RU2586285C1 - Aqueous composition for instillation in bladder - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: described is aqueous composition for instillation in urinary bladder based on hyaluronic acid and chondroitin sulphate. Composition also contains mannitol or sorbitol in relation to sum of hyaluronic acid and chondroitin sulphate, equal to 0.014-4.2. Said composition can additionally contain zinc chloride.

EFFECT: invention provides high protective effect on urothelium cells after oxidative stress and increased mediated antimicrobial action due to inclusion of mannitol or sorbitol in composition, high direct antimicrobial action and protection of urothelium due to inclusion of zinc chloride, as well as controlled viscosity due to inclusion of mannitol and zinc chloride.

1 cl, 4 tbl, 6 ex

Description

The invention relates to medicine and the pharmaceutical industry and is a solution for temporary protection of the mucous membrane of the bladder with various kinds of manipulations: with urethrocystoscopy, radiation therapy, in the treatment of cystitis of various etiologies that cause irritation of the bladder wall (chronic cystitis, radiation cystitis interstitial cystitis), etc.

Symptoms of interstitial cystitis and other cystitis of various etiologies arise due to a defect in the glycosaminoglycan (GAG) component of the mucin layer that covers and protects the urothelium of the bladder. The treatment is based on the use of "substitutes" for glycosaminoglycan, for example hyaluronic acid, with intravesical administration. Irritants in the urine, in particular potassium ions, can penetrate through urothelium, causing inflammation, irritation and tissue damage, degranulation of mast cells and depolarization of sensory nerves, which leads to urgent and frequent urination and pain with interstitial cystitis. Glycosaminoglycans are highly hydrophilic and have a protective effect. They provide a stable layer of water between the urothelium and the lumen of the bladder and prevent bacteria, microcrystals, ions, proteins, and other substances in the urine from attaching to and penetrating into the urothelium.

Under the influence of urogenital infections, urothelium of the bladder is damaged with the formation of squamous metaplasia (leukoplakia). In the light of modern research, leukoplakia is a pathological process that is characterized by a violation of the basic functions of the stratified squamous epithelium: the absence of glycogen formation and the occurrence of keratinization, which are normally absent. Infection is a causative factor in damage to urothelium and the formation of metaplasia.

Surgery, inflammatory changes in the bladder and adenoma bed, as well as hormonal imbalances in older men and women violate the protective layer of urothelium. The use of hyaluronic acid preparations in this case will also be pathogenetically justified, since it will make it possible to restore the protective layer of the wall of the bladder and urethra. The resulting viscoelastic protector, or buffer, between urothelium and the lumen of the bladder will help to reduce the irritating effect of urine and its components on the postoperative wound surface and eliminate dysuric phenomena.

Hyaluronic acid, a polysaccharide from the glucosaminoglycan family (mucopolysaccharide), has the ability to bind 1000 times more water than it weighs. Due to its high hydrophilicity, hyaluronic acid forms highly viscous gels with water that moisturize the mucous membrane. Hyaluronic acid is non-toxic. As part of the gels, hyaluronic acid has an antimicrobial and anti-inflammatory effect, promotes the regeneration of the epithelium, and does not cause clinical complications.

A complete analogue of hyaluronic acid - sodium hyaluronate (sodium salt of hyaluronic acid), is soluble in water with the formation of a viscous colorless gel with a pH of 6.0-7.5 (0.1% solution).

Being one of the most important components of connective tissue, hyaluronic acid plays an important role in the process of tissue growth and regeneration, fertilization and reproduction, permeability of cell membranes and in many other processes that ensure the normal functioning of body systems.

There is a large assortment of drugs for external use as an anti-inflammatory agent and for the healing of wounds containing hyaluronic acid (European application 0433817, RF patents 2048817, 2195262, US patent 5604200). These drugs are not used in urology for the treatment of the mucous membrane of the bladder. Hyaluronic acid and chondroitin sulfate are also known drugs used to treat this disease (US Pat. No. 5,591,724, US Pat. No. 6,083,933, US Pat. No. 7,772,210).

Known methods for treating bladder hyperactivity by instillation using various drugs, for example, an anticholinergic drug containing oxybutynin hydrochloride (US Pat. RF 2193401), glycosaminoglycan analogues, which use heparin (US Pat. RF 2390341), a genetically engineered analogue of endogenous cytokine (US Pat. . RF 2361607), a solution of protargol and a solution of silver nitrate (Rod OI Etiology, clinic and treatment of non-chronic urethritis. Medical practice, 1964, No. 10, p. 141).

