDS 398.3 247.0 0.0514 0.1389 0.0262 (20 mg/mouse) MTX + Q-DDS 111.8 12.9 0.0134 0.0112 0.0026 (3 mg/mouse) MIP-2 Negative control 519.3 249.7 — 0.0301 0.0359 (Vehicle DDS) MTX 4.4 1.7 0.0301 — 0.0077 Xeljanz ® 126.8 46.9 0.0359 0.0077 — Q-DDS 3.9 18.0 0.0139 0.0055 0.0452 (1 mg/mouse) Q-DDS 251.5 13.3 0.1257 <0.0001 0.0015 (3 mg/mouse) Q-DDS 98.1 23.2 0.0236 0.000 0.4237 (5 mg/mouse) Q-DDS 17.4 8.5 0.0089 0.0908 0.0034 (10 mg/mouse) Q-DDS 139.2 92.7 0.0550 0.1021 0.8596 (20 mg/mouse) MTX + Q-DDS 28.2 12.9 0.0029 0.0423 0.0024 (3 mg/mouse)
[0226] Through the above process, as a result of measuring the protein amount of four representative inflammation-promoting cytokines (IL-1β, IL-6, IL-17, and TNF-α) in arthritis tissues, IL-1β and IL-6 showed similar patterns, and the naltrexone DDS administration group entirely showed results similar to CAI, a clinical indicator of arthritis activity. In particular, in the naltrexone DDS 5 mg/mouse administration group, the 10 mg/mouse administration group, and the combination administration group, it was shown that pro-inflammatory cytokines were decreased compared to the negative control group, and also compared to the Xeljanz® administration group as a positive control group (
[0227] In the case of IL-17 and TNF-α, the naltrexone DDS administration group showed a decrease compared to the negative control group, and in particular, the naltrexone DDS 5 mg/mouse administration group and 10 mg/mouse administration group showed a decrease compared to the negative control group, and also showed a decrease when compared with the Xeljanz® administration group, which is a positive control.(IL 17: Xeljanz® vs 10 mg/mouse, 4.57 pg/ml±0.99 pg/ml vs 1.78 pg/ml±0.50 pg/ml, p<0.01; TNF-α: Xeljanz® vs 5 mg/mouse vs 10 mg/mouse, 24.52 pg/ml±6.78 pg/ml vs 10.42 pg/ml±1.28 pg/ml vs 3.54 pg/ml±1.10 pg/ml, p<0.01).
[0228] In addition, as a result of measurement of the chemokines MCP-1 and MIP-2, both naltrexone DDS 5 mg/mouse administration group and 10 mg/mouse administration group and the combination administration group showed a decrease compared to the negative control group(Vehicle DDS vs 5 mg/mouse vs 10 mg/mouse vs MTX+Q-DDS, MCP 1:3371.1 pg/ml f 2096.6 pg/ml vs 366.9 pg/ml±57.0 pg/ml ml vs 69.8 pg/ml±9.4 pg/ml vs 111.8 pg/ml±12.9 pg/ml, p<0.05; MIP-2:519.3 pg/ml±249.7 pg/ml vs 98.1 pg/ml±23.2 pg/ml vs 17.4 pg/ml±8.5 pg/ml vs 28.2 pg/ml±12.9 pg/ml, p<0.05), and also showed a decrease when compared with the Xeljanz® administration group, which is a positive control(Xeljanz® vs 10 mg/mouse vs MTX+Q-DDS, MCP 1:207.9 pg/ml±954.6 pg/ml vs 69.8 pg/ml±9.4 pg/ml vs 111.8 pg/ml f 12.9 pg/ml, p<0.01; MIP-2:126.8 pg/ml±46.9 pg/ml vs 17.4 pg/ml±8.5 pg/ml vs 28.2 pg/ml±12.9 pg/ml, p<0.01).
[0229] As a result, the major inflammatory mediators measured in the arthritic tissue showed a pattern very similar to the clinical indicators, especially in the naltrexone DDS 10 mg/mouse administration group, the pro-inflammatory cytokine group A (IL-1β, IL-6, IL). −17, and TNF-α) and all of the inflammatory mediators belonging to the chemokine B group (MCP-1 and MIP-2) showed a decrease compared to the negative control group, and also showed a decrease compared to the Xeljanz® administration group, the standard clinical treatment.
[0230] (2) Biomarker Evaluation Through Analysis of Protein Concentration of Inflammation Mediators in Serum
[0231] Using the Pro-reagent kit V, the mouse blood (serum) obtained after treatment with the test substance in the method of Example 3.1. above was diluted to ¼ with the same amount of protein (2.7 mg/mL) for each administration group, and then incubated in target-binding beads (IL-6 target) and a culture dish for 30 min. After washing with a wash buffer, it was reacted with the target-measurement antibody for 30 minutes, and after washing with a wash buffer again, it was reacted with streptavidin-PE for 10 minutes. Thereafter, the target protein is quantitatively measured for each administration group using the Bio-Plex® 200 system (Bio-Rad). As the target-binding beads, the beads described in Example 3.6. (1) were used.
[0232] For the measurement result, paw tissue weight of the mouse was corrected for the quantified amount of IL-6 protein. For each target joint, the hind paw was mainly used, and in order to overcome the limitation of the amount of protein extracted from the joint tissue, up to 4 inflammatory mediators were measured in one well.
[0233] The measured results were summarized in
TABLE-US-00021 TABLE 21 p-value ELISA values in vs vs vs Serum (pg/mL) Mean SEM Vehicle MTX Xeljanz ® IL-6 Negative control 144.7 92.2 — 0.2065 0.4558 (Vehicle DDS) MTX 39.6 24.3 0.2065 — 0.093 Xeljanz ® 94.5 30.8 0.4558 0.0903 — Q-DDS 79.5 36.1 0.3494 0.2594 0.6416 (1 mg/mouse) Q-DDS 58.8 38.4 0.2293 0.5972 0.2933 (3 mg/mouse) Q-DDS 33.5 19.1 0.0974 0.7898 0.0224 (5 mg/mouse) Q-DDS 7.2 1.7 0.0400 0.0636 0.0006 (10 mg/mouse) Q-DDS 7.2 2.8 0.0400 0.0648 0.0006 (20 mg/mouse) MTX + Q-DDS 5.2 1.9 0.0156 0.0233 0.0001 (3 mg/mouse)
[0234] As can be seen in
[0235] In conclusion, the blood concentration of IL-6 presents in the downstream of the inflammatory response cascade showed results consistent with the histologic and clinical findings of arthritis in the naltrexone DDS 10 mg/mouse administration group and the combination administration group.
Example 4. Efficacy Evaluation of Naltrexone-Containing Microspheres for Rheumatoid Arthritis (Tertiary)
[0236] 1. In Vivo Testing Method of Naltrexone-Containing Microspheres for Rheumatoid Arthritis
[0237] Following the efficacy evaluation in vivo of naltrexone-containing microspheres for rheumatoid arthritis in Example 2 and Example 3, an additional efficacy experiment was conducted by giving a difference to the positive control group.
[0238] A model of murine collagen-induced arthritis prepared in the same manner as in Example 2 (6-10 week-old male mouse of DBA/1J strain; the first immunization on day 0 and secondary immunization on day 21) was used.
[0239] As a material to be tested, naltrexone DDS prepared as described in preparation example 1 was prepared. As a negative control, DDS containing a carrier was used instead of naltrexone, and Humira® (Humira®, Humira® prefilled syringe, 40 mg/0.4 mL, Abbott Korea), a treatment for rheumatoid arthritis, was used as a positive control.
