WO2022066042A1

WO2022066042A1 - Method for treating osteoarthritis and composition of substances for embodying thereof

Info

Publication number: WO2022066042A1
Application number: PCT/RU2021/000253
Authority: WO
Inventors: Oleg Vladimirovich KOLOMYTKIN
Original assignee: Kolomytkin Oleg Vladimirovich
Priority date: 2020-09-24
Filing date: 2021-06-16
Publication date: 2022-03-31
Also published as: RU2020131492A; RU2020131492A3

Abstract

A composition is injected into a painful joint. This composition comprises: (1) the inhibitors of calcium ions exit from the endoplasmic reticulum and other intracellular organelles into the cytosol, (2) the cytoplasmic membrane calcium-channel blockers. The injection of such composition provides synergistically more effective decrease of the concentration of the signal messenger - calcium ions in the cytosol of synovial cells. According to the invention, this results in significant amplification of treating effect of other agents as well as prolongation of composition treating effect.

Description

METHOD FOR TREATING OSTEOARTHRITIS AND COMPOSITION OF SUBSTANCES FOR EMBODYING THEREOF

A group of inventions relates to methods for treating osteoarthritis and to compositions of substances for that treatment, and can be also used for removing inflammation and pain in body tissues.

In osteoarthritis (OA), a cartilage that covers the surface of bone terminals forming a joint is gradually degraded by the action of various enzymes, especially matrix metalloproteinases (MMP) [1, 2]. The MMP are secreted into the synovial fluid by cells that are lining the inner surface of the synovial envelope of the joint. Production of the MMP takes place when those cells are stimulated by various cytokines - pro inflammatory proteins, in particular interleukin- 10 (IL-1 ) and tumor necrosis factor-a (TNF-a) [3]. The destruction of cartilage tissue by metalloproteinases is accompanied with the inflammatory reaction and the joint pain which is the presentation of the osteoarthritis. Despite the availability of a great number of drugs decreasing the pain and inflammation of the joint at arthritis, effective methods for treating osteoarthritis are absent at present time [4].

Clinical efforts are directed toward relief of symptomatic pain. The variety of agents are used for that purpose: analgesics, non-steroidal antiinflammatory drugs, aspirin, naproxen, ibuprofen, COX 2 inhibitors (CELEBREX or VIOXX et al.). It is used interarticular injection of the following agents: steroids, corticosteroids, glucosamines, chondroitins, penicillin and other antibiotics, immunomodulators, viscosupplementation such as hyaluronic acid and derivatives thereof [5]. For some patients, moderate physical exercises can be helpful, as well as physiotherapy such as local heating of the joint for temporal pain relief. In the case of severe osteoarthritis, a joint replacement surgery may be the only option. From the state of the art, there are known methods for treating osteoarthritis by means of injection into the diseased joint a single regulator of a calcium ion channel. That regulator is chosen from the following list: verapamil, nifedipine, diltiazem which are the blockers of L-type Ca²⁺-channel located in the cytoplasmic membrane of the synovial cell [6-8]. In one of those patents, it is suggested to treat the OA by means of a single ionic channel regulator chosen from the following list: tetrodotoxin, procainamide which are blockers of sodium ion channel [8].

However, according to our investigation and experimental data of the abovementioned patents, that method could not decrease the production of MMP and IL- 1 to low level in biopsies of many patients. It is the reason why the medical effect of the methods described in the known patents is weak for many potential patients.

Thus, no one of known methods for treating osteoarthritis could not be named as completely satisfactory. Known methods are directed toward relief of OA symptoms instead of treating the cause of the disease. This is the general disadvantage of known methods. Therefore there is a great need in developing novel methods that treat the cause of osteoarthritis instead of treating its consequences and symptoms.

Patent [6] can be chosen as the closest analog (prototype) of the invention. It has also the essential disadvantage mentioned in the previous paragraph.

The same patent [6] can be chosen as the prototype of the second invention of the group, related to the composition of substances practised for treating osteoarthritis.