A known therapeutic composition for the treatment of cystitis based on sodium hyaluronate diluted with saline (P.V. Glybochko, Yu.G. Alyaev, Z.K. Gadzhieva, Mirkin Ya.B. Use of hyaluronic acid in the treatment of chronic cystitis. EF. Urology. 5/2011, pp. 30-35). The disadvantage of this composition is the short-term adhesion to urothelium of the bladder, which reduces the possibility of evacuation of pathogenic microorganisms that cause cystitis, requires frequent administration of the drug and does not provide a long-term effect.

Known medicinal composition for the treatment of cystitis (US Pat. RF 2519010). The composition contains 0.8-2.0% wt. sodium hyaluronate, 1-3% wt. sodium alginate and distilled water to 100% of the mass. The composition has insufficient prolonged adhesion. The presence of relapses of cystitis was noted.

Currently, Uro-Gial is widely used, having the following composition: sodium hyaluronate - 0.8 mg, a buffer solution including sodium dihydrogen phosphate monohydrate - 0.45 mg, anhydrous disodium phosphate - 2 mg, sodium chloride - 8.5 mg ( ± 0.05), and deionized water. The preparation is an elastic viscous buffered physiological solution of sodium hyaluronate of non-animal origin. It is used in urology for the treatment of chronic recurrent and interstitial cystitis, radiation cystitis, and painful bladder syndrome. Uro-Gial is a viscoelastic protector of the intercellular substance of urothelium of the bladder, urethra. When introduced into the cavity of the bladder, it creates a viscoelastic film on the surface of the mucous membrane that protects it from external influences during various kinds of manipulations (urethroscopy, cystoscopy, TUR of the prostate and bladder, radiation therapy of the pelvic organs, etc.). A disadvantage of the known preparation: incompatibility between sodium hyaluronate and quaternary ammonium salts, such as benzalkonium chloride, is noted. Uro-Gial should not be allowed to come into contact with such drugs or with medical and surgical instruments treated with such drugs. Dosage and administration: Intravesically, once a week during the first month, then once every two weeks during the second month, then once a month until the symptoms disappear. The course is from 4 to 12 instillations.

A known aqueous composition containing hyaluronic acid, chondroitin sulfate and a hydrochloric acid salt of a divalent metal, namely calcium chloride (WO 2007138014). The composition is intended for use in the treatment of interstitial cystitis for instillation into the bladder. The disadvantage of this composition is the complexity of viscosity control. This composition is selected as a prototype.

The above indicates the feasibility of developing a new drug with a wider spectrum of action based on the main pathogenetic features of diseases of an infectious and inflammatory nature, accompanied by destructive processes or a predisposition to them.

The technical task of the invention is the creation of a new aqueous composition with increased bacteriostatic and anti-inflammatory effects, with controlled viscosity, pronounced pharmacological activity for the treatment of chronic recurrent and interstitial cystitis, radiation cystitis, painful bladder syndrome, which has a high therapeutic activity, allowing to reduce unwanted side effects by providing prolonged adhesion of the created composition and to the glycosaminoglycan layer of urothelium, as well as creating obstacles to the adhesion of bacteria to damaged tissues by covering urothelium with a biopolymer film.

The technical result of the invention is the creation of a new aqueous composition, which has:

- increased protective effect against urothelium cells after oxidative stress and increased indirect antimicrobial effect due to the inclusion of mannitol or sorbitol in the composition;

- increased direct antimicrobial action and protection of urothelium due to the inclusion of zinc chloride;

- controlled viscosity due to the inclusion of mannitol or sorbitol and zinc chloride.

The specified technical result is achieved when using an aqueous composition for instillation into the bladder based on hyaluronic acid and chondroitin sulfate, characterized in that the composition additionally contains mannitol or sorbitol in the ratio to the sum of hyaluronic acid and chondroitin sulfate equal to 0.014-4.2, in the following the content of components in an aqueous solution, mass. %:

hyaluronic acid - 0.05-1.6

chondroitin sulfate - 0.07-2.0

mannitol or sorbitol - 0.05-0.5

water - the rest is up to 100%

In addition, the aqueous composition further comprises zinc chloride, taken in an amount of 0.7-1.9 mass. %

Chronic cystitis is a disease that is characterized by a long-existing inflammatory process in the tissue of the bladder, both bacterial and abacterial in nature. Chronic cystitis can occur in the form of both exudative and productive forms of inflammation. With exudative inflammation, a proliferation or reparative regeneration phase begins. But, very often in the tissues of the bladder can develop several tricks of exudative inflammation at small intervals, i.e. there is a layering of the productive phase of the previous episode of acute exudative inflammation on the exudative phase of the subsequent.