[0240] Administration information for each administration group is as described in Table 22 below.
TABLE-US-00022 TABLE 22 Regimen and dose of Administration administration Adminis- group Adminis- (based on the tration (Each group, tration Route of active volume n = 10) drug administration ingredient) (μl) 1 Vehicle Subcutaneous Administration 140 (Negative DDS injection once on the control) 23.sup.th day, 3 mg/mouse, N/A 2 Humira ® Subcutaneous Twice/week, 100 (Positive injection 10 mg/kg body control) weight/each time 3 Q-DDS Subcutaneous Administration 500 (Test group) injection once on the 23.sup.th day, 10 mg/mouse
[0241] Based on the blind evaluation data for each evaluation index, statistical analysis between the negative control group and the test group or between the two test groups was performed through SPSS. For comparison between the two groups, Student's t-test or Mann Whitney U test was used. To compare differences between treatment groups at multiple time points, repeated measures ANOVA with Turkey's post-hoc test was used. The significance level was defined as a p-value of 0.05 or less.
[0242] 2. Clinical Evaluation of Arthritis Activity
[0243] After administration of the test substance to the collagen-induced arthritis mouse model, the occurrence and level of inflammation were regularly observed from the date of group separation to the end of the experiment (the 41.sup.th day). Inflammation level was given a score of 0 to 4 for each paw according to the criteria in Table 4 above, and the sum was used as a clinical arthritis index (CAI).
[0244] When the clinical index of each paw was 2 or more scores, it was found that arthritis occurred, and the incidence was defined as 100% when arthritis occurred in the four toes.
[0245] Toe images of each administration group were shown in
TABLE-US-00023 TABLE 23 CAI CAI-AUC Group Mean SEM p-value Mean SEM p-value Vehicle DDS 10.55 1.44 — 130.48 15.24 — Humira ® 5.90 2.67 <0.0001 70.28 11.61 <0.0001 Q-DDS (10 mg/mouse) 5.15 1.27 <0.0001 71.88 14.38 <0.0001
[0246] As shown in
[0247] The incidence (%) of arthritis over the time (days) after administration of the test substance was shown in
TABLE-US-00024 TABLE 24 Incidence (%) Incidence-AUC Group Mean SEM p-value Mean SEM p-value Vehicle DDS 62.50 13.18 — 678.8 167.7 — Humira® 25.00 31.18 <0.0001 155.0 186.1 <0.0001 Q-DDS 17.50 12.08 <0.0001 156.3 108.1 <0.0001 (10 mg/mouse)
[0248] As shown in
[0249] 3. Histological Estimation
[0250] Mice were sacrificed on the 41.sup.th day, which is the end of the experiment, and the tissues of the hind paws were stained with hematoxylin/eosin. Hematoxylin/eosin staining was used to evaluate the activity of inflammation in the arthritic tissue, and toluidine blue staining was performed to confirm the histological therapeutic effect on cartilage destruction.
[0251] (1) Hematoxylin/Eosin Staining
[0252] Hematoxylin/eosin staining was performed and evaluated in the same manner as in Example 2.3. (1) above, and the score for items of synovial hyperplasia, pannus formation, cartilage destruction, and bone erosion was calculated by scoring each tissue as the average of the score of 4 sites based on Table 7 above.
[0253] The image of the tissue stained with hematoxylin/eosin was shown in
TABLE-US-00025 TABLE 25 Histochromatographic p-value parameters Mean SEM vs Vehicle vs Q-DDS Synovial Negative control 2.89 0.17 — <0.0001 hyperplasia (Vehicle DDS) Humira ® 1.05 0.41 <0.0001 0.2498 Q-DDS 0.86 0.17 <0.0001 — (10 mg/mouse) Pannus Negative control 2.55 0.32 — <0.0001 formation (Vehicle DDS) Humira ® 0.36 0.60 <0.0001 0.2166 Q-DDS 0.08 0.11 <0.0001 — (10 mg/mouse) Cartilage Negative control 2.50 0.29 — <0.0001 destruction (Vehicle DDS) Humira ® 0.80 0.44 <0.0001 0.7799 Q-DDS 0.75 0.12 <0.0001 — (10 mg/mouse) Bone Negative control 2.45 0.38 — <0.0001 erosion (Vehicle DDS) Humira ® 0.28 0.52 <0.0001 0.2315 Q-DDS 0.05 0.09 <0.0001 — (10 mg/mouse)
[0254] As shown in
[0255] (2) Toluidine Blue Staining
[0256] In the same manner as in Example 2.3. (2) above, the hind paw tissue of the mouse was stained with toluidine blue, and items such as photographing and matrix staining, surface regularity, and cartilage thickness were evaluated. The scoring criteria for each item is described in Table 9, and the average score of 4 sites was calculated as the score of each tissue.
[0257] The image of the tissue stained with toluidine blue was shown in
TABLE-US-00026 TABLE 26 Histochromatographic p-value parameters Mean SEM vs Vehicle vs Q-DDS Matrix Negative control 2.91 0.11 — <0.0001 staining (Vehicle DDS) Humira ® 1.31 0.59 <0.0001 0.2249 Q-DDS 1.00 0.37 <0.0001 — (10 mg/mouse) Surface Negative control 2.59 0.24 — <0.0001 regularity (Vehicle DDS) Humira ® 0.72 0.76 <0.0001 0.3335 Q-DDS 0.44 0.25 <0.0001 — (10 mg/mouse) Cartilage Negative control 1.69 0.15 — <0.0001 thickness (Vehicle DDS) Humira ® 0.34 0.43 <0.0001 0.5824 Q-DDS 0.25 0.16 <0.0001 — (10 mg/mouse)
[0258] As shown in
[0259] Taken together with the histological estimation results, naltrexone DDS 10 mg/mouse administration group not only had an anti-inflammatory effect compared to the negative control group in the evaluation of the activity of inflammation of arthritic tissues and the severity of joint damage but also showed a more effective arthritis inhibitory effect rather than that of Humira which is an antirheumatic agent used in a clinic.
[0260] 4. Safety Assessment In Vivo
[0261] The safety in vivo of the test substance was evaluated by measuring the body weight of mouse in each administration group of Example 4.1.
[0262] For the safety evaluation of the test substance, the body weight of mouse was measured daily from before the start of administration (The 23.sup.th day) to just before the end of administration (The 41.sup.th day), and the body weight (%) of the mouse for each administration group over the time (day) and the body weight on the 41.sup.th day were shown in
TABLE-US-00027 TABLE 27 Change of body weight D23 body D41 body (%, D23 body weight 100%) Change of weight weight p-value body weight Mean SEM Mean SEM Mean SEM (vs Vehicle) Negative control 22.3 1.2 21.2 0.9 95.06 5.29 — (Vehicle DDS) Humira ® 22.2 1.6 22.6 1.9 102.11 7.67 0.0279 Q-DDS 23.6 1.4 24.4 1.8 106.39 3.79 <0.0001 (10 mg/mouse)
[0263] As shown in
[0264] 5. Imaging Evaluation of Arthritis Using Micro-CT
[0265] The paw tissue representing each group's results was fixed after the treatment with the test substance in Example 4.1 and was photographed with micro-CT scanners (Quantum FX, Perkin Elmer, MA). The radiographs were 3D rendered using the Quantum FX μCT imaging system (Perkin Elmer, MA), and then the radiological score was evaluated, and the bone volume ratio (Bone volume [BV]/Tissue volume; TV) and bone surface density (Bone surface area (BS)Bone volume (BV)), and cortical bone thickness were also measured. Two or more researchers performed blind evaluation for joint destruction, and the indicators in Table 17 above were used as the scoring criteria. The average value of the researchers' blind evaluation data was used as the score, and the average score of the paw tissue was calculated as the score of each group.