The object of both proposed inventions (method and composition) consists in developing such method and composition of substances for treating OA that would remove the prime cause of the disease or significantly weaken it at least. For solving said problem, in the method comprising a step of injecting, into a diseased joint, a regulator (blocker) of L-type calcium ion-channel located in a cytoplasmic cell membrane, in accordance with the invention, one more regulator, a blocker of another type of calcium channel located in a membrane of the endoplasmic reticulum is introduced into the joint additionally.

Besides that, the number of injections during one course of treatment being determined from 1 to 3 with an interval from 1 to 3 weeks adopted to a patient.

A composition of substances for treating osteoarthritis, said composition comprising blockers of calcium ion-channel located in a cytoplasmic cell membrane, in accordance with the invention, said composition comprising additionally blockers of calcium ion exit from endoplasmic reticulum and other intracellular organelles.

Besides that, the composition comprising the inhibitors from 0.001 to 2.5 mg and amount of the additional blockers is from 0.001 to 2.5 mg.

Besides that, the composition comprising additionally a pharmaceutically acceptable carrier from 0.1 to 5 ml.

Besides that, the composition comprising dantrolene from 0.001 to 2.5 mg as the inhibitor of calcium ion exit from endoplasmic reticulum.

Besides that, the composition comprising procaine from 0.001 to 2.5 mg as the inhibitor of calcium ion exit from endoplasmic reticulum.

Besides that, the composition comprising nifedipine from 0.001 to 2.5 mg as the blocker of calcium ion-channels of cytoplasmic membrane.

Besides that, the composition comprising verapamil from 0.001 to 2.5 mg as the blocker of calcium ion-channels of cytoplasmic membrane.

Besides that, the composition comprising diltiazem from 0.001 to 2.5 mg as the blocker of calcium ion-channels of cytoplasmic membrane.

Besides that, the composition comprising additionally physiological saline solution from 0.1 to 5 ml as a carrier. Besides that, the composition comprising additionally viscosupplement from 0.01 to 1 g/1 as a carrier.

The present invention shows that, in order to obtain good evident curing effect in treating osteoarthritis, it is necessary to apply simultaneously two blockers of two different types of Ca²⁺-channels located in two different membranes, in the endoplasmic reticulum and in the cytoplasmic membrane.

A usage of two Ca²⁺-channel blockers in the present invention results in significant amplification of their curing effect to much greater level than maximal effect which is possible to achieve using only one ionic channel regulator as it is suggested in the prototype for example [6].

The usage of the blocker of L-type Ca²⁺-channels located in the cytoplasmic membrane is already known. However, in known patents ([8] for example), this blocker is used alone by itself without simultaneous usage with the regulators - inhibitors of different channel types. By other words, in contrast to the present invention, no one of the previous patents do not use the simultaneous action of regulators of all pathways of calcium (Ca²⁺) entry into the cytosol.

The present group of the inventions uses a combination of the regulators of all possible pathways of calcium ion entry into the cytosol. Consequent amplified effect, arising herewith, in decreasing the Ca²⁺ concentration in the cytosol results in principally new amplified curing effect in comparison with known methods of osteoarthritis treatment.

The essence of the present group of the inventions consists as follows.

It is known that the reason of osteoarthritis is a pathological circle of reactions with positive feedback. Synovial cells of B-type lining the inner side of the synovial membrane have specific receptors of proteins-cytokines IL-1 and TNF-a on their surface. Said signaling protein molecules bind to the receptors with high affinity (at pM concentration) and induce a synthesis of metalloproteinases (MMP) and cytokines, particularly IL- 10, by the synovial cells. In norm, small concentration of MMP in the synovial fluid of the joint slowly degrades the worn and damaged cartilage and helps in its regeneration, i.e. substitution for a new tissue. In osteoarthritis, the synovial cells secrete excessive amount of MMP and IL-lp into the synovial fluid. An excess concentration of MMP degrades the cartilage faster than a new cartilage tissue can be synthesized. It causes osteoarthritis syndromes. The excessive concentration of IL-1 (3 in the synovial fluid results in greater production and secretion of IL-lp and MMP by synovial cells. The synovial lining cells cannot stop or decrease the MMP production due to this positive feedback. It leads to accelerated degradation of the cartilage and fast progression of severe osteoarthritis.