Thus, in chronic cystitis of various etiologies, including the bacterial nature of the disease, as well as abacterial forms of chronic cystitis, including post-radiation cystitis, chronic cystitis in postmenopausal women, changes in the mucous membrane of the bladder are of the same nature. The basis of inflammatory infiltrate in the mucous membrane and submucosal layer of the bladder wall is damage to the interstitial substance of the connective tissue due to various reasons. Regardless of these reasons, the outcome is a violation of the physiological regeneration of urothelium of the bladder, with a violation of the barrier function of the transitional epithelium. These changes lead to dysregenerative processes in urothelium followed by a change in the physiologically substantiated distribution of subpopulations of mononuclear cells. These changes are interpreted as interstitial inflammation with a manifestation of glycosaminoglycan deficiency.

It seems promising to develop a new composition with the inclusion of an additional substance that can reduce the effect of free radicals on hyaluronic acid and chondroitin sulfate under conditions of oxidative stress in order to prolong the action of hyaluronic acid and chondroitin sulfate in the bladder and increase the clinical effectiveness of treatment. Accordingly, a composition having a prolonged action will exhibit improved anti-inflammatory and antimicrobial properties, which should directly increase the effectiveness of the treatment of chronic recurrent cystitis, interstitial cystitis, radiation cystitis, chronic cystitis in menopausal women, as well as painful bladder syndrome.

Additionally, it is advisable to include in the composition a component that could enhance the antimicrobial effect of the composition due to the direct antimicrobial effect.

Chondroitin sulfate binds water molecules on the surface of the urothelium of the bladder, creating a protective waterproof barrier, and hyaluronic acid, in turn, increases the number of bound water molecules, thereby creating conditions for the rapid restoration and enhancement of the protective function of the glycosaminoglycan layer.

Moreover, chondroitin sulfate provides an anti-inflammatory effect, inhibits the activity of enzymes that can destroy hyaluronic acid, thereby prolonging the therapeutic effect.

Chondroitin sulfate is involved in the biosynthesis of connective tissue and, due to this, helps prevent the destruction of connective tissue, stimulates tissue regeneration and has an anti-inflammatory effect. Included in the structure of urothelium of the bladder. It is proved that chondroitin sulfate stimulates the formation of hyaluron, the synthesis of proteoglycans, inhibits the activity of elastase, hyaluronidase. Chondroitin sulfate inhibits the collapse of connective tissue.

In any local infectious and inflammatory processes due to the activity of microorganisms, a number of toxic substances are released - exo and endotoxins, enzymes, mitogens, lipopolysaccharides, etc., which leads to tissue destruction, destruction of connective tissue, and an increase in the reaction of lipid peroxidation. To stop these processes, the presence of auxiliary compounds in the composition is required.

As auxiliary substances that provide the necessary rheological properties of the solution, contributing to the prolongation of the effect of the main components, the authors used mannitol, sorbitol. Mannitol and sorbitol are hexatomic alcohols and have antioxidant properties that contribute to better preservation of the composition, its physical and biological parameters. Additionally, mannitol and sorbitol prevent the destruction of hyaluronic acid under the influence of hyaluronidase, thereby prolonging the effect of the drug.

It is further preferred to use zinc chloride to lower the viscosity of the hyaluronic acid-based aqueous solution. This substance has an antimicrobial effect, which has a positive effect on the treatment of damage to urothelium, concomitant with the development of pathogenic microflora.

The combined use of these components in the inventive solution, as shown by experiments, improves the physical, in particular rheological properties of the drug and helps prolong the action.

The possibility of carrying out the invention is confirmed by the following examples.

Example 1. The method of preparation of the solution

To prepare 100 ml of a solution, 1.3 g of zinc chloride and 0.1 g of mannitol are dissolved in 80 ml of purified water (stirred for at least 15 minutes). To the resulting solution was added 0.08 g of hyaluronic acid, 0.1 g of chondroitin sulfate and the remaining water to 100 ml. The mixture is thoroughly mixed to a homogeneous mass (about 4 hours), filtered through a 0.8 μm filter. Sterilize in an autoclave for 15 minutes at a temperature of 121 ° C. Table 1 shows all the compositions of the claimed composition.