[0266] Representative images for each group photographed with micro-CT were shown in FIG. 17A, and in particular, images magnifying joint portions were shown in
TABLE-US-00028 TABLE 28 p-value Radiological analysis values Mean SEM vs Vehicle vs Q-DDS Bone Negative control 16.53 1.50 — 0.0209 volume/ (Vehicle DDS) tissue Humira ® 24.21 3.83 0.0209 0.8845 volume Q-DDS 25.71 0.48 0.0209 — (%) (10 mg/mouse) Bone Negative control 6.88 0.71 — 0.0202 surface group width/Bone (Carrier) volume Humira ® 6.08 0.09 0.0202 0.2944 (mm.sup.−1) Q-DDS 5.97 0.15 0 0202 — (10 mg/mouse) Cortical Negative control 0.62 0.06 — 0.0209 bone group thickness (Carrier) (mm) Humira ® 0.78 0.03 0.0202 0.7674 Q-DDS 0.78 0.03 0.0209 — (10 mg/mouse)
[0267] As noted in
[0268] As a result of measurement of bone surface density, the positive control group and the naltrexone DDS administration group showed lower levels than the negative control group, and the naltrexone DDS administration group showed a level similar to the positive control group (Vehicle DDS vs Humira vs Q-DDS, 6.88 min.sup.−1±0.71 mm.sup.−1 vs 6.08 mm.sup.−1±0.09 mm.sup.−1 vs 5.97 mm.sup.−1
[0269] As a result of quantification of cortical bone thickness, it was found that the negative control group had a thinner cortical bone thickness due to bone erosion, but the positive control group showed a greater thickness than the negative control group. The naltrexone DDS administration group was thicker than the negative control group (Vehicle DDS vs Q-DDS, 0.62 mm±0.06 mm vs 0.78 mm±0.03 mm p<0.05), and exhibited a thickness similar to the Humira administration group.
[0270] In the radiological score, the positive control group and the naltrexone DDS administration group showed a decrease compared to the negative control group, and as a result, through imaging analysis, it was confirmed that naltrexone DDS not only treats arthritis inflammation, but also inhibits the progression of joint destruction as a result.
[0271] 6. Multiplex Protein Immunoassay on Arthritic Tissue and Serum
[0272] In order to evaluate the inflammatory activity and confirm the mode of action of the test substance through quantitative analysis of inflammatory substances in mouse arthritis tissue and serum, inflammatory mediators were investigated at the protein level. The same multi-analyte-EL ELISA (multi-analyte-ELISA) method based on Luminex technology as in Example 3.6. above was used.
[0273] (1) Biomarker Evaluation Through Analysis of Protein Concentration of Inflammation Mediators in Arthritic Tissues
[0274] The mouse joint tissue obtained after treatment with the test substance in the method of Example 4.1. above was pulverized through ceramic beads, and all proteins were extracted using a lysis buffer (Cat. 1713040111, Bio-Rad). Using Pro-reagent kit V, the same amount of protein (2.7 mg/mL) for each administration group was diluted ¼ and reacted with the target-binding beads in a culture dish for 30 minutes. After washing with a wash buffer, it was reacted with the target-measurement antibody for 30 minutes, and after washing with a wash buffer again, it was reacted with streptavidin-PE for 10 minutes. Thereafter, the target protein is quantitatively measured for each administration group using the Bio-Plex® 200 system (Bio-Rad). The target-binding beads were composed of a set that can be combined as needed from Bio-Plex Mouse cytokine 4-plex (IL-1β, IL-6, IL-17A, TNF-α; Cat. 171 G5002M), Bio-Plex Pro Mouse Cytokine 1-plex (MCP1; Cat. 171G5019M), Bio-Plex Pro Mouse Cytokine II 1-plex (MIP2; Cat. 171 G6006M), and Bio-Plex Pro Mouse Cytokine 1-plex (IL-6; Cat. 171G5007M) (all products are from Bio Rad) and were used for measurement.
[0275] For the measurement result, paw tissue weight of the mouse was corrected for the quantified target protein. For each target joint, the hind paw was mainly used, and in order to overcome the limitation of the amount of protein extracted from the joint tissue, up to 4 inflammatory mediators were measured in one well.
[0276] On the other hand, as the target protein to be measured, a representative inflammation-promoting cytokine group A (IL-1β, IL-6, IL-17, and TNF-α) among inflammatory mediators, and IL-2 and a chemokine group B (IL-2, MCP-1 and MIP-2) among inflammatory mediators were selected to carry out the experiment. The measurement results were summarized in
TABLE-US-00029 TABLE 29 ELISA values in Arthritis p-value Paws (pg/mL) Mean SEM vs Vehicle vs Q-DDS IL-1β Negative control 56.80 8.43 — <0.0001 (Vehicle DDS) Humira ® 4.09 1.90 <0.0001 0.0018 Q-DDS 1.67 0.42 <0.0001 — (10 mg/mouse) IL-6 Negative control 397.25 247.40 — 0.0002 (Vehicle DDS) Humira ® 10.75 12.57 0.0003 0.0589 Q-DDS 2.20 1.02 0.0002 — (10 mg/mouse) IL-17 Negative control 40.56 11.83 — <0.0001 (Vehicle DDS) Humira ® 9.93 1.01 <0.0001 0.0001 Q-DDS 7.50 1.02 <0.0001 — (10 mg/mouse) TNF-α Negative control 105.64 20.60 — <0.0001 (Vehicle DDS) Humira ® 18.93 7.64 <0.0001 0.0299 Q-DDS 12.49 2.77 <0.0001 — (10 mg/mouse)
TABLE-US-00030 TABLE 30 ELISA values in Arthritis p-value Paws (pg/mL) Mean SEM vs Vehicle vs Q-DDS IL-2 Negative control 182.26 170.68 — 0.0093 (Vehicle DDS) Humira ® 36.59 16.19 0.0215 0.0016 Q-DDS 14.00 7.56 0.0093 — (10 mg/mouse) MCP-1 Negative control 7808.4 3280.5 — <0.0001 (Vehicle DDS) Humira ® 281.3 204.6 <0.0001 0.0536 Q-DDS 138.4 22.4 <0.0001 — (10 mg/mouse) MIP-2 Negative control 12373.11 3255.29 — <0.0001 (Vehicle DDS) Humira ® 564.83 393.77 <0.0001 0.0165 Q-DDS 211.31 43.55 <0.0001 — (10 mg/mouse)
[0277] Through the above process, as a result of measuring the protein amount of four representative pro-inflammative cytokines (IL-1β, IL-6, IL-17, and TNF-α) in the arthritic tissue, the positive control group and the naltrexone DDS administration group showed a decrease compared to the negative control group, and in particular, for IL-1β, IL-17, and TNF-α, the naltrexone DDS administration group showed a decrease even when compared to the Humira-administered group, a positive control group (IL 1β: Humira vs Q-DDS, 4.09 pg/ml±1.90 pg/ml vs 1.67 pg/ml±0.42 pg/ml, p<0.01; IL-17: Humira vs Q-DDS, 9.93 pg/ml±1.01 pg/ml vs 7.50 pg/ml±1.02 pg/ml, p<0.05; TNF-α: Humira vs Q-DDS, 18.9.3 pg/ml±7.64 pg/ml vs 12.49 pg/ml±2.77 pg/ml, p<0.05).