According to the present invention, decreasing the Ca²⁺-concentration in the cytosol of the synovial cells results in disruption of the abovementioned pathological circle of reactions with positive feedback. Owing to this process, a decrease of MMP and cytokine secretion takes place, which leads to curing the OA. It is possible to decrease the Ca²⁺-concentration in the cytosol by means of the blockers of all Ca²⁺-channels through which Ca²⁺-ions enter the cytosol. In known patents, it is possible to block only one pathway of Ca²⁺ entry into the cytosol - from external medium through ionic channels in the cytoplasmic membrane. However, there is one more pathway which is impossible to be regulated by means of the method of the previous patents. It is Ca²⁺ entry into the cytosol from intracellular organelles.

Our new experiments with fragments of synovial biopsies from the human knee joints in vitro allowed us to discover a new astonishing phenomenon that was impossible to figure out theoretically from the known data. A combined usage of two types of substances-regulators of calcium ion concentration in the cytosol resulted in more intensive decreasing the MMP production by synovial cells of OA patients. Said substances-regulators were related to the following two classes: (1) an inhibitor of Ca²⁺ entry into the cytosol from endoplasmic reticulum, (2) a blocker of the Ca²⁺-channels in the cytoplasmic membrane. Tables 1, 2 present experimental results. It was found that a combined application of two types of blockers results in decreasing the MMP production to a level significantly lower in comparison with the methods suggested in the previous patents where only one ionic channel blocker was used [6, 7, 8].

In the present inventions, based on the obtained experimental data, in treating osteoarthritis, the Ca²⁺-ion concentration in the cytosol is decreased using the combined action on two types of Ca²⁺-channels regulating the abovementioned concentration: (1) we inhibit Ca²⁺ entry into the cytosol from the endoplasmic reticulum and other intracellular organelles, (2) we block the Ca²⁺-channels in the cytoplasmic membrane preventing Ca²⁺ entry into the cytosol from external medium.

Two types of the substances providing the above actions have to be introduced together into a diseased joint inside the synovial envelope.

Decreasing the cytosolic Ca²⁺ results in inhibition of the MMP production. The combined injection of two types of substances-regulators into a diseased joint provides stronger curing effect by means of greater decreasing the Ca²⁺-ion concentration in the cytosol of the synovial cells.

According to the present invention, a molecular mechanism of curative action of the method can be as follows. It is known that Ca²⁺ plays a role of signal messenger in intracellular signaling cascade [10, 12]. Therefore according to the invention, it is possible to regulate cell functioning by means of regulation of Ca²⁺-concentration in the cytosol. It is known from the abovementioned references that the synovial lining cells have highly specific receptors of IL- 10 on their surface [9, 10, 12], The receptor binds IL- 10 if it is present in the synovial fluid and then activates a signal protein proteinkinase C (PKC) by means of diacylglycerol (DAG) [15]. It is considered that the PKC switches on expression of MMP, IL- 10, and TNF-a gens by means of purely studied signal transduction pathway [9, 12]. The only IL- 10 is not sufficient for activating the PKC. Ca²⁺ has to be present in the cytosol because Ca²⁺ is the second signaling messenger for PKC in the synovial cells [10, 12].

Therefore, when decreasing the Ca²⁺ concentration in the cytosol, the PKC is deactivated, and thereby the expression of MMP, IL- 10, TNF-a genes is inhibited. As a result, the synovial cell stops to produce those proteins. It results in elimination of cartilage tissue erosion, inflammation decreasing, and cure from OA comes gradually.