Example 2. Determination of antibacterial activity

The specific activity of the pharmacological agent is determined by diffusion into agar. 0.1 ml of E. coli is poured into a Petri dish with meat-peptone agar, the suspension of gram-negative microorganisms is evenly distributed, discs are applied to the surface of the seeded medium, and the proposed solution is applied to the discs (composition 1). The cups are incubated for 24 hours at a temperature of 37 ° C. The results are taken into account by measuring zones of growth inhibition. When determining the antibacterial properties of the solution in relation to ml of E. coli, the presence of growth inhibition zones was detected, the diameter of the growth inhibition zone ranged from 17.6 to 35.0 mm, which indicates the sensitivity of this microorganism to the test drug. The presence of zinc chloride in the solution leads to an increase in the suppression effect of ml of E. coli.

To confirm the best adhesion to urothelium of the proposed solution in the treatment of cystitis, the following studies were carried out.

Example 3. Confirmation of adhesion to urothelium

To determine the adhesion of the proposed solution to biological tissues, thin collagen-elastin films (collagen and elastin, the basis of connective tissue) were cast using a specially developed technique. The films were dried under pressure at 25-35 ° C, after which an aqueous composition of hyaluronic acid, chondroitin sulfate, mannitol, zinc chloride (in the ratio specified in the formula) was applied to the outer side of the film 1 and, in parallel, for comparison, with the Uro-Gial preparation . Then the film with a layer of polymer composition coated with a second collagen film, kept under pressure for 10, 20, 30, 40, 50, 60 minutes (6 samples for each composition). Then, the films joined by the composition were installed between the fixed lower clamp and the movable upper and the work of separation of the films from each other was determined. The tests were performed on a Brookfield CT3 texture analysis device. The experiments showed that the proposed composition adhesion compared with the drug Uro-Gial is 2-4 times higher. To determine the degree of protection of urothelium from the aggressive effects of the bladder medium, a seven-layer membrane was assembled from pre-cast collagen films, the proposed composition was applied to it, it was kept for 15 to 60 minutes, it was exposed to alkali solutions and the degree of membrane destruction was observed. In parallel, an experiment was conducted with the drug Uro-Gial.

The experiments showed that the proposed aqueous composition based on hyaluronic acid provides protection of urothelium to an aggressive environment of the bladder by 25% compared with the drug Uro-Gial.

Example 4. The study of antioxidant activity

To study the antioxidant activity of the composition, the solution was subjected to oxidative stress generated by the addition of hydrogen peroxide at a concentration of 2.5%. The rheological properties of the solutions were studied after exposure to oxidative stress of variable duration at room temperature on a Haake viscotester 7 plus instrument (Thermo scientific, Germany). Viscosity was measured using a cone plate of a rheometer at 25 ° C (see table 2). Without mannitol, hyaluronic acid quickly decomposes under the influence of peroxide, which is characterized by a decrease in viscosity (composition 2).

The rheological parameters of a solution of hyaluronic acid (0.08%) and chondroitin sulfate (0.1%) containing 0.1% mannitol did not change in the presence of hydrogen peroxide for 30 minutes (composition 1).

Thus, the presence of mannitol provides the antioxidant properties of the solution, does not allow free radicals to destroy the molecules of hyaluronic acid and chondroitin sulfate, stabilizes the viscosity of the solution, prevents the destruction of the glucosaminoglycan film on the inner wall of the bladder, which ensures a prolonged therapeutic effect.

Example 5. Research on animal models

In order to evaluate the effectiveness of a composition containing hyaluronic acid, chondroitin sulfate, mannitol or sorbitol and, preferably, zinc chloride in the claimed intervals for the restoration of urothelium of the bladder, an experimental part was developed consisting in modeling the pathological process in the bladder wall that occurs against the background of a long-existing inflammatory process and oxidative stress with interstitial cystitis.

Its essence was to model oxidative stress and the formation of extremely reactive free radicals that can induce the destruction of urothelial cells of the bladder due to the destruction of the glycosaminoglycan layer and, at the same time, reduce the effectiveness of intravesical therapy with glycosaminoglycine substitutes, because destroy the glycosaminoglycan molecule, which is part of the drug.

A tampon was injected into the bladder of rats (15 rats, five rats in each group) for 10 seconds moistened with a solution of hydrogen peroxide at a concentration of 10 mM, and a pathological lesion was simulated, the morphological changes in which can be interpreted as focal interstitial cystitis. Then the animals were divided into three groups. After 24 hours, the following solutions were injected through a catheter into an empty animal bladder:

1st group (n = 5): test composition (examples 1, 5, 6, table. 1),

2nd group (n = 5): a solution of the drug "Uro-Gial",

3rd group (control, n = 5): physiological saline.