[0278] In the case of IL-6, the naltrexone DDS administration group showed a decrease compared to the negative control group.
[0279] In addition, as a result of measurement of the chemokines MCP-1 and MIP-2, a pattern similar to that of pro-inflammatory cytokines of group A was found. Specifically, the IL-2 concentration in the naltrexone DDS administration group was lower than that of the negative control group and was measured at a lower level than Humira-administered group as the positive control group (Humira vs Q-DDS, 36.59 pg/ml±16.19 pg/ml vs. 14.00 pg/ml±7.56 pg/ml, p<0.01). MIP-2 and MCP-1 measurement results also showed a decrease in the naltrexone DDS administration group compared to the negative control group.
[0280] As are sult, the major inflammatory mediators measured in arthritic tissues showed a pattern very similar to the previously measured clinical indicators, and in the naltrexone DDS administration group, all of IL-1β, IL-6, IL-17, TNF-α, IL-2, MCP-1, and MIP-2 not only showed a decrease compared to the negative control group, but also showed an inhibition compared to Humira, a standard treatment. The major inflammatory cytokines and chemokines in these arthritic tissues are important biomarkers of arthritis. Therefore, from the above result that the main causative agents of inflammation were more inhibited in the naltrexone DDS administration group than in the Humira administration group, it could be confirmed that the naltrexone DDS of the present disclosure effectively inhibits, alleviates, or modulates both the symptoms and inflammation of arthritis in the treatment of arthritis.
[0281] (2) Biomarker Evaluation Through Analysis of Protein Concentration of Inflammation Mediators in Serum
[0282] Using the Pro-reagent kit V, the mouse blood (serum) obtained after treatment with the test substance in the method of Example 4.1. above was diluted to ¼ with the same amount of protein (2.7 mg/mL) for each administration group, and then incubated with target-binding beads (IL-6 target) and a culture dish for 30 min. After washing with a wash buffer, it was reacted with the target-measurement antibody for 30 minutes, and after washing with a wash buffer again, it was reacted with streptavidin-PE for 10 minutes. Thereafter, the target protein is quantitatively measured for each administration group using the Bio-Plex ri 200 system (Bio-Rad). As the target-binding beads, the beads described in Example 4.6. (1) were used.
[0283] For the measurement result, paw tissue weight of the mouse was corrected for the quantified amount of IL-6 protein. For each target joint, the hind paw was mainly used, and in order to overcome the limitation of the amount of protein extracted from the joint tissue, up to 4 inflammatory mediators were measured in one well.
[0284] The measured results were summarized in
TABLE-US-00031 TABLE 31 ELISA, values in p-value serum (pg/mL) Mean SEM vs Vehicle vs Q-DDS IL-6 Negative control 74.72 20.33 — <0.0001 (Vehicle DDS) Humira ® 29.02 21.74 0.0003 0.0219 Q-DDS 10.45 2.85 <0.0001 — (10 mg/mouse)
[0285] As can be seen in
[0286] In conclusion, the blood concentration of IL-6 consists of the downstream of the inflammatory response cascade was shown to be decreased, consistent with the histological and clinical findings of arthritis in the naltrexone DDS administration group. The naltrexone DDS of the present disclosure exhibits more reducing effect on the concentration of IL-6 in the blood compared to the positive control Humira, which means that the present disclosure reduces IL-6 not only in arthritic tissues but also in a systemic level. In addition, since IL-6 is a cytokine substantially showing high levels in the serum of arthritis patients and reflects disease activity, this result means that naltrexone DDS can be effective in the treatment of arthritis.
[0287] 7. NK Cell Immunostaining in Arthritic Tissue
[0288] In order to verify the etiological mechanism according to the severity of arthritis, the infiltration of NK cells in the mouse paw tissue was measured after administration of the test substance in the method of Example 4.1.
[0289] Specifically, after de-paraffinization and rehydration on a paraffin-embedded tissue piece slide, the antigen in the tissue is exposed through heat-induced antigen retrieval. After the serum blocking step, the slides were reacted with a CD56 target the first antibody (rabbit anti-CD56 antibody, Abcam, ab220360) for 1 hour, and then peroxidase blocking was performed for 15 minutes. After reacting with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG-HRP, Jackson lab, #111-035-144) for 30 minutes, OPAL 690 solution pack (Akoya Biosciences, CA) was reacted for 10 minutes to proceed with binding to the secondary antibody. After nuclear staining by reaction with DAPI for 3 minutes, the slides were mounted with Vector Shield (Vector Lab, CA) medium having a fluorescence preservation formula, and photographed with confocal microscopy; C2 plus Ti2-E, Nikon, NY). After photographing 3 areas per slide (400 magnification), the number of cells stained with CD56 antibody (NK cells) was quantitatively evaluated. The images taken according to the magnification were shown in
TABLE-US-00032 TABLE 32 The number of CD56+ cell p-value (number/HPF) Mean SEM vs Vehicle vs Q-DDS Negative control 10.25 3.25 — <0.0001 (Vehicle DDS) Humira ® 2.00 1.41 <0.0001 0.7841 Q-DDS 1.83 1.53 <0.0001 — (10 mg/mouse)
[0290] As can be seen in
[0291] As a result, in the case of CD56-positive NK cells in the development of arthritis, infiltration into the inflamed tissue occurs as the arthritis becomes worse, but it was confirmed that the infiltration is clearly inhibited by administration of naltrexone DDS, and compared to Humira as an antirheumatic agent used in clinical practice, it was further inhibited.
Example 5. Efficacy Evaluation of Naltrexone-Containing Microspheres for Multiple Sclerosis
[0292] 1. In Vivo Test Method of Naltrexone-Containing Microspheres for Multiple Sclerosis
[0293] In vivo efficacy was evaluated to determine whether naltrexone-containing microspheres, i.e., the naltrexone drug delivery systems, have therapeutic efficacy for multiple sclerosis.
[0294] As an experimental autoimmune encephalomyelitis mouse model (Mouse Experimental Autoimmune Encephalomyelitis: EAE), 6 to 10 week-old female mice of C57BI./6 strain were prepared. Mice were kept and tested in a specific pathogen free (SPF) laboratory under an environment of a temperature of 21° C. to 23° C. and a relative humidity of 40% to 50%. Experimental animals were kept per cage in the number of 4 or less, and a cage was exchanged 2 to 3 times a week and feed was supplied.
[0295] Myelin-oligodendrocyte glycoprotein (MOG) was dissolved in phosphate-buffered saline (PBS) to be 1 mg/mL, and was emulsified by mixing at 1:1 (v/v) with 5 mg/mL Complete Freund's adjuvant. 100 μl of this was subcutaneously injected into both sides of the mouse, and then 400 μl of 2 pg/mL pertussis toxin was intraperitoneally injected (first immunization, day 0). On the second day, 400 μl of 2 μg/mL pertussis toxin was intraperitoneally injected a second time. On the third day after the first immunization, the test was performed by separating the experimental group.
[0296] As a material to be tested, naltrexone DDS prepared as described in preparation example 1 was prepared. As a negative control, DDS containing a carrier was used instead of naltrexone, and as a positive control, Fingolimod (Fytarex® capsule, 0.5 mg, Novartis Korea) and naltrexone hydrochloride (Revia® tablet, 50 mg, Jell Pharmaceutical) were used.