It should be noted that, besides the role in the synovial cells, IL- 10 is the main signaling-cytokine initiating inflammation reaction. Therefore, decreasing the expression and, consequently, the synthesis of IL- 10 by synovial cells will result in decreasing the inflammation of OA joint.

The therapeutic action of the group of inventions is based not only on the described mechanism, but is confirmed by experiments shown below. Therefore, the inventions will remain true during the refinement and development of the theory described above.

The particular composition of drugs injected into a joint and providing the high treating effectiveness can comprise the following substances: (1) dantrolene (or procaine) - the inhibitor of Ca²⁺-ion exit from the endoplasmic reticulum, in amount from 0.001 to 2.5 mg, (2) verapamil (or nifedipine) - the blocker of Ca²⁺-ions entry from external medium, in amount from 0.001 to 2.5 mg. The blocking properties of the dantrolene, procaine, verapamil, nifedipine are well known [10, 11, 12, 13].

According to the invention, one method for preparing a pharmaceutical form is as follows. 0.5 mg dantrolene, 0.5 mg verapamil are dissolved in 1 ml physiological solution. The pH-buffer, for example, from 1 to 3 mM HEPES is added into the solution, and pH is adjusted to the neutral value. The prepared solution is poured into an ampulla and sealed at sterile conditions. The solution from the ampulla is used for injection by a syringe into the joint of the patient. The methods for preparing and administering, as well as the composition formulation are not limited to the abovementioned. They can be changed if it is necessary. One course of treatment includes from 1 to 3 injections of the composition into the joint with an interval from 1 to 3 weeks. The number of injections and the doses should be adapted to a specific patient.

Substances used in the present inventions do not show significant adverse effects when using in the above concentrations. This follows from the fact that they are already applied in medical practice for treating other diseases. For example, dantrolene is used for stopping malignant hyperthermia and treating muscle hypertonia [11], verapamil is used for treating cardiac diseases [13].

The invented composition of substances can be injected into the joint alone or together with other useful additives, including but not limited to injectable agents such as: saline solution, viscosupplements, liposomes, steroid hormones, pain relief and antibacterial agents. The combined introduction of the invented composition together with other components results in amplification of the treating effect of other agents, as well as prolongation of treating action of the invented composition.

The present inventions have been confirmed by experiments with synovial tissue biopsies from human knee joints of six patients. The synovial tissue biopsies were taken from the patients having osteoarthritis of grade IV (Kellgren-Lawrence scale) during knee replacement surgery. Each fragment of the synovial tissue of the standard mass about 20 mg was placed into a Petri dish with 1 ml nutrition medium. Then, the invented biologically active substances were added into the Petri dish, and the dishes were incubated for 48 hours at 37 °C. After incubation, the enzyme activity of the matrix metalloproteinases in the medium was measured by the method that we have developed previously [14]. Each experiment value of MMP enzyme activity was obtained by averaging results of three identical experiments with three different synovial biopsies from the same patient. For each Petri dish, the MMP activity was measured in triplicate, and the results were averaged. The experimental value of MMP enzyme activity was present as the mean value ± the standard error.

High MMP enzymatic activity means that a cartilage layer in the joint will be degraded quickly in the same way as it happens at osteoarthritis. Low MMP enzymatic activity means that the cartilage layer in the joint is not degraded and remains intact in the same way as it happens in the healthy joint.

EXAMPLE 1. Patient: 74 years old female, 02.03.2015.

EXAMPLE 2. Patient: 77 years old male, 02.10.2015.

EXAMPLE 3. Patient: 70 years old female, 03.24.2015.

EXAMPLE 4. Patient: 69 years old female, 04.14.2015.

EXAMPLE 5. Patient: 63 years old female, 04.21.2015.

EXAMPLE 6. Patient: 67 years old male, 05.12.2015.

The control Trypan Blue dye test has confirmed that the used blockers did not kill the synovial cells.

For each patient, two control experiments were carried out: (1) Petri dish was filled with the nutrition medium alone without any other additives, (2) 100 ng/1 IL-1 p was added into the Petri dish. For each patient, the MMP enzymatic activity in the second case was taken as 100%. In the first experiment without IL-1 p, the MMP enzymatic activity was less than 7% for all 6 patients.