These solutions were administered 1 time per day for 5 days, the exposure of these substances was in the bladder of animals for 30 minutes. The rats were then sacrificed with an overdose of sodium thiopental and the bladders were removed.

The result was assessed by the degree of damage to urothelial cells of the bladder. Tissue samples were stained with 1% Janus Green B dye solution (Sigma, Germany) for 90 seconds. Janus green dye B selectively stains areas damaged by the simulated oxidative stress process, and the degree of staining (degree of metachromasia) depends on the degree of tissue damage. An electron microscopic examination also revealed the destruction of collagen fibers with the loss of their transverse striation, as well as the state of arterioles and venules in the interstitial connective tissue of animals.

The most pronounced lesions were observed in the control group (saline). Pronounced Janus green color was present in histological samples of animals treated with Uro-Gial. The smallest damage and stabilization of collagen fibers during electron microscopic examination was observed in a histological sample of animals treated with the studied composition, table. 3. Stabilization of collagen fibers was characterized by partial preservation of their transverse structure.

The greatest tendency to increase the average size of arterioles and venules (Table 4) was observed in the group of animals treated with the studied composition. This trend is positive, as we can speak of an increase in the activity of the vessels of the microvasculature under the action of a composition including hyaluronic acid, chondroitin sulfate and mannitol or sorbitol.

Thus, the effect of the studied composition in comparison with the well-known Uro-Gial drug and the control group in experimental modeling of oxidative stress was to reduce the effect of active radicals on urothelium cells due to the longer action of the formed protective film, because the effect of free radicals on glycosaminoglycans is reduced.

Example 6. The use of the proposed composition

Patient A., 36 years old, suffers from recurrent bacterial cystitis, the frequency of relapses 10-12 relapses per year. I had extensive experience in treating my disease in the form of a standard conservative treatment, but without a satisfactory effect. I received treatment with the claimed composition. A course of intravesical instillations for 8 weeks. The first month once a week. Further, the drug is administered once every 2 weeks for another month, and after a course of therapy with this composition, no relapse was observed for 1 year. Treatment efficacy was evaluated clinically, endoscopically and morphologically. Against the background of the patient’s treatment, a significant improvement in well-being was noted: the pain behind the bosom stopped, urgent urination, and pollakiuria disappeared. During control cystoscopy with biopsy, a regression of leukoplakia and squamous metaplasia was observed. The drug was well tolerated.

In the treatment of patients included in our study with the use of the proposed composition, the severity of the reaction of edema of the walls of the bladder is significantly reduced, the capacity of the bladder increases by an average of 40 ml, after treatment there is a pronounced positive dynamics: in some patients the pain stopped completely, the remaining patients noted a significant reduction of pain. The indicator of the number of cystitis relapses decreased significantly - more than 2 times, which had a positive effect on the quality of life of patients, the psychoemotional state improved.

FINDINGS. Analyzing the obtained experimental data, we can conclude that due to the presence in the proposed composition of a combination of hyaluronic acid, chondroitin sulfate, mannitol or sorbitol, and preferably zinc chloride, the therapeutic activity of the drug is enhanced, which allows this solution to counteract the spread and multiplication of bacteria, reduce microbial contamination of the mucous membrane , cleanse it of necrotic plaque, accelerate epithelization and maturation of granulation tissue, reduce swelling, iperemiyu effectively reduce inflammatory response. The addition of a polyhydric alcohol (mannitol or sorbitol) prevents exposure to free radicals. The presence of zinc chloride in the solution provides a lower viscosity of the hyaluronic acid solution. The resulting solutions have new viscoelastic properties and a prolonged effect.

The resulting solutions have an optimal viscosity, which allows the solution to be applied through a catheter and ensures the formation of a film on the inner wall of the bladder and it remains stable when exposed to free radicals.

Claims (2)

1. An aqueous composition for instillation into the bladder based on hyaluronic acid and chondroitin sulfate, characterized in that the composition further comprises mannitol or sorbitol in the ratio to the sum of hyaluronic acid and chondroitin sulfate equal to 0.014-4.2, with the following content of components in aqueous solution, wt.%:
hyaluronic acid 0.05-1.6 chondroitin sulfate 0.07-2.0 mannitol or sorbitol 0.05-0.5 water the rest is up to 100% 2. The aqueous composition according to p. 1, characterized in that it further comprises zinc chloride, taken in an amount of 0.7-1.9 wt.%.