[0297] Administration information for each administration group is as described in Table 33 below. The animal model preparation and administration schedule were shown in
TABLE-US-00033 TABLE 33 Regimen and dose of Administration administration Adminis- group Adminis- (Baseline of tration (Each group, tration Route of the active volume n = 6) drug administration ingredient) (μl) 1 Vehicle Subcutaneous Administration (Negative DDS injection once on the control) third day, N/A 2 Fytarex ® Oral Once/day, (Positive 3 mg/kg body control) weight/each time 3 Revia ® Oral Once/day, (Positive 1 mg/kg/time control) 4 Naltrexone Sub- Administration (Test group) DDS cutaneously once on the (Q-DDS) third day, 1 mg/mouse
[0298] Based on the blind evaluation data for each evaluation index, statistical analysis between the negative control group and the test group or between the two test groups was performed using SPSS. For comparison between the two groups, Student's t-test or Mann Whitney U test was used. The significance level was defined as a p-value of 0.05 or less.
[0299] 2. Clinical Evaluation of the Occurrence and Severity of Neurological Symptoms
[0300] The experimental autoimmune encephalomyelitis mouse model was prepared, and from the third day after immunization, administered with carrier-containing DDS as a negative control, and Q-DDS as the test substance by subcutaneous injection once at 1 mg/mouse, and Fytarex® and Revia® as the positive control group were administered orally daily with the corresponding dose. Then, the occurrence and extent of clinical symptoms were regularly observed until the end of the experiment (The 30.sup.th day). The clinical symptom level was assigned a score of 0 to 5 after evaluating the functionality of the tail and four legs according to the criteria in Table 34 below, and was used as the EAE clinical score.
TABLE-US-00034 TABLE 34 Score Clinical neurologic findings 0 No obvious changes in motor function compared to non- immunized mice. 0.5 Tip of tail is limp. 1 Limp tail. 1.5 Limp tail and hind lig inhibition. 2 Limp tail and weakness of hind legs. 2.5 Limp tail and dragging of hind legs. 3 Limp tail and complete paralysis of hind legs (most common). 3.5 Limp tail and complete paralysis of hind legs. In addition to: Mouse is moving around the cage, but when placed on its side, is unable to right itself. Hind legs are together on one side of body. 4 Limp tail, complete hind leg and partial front leg paralysis. 4.5 Complete hind and partial front leg paralysis, no movement around the cage. Moue is not alert. 5 Mouse is spontaneously rolling in the cage (euthanasia is recommended).
[0301] The clinical score of EAE mouse over the time (days) after administration of the test substance was shown in
TABLE-US-00035 TABLE 35 Clinical score (D28) Clinical score-AUC p-value p-value vs vs Group Mean SEM Vehicle Mean SEM Vehicle Vehicle DDS 3.33 0.47 — 44.50 3.88 — Fytarex ® 1.67 0.13 0.002 20.67 2.16 0.0001 Revia ® 2.00 0.16 0.008 21.08 4.37 0.0006 Q-DDS (1 mg/mouse) 1.92 0.25 0.008 21.71 2.42 0.0001
[0302] As shown in
[0303] The analyzed results of the area under the curve (AUC) of the clinical scores also showed a similar pattern to the time course data of the clinical scores, and the positive control group (Fytarex® and Revia®) and Q-DDS 1 mg/mouse administration group showed a decrease compared to the carrier-containing DDS group. Through the above results, it was confirmed that a low-dose Q-DDS (1 mg/mouse) exhibited a reducing effect in clinical index similar to the standard treatment used in clinical practice. In addition, in the case of the sustained-release injection naltrexone DDS administration group, despite administration once a month, the results were almost similar to the results of Revia tablet administration group which is daily orally administered, and from this, it was confirmed that sustained-injection of the present disclosure was better in terms of formulation.
[0304] 3. Histological Estimation
[0305] After mouse euthanasia, spinal cord tissue was extracted, hematoxylin/eosin staining and Luxol Fast Blue staining were performed, and MBP-targeted immunohistochemical staining was performed, and histological evaluation was performed through blind evaluation.
[0306] (1) Hematoxylin/Eosin Staining
[0307] On the 30th day which is the last day of the experiment, mouse was sacrificed, and the mouse spinal cord tissue was extracted and paraffin-embedded slides were prepared and then stained with hematoxylin/eosin, and the spinal cord tissue was photographed at 50 and 200 magnifications. Then, histological scores were measured according to the histological evaluation criteria of Table 36 below to evaluate the distribution of inflammatory cells.
TABLE-US-00036 TABLE 36 Score Inflammation scoring 0 No inflammation 1 Cellular infiltrate only in the perivascular areas and meninges 2 Mild cellular infiltrate in parenchyma: Less than one third part of the white matter is infiltrated with inflammatory cells 3 Moderate cellular infiltration in parenchyma: More than one third part of the white matter is infiltrated with inflammatory cells 4 Severe cellular infiltration in parenchyma: Infiltration of inflammatory cells are observed in the whole white matter
[0308] The image of the tissue stained with hematoxylin/eosin was shown in
TABLE-US-00037 TABLE 37 p-value Administration group Mean SEM vs Vehicle vs Fytarex vs Revia Vehicle DDS 3.0 0.35 — Fytarex ® (3 mg/kg) 1.08 0.29 0.0005 Revia ® (1 mg/kg) 1.42 0.46 0.0074 0.4702 Q-DDS (1 mg/mouse) 1.13 0.44 0.0046 0.5737 0.8753
[0309] As shown in
[0310] (2) Luau Fast Blue Staining
[0311] On the 30th day which is the last day of the experiment, mouse was sacrificed, and the mouse spinal cord tissue was extracted and paraffin-embedded slides were prepared and then stained with Luxol Fast Blue, and the spinal cord tissue was photographed at 50 and 200 magnifications. In Luxol Fast Blue staining, myelin is stained from blue to green, and nerves are stained purple, and through this, the degree of demyelination was analyzed in the EAE model by confirming the neural structure in the spinal cord. Demyelination was analyzed according to the criteria described in Table 38 below and measured as a score.
TABLE-US-00038 TABLE 38 Score Matrix staining results for demyelination scoring 6 No demyelination foci or lesion 1 Mild demyelination 2 Moderate demyelination 3 Severe demylelination
[0312] The image of the tissue stained with Luxol Fast Blue was shown in
TABLE-US-00039 TABLE 39 p-value Administration group Mean SEM vs Vehicle vs Fytarex vs Revia Vehicle DDS 1.72 0.51 — <0.0001 <0.0001 Fytarex ® (3 mg/kg) 0.14 0.12 <0.0001 — 0.0120 Revia ® (1 mg/kg) 0.61 0.36 <0.0001 0.0120 — Q-DDS (1 mg/mouse) 0.56 0.34 <0.0001 0.0196 0.8136
[0313] As shown in
[0314] As a result, in the evaluation of the inflammatory activity of myelitis tissue and the severity of myelin sheath, the Q-DDS 1 mg/mouse administration group showed a therapeutic effect compared to the negative control group, and also showed a similar level of therapeutic effect compared to Fytarex®, the clinically most widely used standard multiple sclerosis treatment. In addition, in the case of the sustained-injection naltrexone DDS administration group, despite administration once a month, the results were better than the results of Revia tablet administration group which is daily orally administered, and from this, it was confirmed that sustained injection of the present disclosure was better in terms of efficacy and formulation.