The last result means that replacement of the synovial fluid in the OA joint for saline solution without bioactive agents could produce the effect like curing one because it decreased MMP production. However, such “curing effect” was weak and unstable because it was reversible. Addition of IL-ip again stimulated MMP production as it had been shown by the second control experiment.

Contrary to the saline solution, the invented composition of substances produces strong irreversible curing effect even in the presence of IL-ip as shown in Tables 1 and 2. Table 1.

Table 1 presents the results of experiments at examples (1, 2, 3), which experiments were carried out in the presence of 100 ng/1 IL-ip with the following two Ca²⁺-channel blockers: 25 mg/1 dantrolene and 25 mg/1 verapamil.

Table 2 presents the results of experiments at examples (4, 5, 6), which experiments were carried out in the presence of 100 ng/1 IL-ip with the following two Ca²⁺-channel blockers: 25 mg/1 procaine and 25 mg/1 verapamil. In all those experiments for all six patients, the MMP enzymatic activity was in the interval from 19 to 23%.

Table 2.

In order to confirm the amplified effect of the composition of two blockers, we carried out experiments with separate substances in the presence of IL- 10. Three series of experiments with the following additives into the Petri dishes: (1) 100 ng/1 IL- 10, 50 mg/1 dantrolene; (2) 100 ng/1 IL- 10, 50 mg/1 procaine; (3) 100 ng/1 IL- 10, 50 mg/1 verapamil have shown the decrease of MMP enzymatic activity to values presented in Tables 1 and 2. We used saturating concentrations of dantrolene, procaine and verapamil. Increasing their concentrations did not decrease MMP activity.

It follows from the Tables that the composition of substances decreased the MMP production to a lesser level in comparison with what can be achieved in using them separately. Since this effect took place in the presence of IL- 10, then a strong and stable curing effect had been achieved.

It followed from the experimental data that administering the blocker of L-type Ca²⁺-channel located in the cytoplasmic membrane (verapamil) alone to abovementioned patients is not enough to inhibit the MMP production by synovial cells to a low level. For this purpose it is necessary to inhibit the Camchannels in the cytoplasmic membrane and Ca²⁺ exit from the endoplasmic reticulum simultaneously.

Therefore, the experimental results confirm the amplified curing effect of the invention and show its advantage in comparison with the previous patents.

References

1. Glyn-Jones S, Palmer AJ, Agricola R, Price AJ, Vincent TL, Weinans H, Carr AJ. "Osteoarthritis". Lancet. 2015, 386 (9991): 376-387.

2. Osteoarthritis, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, NIH Publication No. 02-4617. Jul. 2002.

3. Baratz, M. E., H. I. Georgescu, and C. H. Evans. Studies on the autocrine activation of a synovial cell line. J. Orthop. Res. 1991, 9:651-657. 4. Oo, Win Min; Yu, Shirley Pei-Chun; Daniel, Matthew Sean; Hunter, David John. Disease-modifying drugs in osteoarthritis: current understanding and future therapeutics. Expert Opinion on Emerging Drugs. 2018, 23 (4): 331- 347.

5. Zhang W, Moskowitz RW, Nuki G, Abramson S, Altman RD, Arden N, Bierma-Zeinstra S, Brandt KD, Croft P, Doherty M, Dougados M, Hochberg M, Hunter DJ, Kwoh K, Lohmander LS, Tugwell P. OARSI recommendations for the management of hip and knee osteoarthritis, part I: critical appraisal of existing treatment guidelines and systematic review of current research evidence. Osteoarthritis and Cartilage. 2007, 15 (9): 981-1000.