[0315] (3) Lmmunohistochemistry (IHC) Staining Using Anti-Myelin Basic Protein (MBP) Antibody
[0316] On the 30th day which is the last day of the experiment, mouse was sacrificed, and the mouse spinal cord tissue was extracted and paraffin-embedded slides were prepared and then myelin basic protein (MBP) was stained immunohistochemically, and the spinal cord tissue was photographed at 50 and 200 magnifications. This is to confirm demyelination through immunohistochemical analysis using an antibody against myelin basic protein (MBP), a component of myelin.
[0317] The immunohistochemical staining image of the tissue was shown in
TABLE-US-00040 TABLE 40 p-value Administration group Mean SEM vs Vehicle vs Fytarex vs Revia Vehicle DDS 39.72 8.46 — 0.0237 0.0251 Fytarex ® (3 mg/kg) 58.47 8.12 0.0237 — 0.9029 Revia ® (1 mg/kg) 58.23 5.67 0.0251 0.9029 — Q-DDS (1 mg/mouse) 56.41 5.99 0.0454 0.4661 0.51514
[0318] As shown in
[0319] 4. Safety Assessment In Vivo
[0320] The in vivo safety of the test substance was evaluated by measuring the body weight of mouse in each administration group of Example 5.1.
[0321] For the safety evaluation of control substance, the test substance, the body weight of mouse was measured daily from before the start of administration (0 day) to just before the end of administration (The 30th day), and the body weight (%) of the mouse for each administration group over the time (day) was shown in
[0322] As shown in
Example 6. Study on the Mechanism of Action of Low-Dose Naltrexone-Containing Microspheres Related to Autoimmune Diseases
[0323] Toll-like receptor 4 (TLR4) is closely related to innate immunity, which is the first step of the immune response in the body, and is a representative receptor responsible for regulating the initial immune response and inflammation response, when there is damage or infection in cells or the body. When TLR4 is activated due to injury or inflammatory response, NF-κB, a sub-regulator of TLR4, is activated to increase the expression of inflammatory cytokines and secrete inflammatory cytokines into the body (J. Med. Chem. 2020, 63, 22), 13466-13513).
[0324] The present inventors conducted the following experiment to confirm whether low-dose naltrexone exerts an antagonistic effect by regulating the activity of TLR4.
[0325] 1. Confirmation of Direct Binding of Naltrexone to MD2
[0326] Naltrexone was purchased from USP (Rockville, Md., USA), and MD2 was purchased from R&D systems (Minneapolis, Minn., USA). It was confirmed through surface plasmon resonance analysis that naltrexone binds to the MD2 protein on TLR4 signaling. As the concentration of naltrexone increased, the binding to MD2 gradually increased (
[0327] In the present disclosure, it was revealed for the first time that low-dose naltrexone binds to MD2, the major active site of TLR4, and these results show that naltrexone may inhibit TLR4 activity by directly binding to MD2, which is an active central factor in TLR4 signaling. From these results, it is expected that naltrexone has a mechanism of curing autoimmune diseases and inflammatory diseases caused by abnormal immune responses by targeting a novel target TLR4 signaling molecule and blocking immune abnormalities or immune overreaction. A schematic diagram of the mechanism by which low-dose naltrexone affects TLR4 signaling was shown in
[0328] Hereinafter, it was confirmed whether a low-dose naltrexone capable of blocking TLR4 signal transduction could inhibit TLR4-mediated inflammatory factors increased by LPS in actual cells.
[0329] 2. Effect of Low-Dose Naltrexone on Expression of Pro-Inflammatory Cytokines (TNF-α, IL-1β, IL-6, IL-1β, IL-17, iNOS) in Human Synovial Cell Lines
[0330] The SW982 cell line (ATCC, Manassas, Va., USA), a human synovial cell line, was placed in RPM 1640 medium (Welgene, Republic of Korea) to which 10% fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 μg/ml streptomycin (Thermo Fisher Scientific Inc.) was added, and cultured in a culture system (Sanyo, Japan) under 37° C. and 5% CO2 conditions. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, Mo., USA).
[0331] After treating SW982 cells with LPS (1 ug/ml) and naltrexone 200 or 500 ug/ml for 6 hours, RNA was extracted and cDNA was synthesized. As a result of performing RT-qPCR on the synthesized cDNA, it was confirmed that the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-17, iNOS) increased by LPS was significantly inhibited by 200 or 500 ug/ml of naltrexone (see
[0332] 3. Effect of Low-Dose Naltrexone on Activity of NF-κB in Human Synovial Cell Lines
[0333] After treating SW982 cells with 200 or 500 ug/ml of naltrexone, Western blotting was performed to confirm whether the activity of NF-κB was inhibited. 1×Cell lysis buffer (Cell signaling, CA, USA) was mixed with protease and a phosphatase inhibitor and added to SW982 cells. After extracting the protein from the cells, the concentration was measured, and the same amount of protein was developed on an SDS-polyacrylamide gel, and transferred to an ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., Piscataway, N.J., USA) and blocked with nonfat dried milk for 1 hour. Thereafter, the reaction was performed with the first antibody (p65 NF-κB, phospho p65 NF-κB or calnexin; Cell signaling, CA, USA), while shaking at 4° C. for 24 hours. Then, after shaking and washing 3 times for 10 minutes with PBST buffer, the secondary antibody was incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibody for 1 hour at room temperature, and reacted with the protein band on the membrane using Supersignal west pico ECL solution (Thermo Fisher Scientific Inc.). The results were visualized with the Bio-Rad Gel Documentation system (Bio-Rad Laboratories, Hercules, Calif., USA), and the results were shown in
[0334] As shown in
[0335] 4. Effects of Low-Dose Naltrexone on Activity of Mitogen-Activated Protein Kinases (MAPKs) in Human Synovial Cell Lines
[0336] After treating SW982 cells with 200 or 500 ug/ml of naltrexone, western blotting was performed to confirm whether the activity of MAPKs was inhibited. Western blotting was performed in the same manner as in Example 6.3. The first antibody used at this time is an antibody against p-ERK, ERK, p-JNK, JNK, p-p38, p38 and calnexin (Cell signaling, CA, USA), and the secondary antibody is anti-mouse or anti-rabbit HRP-binding secondary antibody. The results obtained by performing Western blotting were shown in
[0337] As shown in
[0338] 5. Effect of Low-Dose Naltrexone on Expression of Pro-Inflammatory Cytokines (TNF-α, IL-1β, IL-6, IL-17, iNOS) in murine macrophage cell
[0339] The Raw 264.7 cell line (ATCC, Manassas, Va., USA), murine macrophage cell line, was placed in DMEM medium (Welgene, Republic of Korea) to which 10% fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 μg/ml streptomycin (Thermo Fisher Scientific Inc.) was added, and cultured in a culture system (Sanyo, Japan) under 37° C. and 5% CO2 conditions. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, Mo., USA).