6. US Patent N° 7767710, Int. Cl. A61K 31/00, 2010.

7. US Patent Ns 8329746, Int. Cl. A61K 31/455, 2012.

8. US Patent Na 8557865, Int. Cl. A61K 31/353, 2013.

9. Kolomytkin O.V., Marino A.A., Sadasivan K.K., Wolf R.E., Albright J.A. Interleukin- IB switches electrophysiological states of synovial fibroblasts. American Journal of Physiology (Regulatory, Integrative and Comparative Physiology). 1997, 42: R1822-R1828.

10. Sadasivan K., Kolomytkin O., Marino A., Wolf R., Albright J. Interleukin- ip is transduced by synovial fibroblasts via a pathway involving protein kinase C and Ca²⁺ influx. The FASEB Journal, 1998, v. 12, N4, Abstracts, part 1, Experimental Biology 98, San Francisco, CA, p. A437.

11. Krause T, Gerbershagen MU, Fiege M, Weisshom R, Wappler F. Dantrolene - a review of its pharmacology, therapeutic use and new developments. Anaesthesia. 2004, 59 (4): 364-373.

12. Kolomytkin O.V., Marino A.A., Sadasivan K.K., Wolf R.E., Albright J.A. Intracellular signaling mechanisms of Interleukin- IB in synovial fibroblasts. American Journal of Physiology (Cell Physiology). 1999, 45:C9- C15. 13. Hughes A. Calcium channel blockers. In Bakris G, Sorrentino M. (eds.). Hypertension: a companion to Braunwald's heart disease (Third ed.). Philadelphia, PA: Elsevier Health Sciences. 2017, pp. 242-253.

14. Kolomytkin O.V., Marino A. A., Waddel D.D., Mathis J.M., Wolf R.E., Sadasivan K.K., Albright J.A. IL-10-induced production of metalloproteinases by synovial cells depends on gap junction conductance. American Journal of Physiology (Cell Physiology). 2002, 282:C1254-C1260.

15. Mellor H, Parker PJ (Jun 1998). "The extended protein kinase C superfamily". The Biochemical Journal. 332. 332 (Pt 2): 281-92.

Claims

1. A method for treating osteoarthritis comprising a step of injecting, into a diseased joint, a blocker of L-type calcium ion-channel located in a cytoplasmic cell membrane, wherein one more regulator, a blocker of another type of calcium channel located in a membrane of endoplasmic reticulum being introduced into the joint additionally.

2. The method of claim 1, wherein the number of injections during one course of treatment being determined from 1 to 3 with an interval from 1 to 3 weeks adopted to a patient.

3. A composition of substances for treating osteoarthritis, said composition comprising blockers of calcium ion-channel located in a cytoplasmic cell membrane, wherein said composition comprising additionally blockers of calcium ion exit from endoplasmic reticulum and other intracellular organelles.

4. The composition of claim 3, wherein amount of the inhibitors in said composition being from 0.001 to 2.5 mg, and amount of the additional blockers being from 0.001 to 2.5 mg.

5. The composition of claim 4, wherein said composition comprising a pharmaceutically acceptable carrier from 0.1 to 5 ml.

6. The composition of claim 3, wherein said composition comprising dantrolene from 0.001 to 2.5 mg as the inhibitor of calcium ion exit from endoplasmic reticulum.

7. The composition of claim 3, wherein said composition comprising procaine from 0.001 to 2.5 mg as the inhibitor of calcium ion exit from endoplasmic reticulum.

8. The composition of claim 3, wherein said composition comprising nifedipine from 0.001 to 2.5 mg as the blocker of calcium ion-channels of cytoplasmic membrane.

9. The composition of claim 3, wherein said composition comprising verapamil from 0.001 to 2.5 mg as the blocker of calcium ion-channels of cytoplasmic membrane.

10. The composition of claim 3, wherein said composition comprising diltiazem from 0.001 to 2.5 mg as the blocker of calcium ion-channels of cytoplasmic membrane.

11. The composition of claim 3, wherein said composition comprising additionally physiological saline solution from 0.1 to 5 ml as a carrier.

12. The composition of claim 3, wherein said composition comprising additionally viscosupplement from 0.01 to 1 g/1 as a carrier.