[0340] After treating Raw 264.7 cells with LPS (1 ug/ml) and naltrexone 200 or 500 ug/ml for 6 hours, RNA was extracted and cDNA was synthesized. As a result of performing RT-qPCR on the synthesized cDNA, it was confirmed that the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-17, iNOS) increased by LPS was significantly inhibited by 200 or 500 ug/ml of naltrexone (see
[0341] 6. Effect of Low-Dose Naltrexone on the Activity of NF-κB in Murine Macrophage Cell
[0342] After treating Raw 264.7 cells with 100, 200, 500 or 1000 ug/ml of naltrexone, Western blotting was performed to confirm whether the activity of NF-icB was inhibited. 1×Cell lysis buffer (Cell signaling, CA, USA) was mixed with protease and a phosphatase inhibitor and added to SW982 cells. After extracting the protein from the cells, the concentration was measured, and the same amount of protein was developed on an SDS-polyacrylamide gel, and transferred to an ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., Piscataway, N.J., USA) and blocked with nonfat dried milk for 1 hour. Thereafter, the reaction was performed with the first antibody (p65 NF-κB, phospho p65 NF-κB or calnexin; Cell signaling, CA, USA), while shaking at 4° C. for 24 hours. Then, after shaking and washing 3 times for 10 minutes with PBST buffer, the secondary antibody was incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibody for 1 hour at room temperature, and reacted with the protein band on the membrane using Supersignal west pico ECL solution (Thermo Fisher Scientific Inc.). The results were visualized with the Bio-Rad Gel Documentation system (Bio-Rad Laboratories, Hercules, Calif., USA), and the results were shown in
[0343] As shown in
[0344] 7. Effects of Low-Dose Naltrexone on Activity of Mitogen-Activated Protein Kinases (MAPKs) in Murine Macrophage Cell
[0345] After treating Raw 264.7 cells with 100, 200, 500 or 1000 ug/ml of naltrexone, Western blotting was performed to confirm whether the activity of MAPKs was inhibited. Western blotting was performed in the same manner as in Example 6.3. The first antibody used at this time is an antibody against p-ERK, ERK, p-JNK, JNK, p-p38, p38 and calnexin (Cell signaling, CA, USA), and the secondary antibody is anti-mouse or anti-rabbit HRP-binding secondary antibody. The results obtained by performing Western blotting were shown in
[0346] As shown in
Example 7. Evaluation of the Inhibitory Effect of Naltrexone and 6β-Naltrexol on Autoimmune Inflammatory Factors and the Regulation of TLR4 Signaling Factors
[0347] 1. Background and outline
[0348] When naltrexone is orally administered to humans, 60-naltrexol as a metabolite, is produced in the body due to the first-pass effect and accordingly, in the case of oral preparations, 6β-naltrexol is relatively produced approximately 10 times more than injections. In addition, when administered orally, the deviation of naltrexone and 6β-naltrexol production rates between patients is large enough to cause a 128-fold difference between individuals (0.73 to 92.00), and when a 60-naltrexol production rate is high, side effects including nausea and headache may occur and this has been reported to affect drug efficacy, toxicity, and patient compliance (Journal of Analytical Toxicology 2014; 38:212-217). Although 6β-naltrexol is known to be active against opioid addiction, which is currently used commercially, it has not been studied whether naltrexone and its metabolite, 60-naltrexol show a difference in efficacy against TLR4 in relation to autoimmune diseases. Accordingly, the present inventors compared the autoimmune or inflammation inhibitory effects of naltrexone and 6β-naltrexol at the cellular level for the first time in order to confirm whether the sustained-injection of the present disclosure exhibits superior effects compared to the oral preparation, and checked whether TLR4 signaling factors (NF-κB, MAPKs) are regulated.
[0349] Toll-like receptor 4 (TLR4) is closely related to innate immunity, which is the first step of the immune response in the body, and is a representative receptor responsible for regulating the initial immune response and inflammation response, when there is damage or infection in cells or the body. When TLR4 is activated due to injury or inflammatory response, NF-κB, a sub-regulator of TLR4, is activated to increase the expression of inflammatory cytokines and secrete inflammatory cytokines into the body (J. Med. Chem. 2020, 63, 22), 13466-13513).
[0350] IL-1β and IL-6 are highly expressed in the synovial membrane, the lesion of rheumatoid arthritis, and this leads to aggravation of the disease. As a result of a recent study in muuse with multiple sclerosis, it has been found that IL-1β activates bystander T cells, which migrate to the spinal cord and release interleukin-17 and interferon-gamma, which are signaling substances that cause autoimmune diseases again and then damage the central nervous system (Nature Communications volume 10, Article number: 709 (2019)).
[0351] Recent biologics block the action of TNF-α or IL-6 receptors, directly interfering with the action of T cells or deplete B cells, and targeted therapies are being developed to prove the effect of RA treatment, T cell inhibition by abatacept and a decrease of cytokine signaling by JAK inhibitors, but unmet needs still remain.
[0352] Biological disease-modifying antirheumatic drugs (BDMARD) developed by targeting TNF-α show excellent effects in the treatment of rheumatoid arthritis, but there are limitations that patients show only a partial response to the treatment, resistance to the treatment, and side effects. Multiple sclerosis treatments have also recently improved the treatment effect due to high efficacy and ease of administration, but they still have a problem in that they lack efficacy for neuroprotection. Because existing treatment methods block only one target among several etiological mechanisms, it is thought that the therapeutic effect decreases or resistance occurs in cases caused by a complex network of mechanisms.
[0353] On the other hand, lipopolysaccharide (LPS) is a large glycolipid molecule located in the outer membrane of Gram-negative bacteria and is a major inducer of TLR4-mediated immune responses. It was known that TLR4 induces Myd88 (myeloid differentiation first response gene 88) and TRIF (Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-13)-dependent pathways, and when MyD88-dependent pathways are initiated, nuclear factor NF-κB and MAPKs are activated to induce the expression of cytokines related to autoimmune diseases (see
[0354] The present inventors confirmed the effect of naltrexone and 6β-naltrexol on the mechanism of TLR4 activation through LPS stimulation, which is a major causative agent of immune-inflammatory mediated response.
[0355] 2. Effect of Naltrexone and 6β-Naltrexol on Expression of Pro-Inflammatory Cytokines (iNOS, IL-1β, IL-6, and TNF-α) in Human Synovial Cell Lines
[0356] The SW982 cell line (ATCC, Manassas, Va., USA), a human synovial cell line, was placed in RPMI 1640 medium (Welgene, Republic of Korea) to which 10% fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 gg/ml streptomycin (Thermo Fisher Scientific Inc.) was added, and cultured in a culture system (Sanyo, Japan) under 37° C. and 5% CO2 conditions. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, Mo., USA).
[0357] After treating SW982 cells with LPS (1 ug/ml) and naltrexone (0.5, 1, 10, 100 uM) or 6β-naltrexol (0.5, 1, 10, 100 uM) for 6 hours, RNA was extracted and cDNA was synthesized. By performing RT-qPCR on the synthesized cDNA, it was confirmed whether the expression of pro-inflammatory cytokines (iNOS, IL-1β, IL-6, and TNF-α) increased by LPS was inhibited, and the results were shown in
[0358] Since IL-1β is the most potent cytokine that induces autoimmune diseases and is also an important marker for clinical indicators of autoimmune diseases such as arthritis, the fact that naltrexone has a higher cytokine inhibitory effect than its metabolite 6β-naltrexol indicates that the low-dose naltrexone sustained-injection of the present disclosure exhibits superior effects compared to the oral naltrexone.
[0359] 3. Effect of Naltrexone and 6β-Naltrexol on Expression of Pro-Inflammatory Cytokines (iNOS, IL-1β, IL-6, and TNF-α) in Murine Macrophage Cell Lines
[0360] The Raw 264.7 cell line (ATCC, Manassas, Va., USA), murine macrophage cell line, was placed in DMEM medium (Welgene, Republic of Korea) to which 10% fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 μg/ml streptomycin (Thermo Fisher Scientific Inc.) was added, and cultured in a culture system (Sanyo, Japan) under 37° C. and 5% CO2 conditions. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, Mo., USA).
[0361] After treating Raw 264.7 cells with LPS (1 ug/ml) and naltrexone (0.5, 1, 10, 100 uM) or 60-naltrexol (0.5, 1, 10, 100 uM) for 6 hours, RNA was extracted and cDNA was synthesized. By performing RT-qPCR on the synthesized cDNA, it was confirmed whether the expression of pro-inflammatory cytokines (iNOS, IL-1β, IL-6, and TNF-α) increased by LPS was inhibited, and the results were shown in
[0362] 4. Effects of Naltrexone and 6β-Naltresol on Activity of NF-κB or Mitogen-Activated Protein Kinases (MAPKs) in Human Synovial Cell Lines
[0363] After treating SW982 cells with LPS (1 ug/ml) and naltrexone (0.5, 1, 10, 100 uM) or 6β-naltrexol (0.5, 1, 10, 100 uM) for 30 minutes, proteins were isolated from the cells, and Western blotting was performed to conform whether the activity of NF-κB and MAPKs was inhibited.
[0364] 1×Cell lysis buffer (Cell signaling, CA, USA) was mixed with protease and a phosphatase inhibitor and added to SW982 cells. After extracting the protein from the cells, the concentration was measured, and the same amount of protein was developed on an SDS-polyacrylamide gel, and transferred to an ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., Piscataway, N.J., USA) and blocked with nonfat dried milk for 1 hour. Thereafter, the reaction was performed with the first antibody (p65 NF-κB, phospho p65 NF-κB, p-ERK, ERK, p-JNK, JNK, p-p38, p38 or calnexin; Cell signaling, CA, USA), while shaking at 4° C. for 24 hours. Then, after shaking and washing 3 times for 10 minutes with PBST buffer, the secondary antibody was incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibody for 1 hour at room temperature, and reacted with the protein band on the membrane using Supersignal west pico ECL solution (Thermo Fisher Scientific Inc.). The results were visualized with the Bio-Rad Gel Documentation system (Bio-Rad Laboratories, Hercules, Calif., USA), and the results are shown in
[0365] As shown in
[0366] That is, as shown in
[0367] 5. Effects of Naltrexone and 60-Naltrexol on Activity of NF-κB or Mitogen-Activated Protein Kinases (MAPKs) in Murine Macrophage Cell Lines
[0368] After treating Raw 264.7 cells with LPS (1 ug/ml) and naltrexone (0.5, 1, 10, 100 uM) or 6β-naltrexol (0.5, 1, 10, 100 uM) for 30 minutes, proteins were isolated from the cells, and Western blotting was performed to conform whether the activity of NF-κB and MAPKs was inhibited. Western blotting was performed in the same manner as in Example 7.4. The results obtained by performing Western blotting were shown in
[0369] As shown in
[0370] Although similar results in both of naltrexone and metabolite 6β-naltrexol were shown, a clear difference (naltrexone showed a distinct decrease compared to 6β-naltrexol) in the cytokine inhibitory effect experiment performed in the item of Example 7.4. suggests that there is an additional autoimmune suppression pathway in addition to the effect through TLR4 in the case of murine macrophage cell line.
[0371] 6. Conclusion
[0372] In this experiment, the effects of naltrexone and 6β-naltrexol on the mechanism of TLR4 activation through LPS stimulation were compared to confirm whether low-dose naltrexone had an antagonist effect by regulating TLR4 activity. As a result, it was found that phosphorylation of p65 NF-κB and MAPKs (ERK1/2, JNK, p38), which are TLR4 signaling subfactors increased by LPS in human synovial cell line (SW982 cell line), was inhibited from 1 uM in the naltrexone-treated group. However, in 6β-naltrexol, an inhibitory effect on the phosphorylation of p65 NF-κB and MAPKs (ERK1/2, JNK, p38) was not observed. That is, it means that the autoimmune inhibitory effect through TLR4 by the orally administered naltrexone is due to non-metabolized naltrexone, not 6β-naltrexol.
[0373] In addition, the result of measuring the production amount of cytokines involved in autoimmunity also showed the same result. When naltrexone was treated compared to 6β-naltrexol, it showed a better inhibitory effect on cytokines such as IL-6, TNF-α, and iNOS related with autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and lupus, and was in particular confirmed that the expression of IL-1β was significantly decreased. The reduction of cytokines such as IL-1β, IL-6, TNF-α, iNOS, etc., by naltrexone treatment is an important result indicating that excellent therapeutic effects may be achieved by inhibiting disease activity of rheumatoid arthritis. Above all, the result of significantly inhibiting the expression of IL-1β means that naltrexone functions as an excellent and effective therapeutic agent for autoimmune diseases, which regulates the activation and inflammatory response of immune cells.
[0374] In addition, in an experiment conducted in murine macrophage cell line (Raw 264.7 cell), blocking of the NF-κB-mediated pathway among the mechanisms acting through TLR4 was observed in both naltrexone and 6β-naltrexol, and it was confirmed that it did not act on the MAPK-mediated pathway. However, specifically, in cytokine inhibition, there was a clear difference between the two substances, and since the distinct difference was shown in the inhibition rate in IL-1β, IL-6, TNF-α, iNOS, etc., it suggests that there are other mechanisms of action that leads to the difference of the inhibition rate between naltrexone and 6β-naltrexol. This fact also indicates that the main substance exhibiting an autoimmune inhibitory effect is naltrexone and not 6β-naltrexol.
[0375] In conclusion, it was confirmed that the substance exhibiting an autoimmune inhibitory effect through TLR4 was naltrexone and not its metabolite 6β-naltrexol, and by confirming the result that naltrexone exhibited a significantly superior autoimmune inflammatory cytokine inhibitory effect compared to 6β-naltrexol, it was found that the sustained injection of the present disclosure, may reduce the lowering of the drug efficacy due to the production of 6β-naltrexol, a representative metabolite occurring in oral drugs due to the first-pass effect, and may reduce side effects, and the difference in metabolic rate between individuals, and maximize the bioavailability and effect of naltrexone in the body, and is superior in terms of safety and effectiveness. In addition, the present disclosure has a very convenient advantage because it eliminates the discomfort and pain of daily administration when using the injection, and may achieve its purpose with a single administration.
[0376] From the results of Examples 6 and 7 above, it was confirmed that it is possible to treat various autoimmune diseases and immune diseases related to TLR4 by using the naltrexone microparticles of the present disclosure that may excellently maintain naltrexone a low dose and effective concentration for a long period of time.
[0377] In addition, if naltrexone is administered at a high dose in a situation with increased inflammation in affected lesion, such as arthritis, the activity of TLR4 in the body might be strongly inhibited and the secretion of inflammatory substances might be temporarily remarkably decreased, but a strong inhibition of TLR4 due to an administration of such a high dose of the naltexone might decrease all of the inflammatory responses in the body, resulting in a sudden loss of immunity. Unlike high-dose administration, the low-dose naltrexone sustained formulation of the present disclosure does not cause a sudden decrease in immunity and maintains a normal immune response and immune system in the patient, even when administered to a patient with an autoimmune disease such as arthritis, while inhibiting TLR4 in the body since it may effectively inhibit the inflammatory response, and thus it will be a safe and excellent method for treating autoimmune diseases.
[0378] From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present disclosure may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.