WO2023063811A1 - Formulation for the management and antiviral, immunomodulatory and anti-inflammatory treatment of infection by sars-cov2 virus (covid-19) and its variants, formulated from glycyrrhizinic acid salt and derivatives of its chemical structure, as well as antitussive agents and antihistamines in syrup form - Google Patents

Abstract

The present invention relates to a gel formulation of ammonium salt of glycyrrhizinic acid as active agent with a thermo-reversible gel former such as polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE) as a vehicle, characterised in that it comprises POE-POP-POE in concentrations of 20%-30% (w/w) and the active agent is in concentrations of 10 mg/ml, 20 mg/ml, 100 mg/ml and 200 mg/ml. The invention further relates to the process for preparing said formulation and to the use thereof in the preparation of a medicinal product for therapeutic use in SARS-CoV-2 infections and pathologies associated with COVID-19.

Description

FORMULATION FOR THE MANAGEMENT AND ANTIVIRAL, IMMUNOMODULATOR AND ANTI-INFLAMMATORY TREATMENT OF THE INFECTION BY THE SARS-COV2 VIRUS (COVID-19) AND ITS VARIANTS, FORMULATED FROM THE SALT OF GLYCYRRHINIC ACID AND ITS DERIVATIVES OF ITS CHEMICAL STRUCTURE, AS WELL AS AGENTS ANTITUSSIVES AND ANTIHISTAMINES IN SYRUP.

FIELD OF THE INVENTION.

The present invention, as expressed in the following statement, refers to the development of a new formulation of the compound glycyrrhizinic acid (AG) or its derivatives in a syrup formulation after dissolving the compound in gel and propylene glycol as a solvent and absorption enhancer, and later the adjustment of the pH for its physiological conditioning and addition of antitussives for its administration to patients with infection and clinical manifestations of CoVid-19.

OBJECT OF THE INVENTION.

The objective of the present invention is to develop a pharmaceutical formulation for oral administration in the therapeutic management of SARS-Cov2 infections and its associated pathology COVID-19. This formulation decreases the viral load due to the inhibition of the penetration of coronaviruses into the target cells, preventing replication, as well as interfering in the viral cycle; changing its evolution. It also has an immunomodulatory effect generating an antiviral immune response. The formulation is added with agents with a proven and recognized antitussive effect such as dextromethorphan hydrobromide and guaifenesin.

BACKGROUND.

The COVID-19 pandemic represents a major public health crisis worldwide, where despite accelerated research studies, there is still no specific antiviral drug for the prevention or treatment of COVID-19. Since the first case was reported, more than two hundred countries have also reported cases of COVID-19 and the number of cases of infections reported worldwide is 183,726,541, which has caused 3,975,915 deaths. In Mexico, with a total of 2,537,457 cases and 233,580 deaths, it is in third place, just below countries like the United States and Brazil.

Coronaviruses are a family of viruses that have been the cause of various epidemic outbreaks, SARS in 2003, MERS in 2012 and currently the COVID19 pandemic. SARS-CoV2 is a new coronavirus that is characterized by being an enveloped virus and containing RNA as genetic material, which comes from bats and is transmitted from human to human mainly through droplets from the respiratory tract, although it can also occur through aerosols; causing a highly infectious and pathogenic disease of the airways of variable severity. The incubation period in most cases is from three to seven days, and can be up to fourteen days, and 95% of patients develop symptoms within thirteen days after exposure. The immune response represents the fundamental mechanism for effective control of the infection; however, a poorly regulated response can trigger greater lung damage and consequently greater patient mortality. The factors that generate this immune response are rapid viral replication, its ability to recruit immune system cells, mainly macrophages and neutrophils, the release of inflammation-mediating cytokines, as well as the decreased number and response of T lymphocytes. During the infection, an exacerbated immune response against the virus begins, which is responsible for the different clinical manifestations and the damage to the respiratory epithelium, which would be the central point of the pathophysiology of COVID19, and the cause of patients presenting syndrome of acute respiratory failure, septic shock, multiple organ failure, and eventually death. Considering the background described and the lack of a specific antiviral treatment, it is necessary to continue research to explore the potential of new compounds derived from natural products, for the prevention and treatment of CoVid-19.

Glycyrrhizal compounds are extracted from the root of a legume called Glycyrrhiza glabra, whose common name is licorice and has been used since ancient times in botanical medicine in Japan, Russia, Spain, India, the Middle East, and China. There are written references to its use from the year 2,100 BC. Glycyrrhizinic acid is a terpenoid glycosan derived from beta-amine, whose chemical name is acid (3beta-20p)-20-carboxyl-11-oxo-30-norolean- 12-en-3-yl-2-OpD-glucopyranuronosyl-aD-glucopyranosiduronic acid; its formula is C42-H62-O16 and its molecular weight is 822.94. Glycyrrhizinic acid has been used as an anti-inflammatory, anti-ulcer, anti-thrombotic, anti-allergenic, and anti-carcinogenic agent in hepatocellular carcinomas. secondary to viral hepatitis and as an antiviral. Its effect is due to the fact that it inhibits the enzymes that metabolize prostaglandins (PGE2 and PGE2a) to their inactive form; in the gastro-duodenal mucosa it acts as an anti-ulcer agent and a possible therapeutic agent against strains of Helycobacter pylori; it is antithrombotic, because it inhibits thrombin; as an anticarcinogen in hepatocellular carcinomas secondary to Hepatitis C and its antiviral activity has been particularly tested against Coronaviruses associated with severe acute respiratory syndrome, respiratory syncytial virus, arboviruses, flaviviruses that cause yellow fever, dengue and Japanese encephalitis, herpes simplex virus, HIV-1, vesicular stomatitis virus, CHIKY and SFV virus, and varicella virus.

The antiviral activity of glycyrrhizinic acid is attributed to the fact that it interacts with the protein structure of the capsid and interferes with the viral cycle, inhibiting the replication of viral DNA and RNA. In the case of Herpes simplex, it irreversibly inactivates the virus and promotes interferon activation. The characteristic of these viruses is that they have the property of infecting the epithelium and mucosa. Regarding its effect on Coronaviruses, several in vitro tests have been reported where its effect has been evaluated compared to antivirals such as ribavirin, 6-azauridine, pyrazofurin and mycophenolic acid; and of all the compounds, glycyrrhizinic acid showed a greater inhibitory potency of the replication activity of the SARS-related viruses, as well as inhibition of the adsorption and penetration of the virus into its target cells, during the early stages of the viral replication cycle. . An effect similar to that of interferon-beta, but without the toxic effects that it can present, due to the higher doses at which the antiviral effect is achieved. Another important mechanism of glycircinic acid is its immunomodulatory effect, and the best known is the antagonistic effect of the mechanisms dependent on Tolk-4 type receptors (TLR4), as a consequence of its antagonistic effect is the reduction of inflammation in various tissues, including the lung. It also leads to a reduction in the expression of these TLR4 receptors in the heart and lungs, this is accompanied by a significant reduction in the release of cytokines (TNFa, IL6 and IL10), consequently it presents a protective effect against respiratory syndrome induced by TLR4. , activated by lipopolysaccharides (LPS). Another important mechanism of action is on the protein "Spike" (S) of coronaviruses, these proteins play an important role in the entry of viruses into host cells mediated by receptors, so an inhibitor of these S proteins would be useful. clinical relevance to inhibit the entry of coronaviruses into cells. Glycyrrhizinic acid has shown a high affinity interaction with the S1 subunit of the Spike protein of SARS-CoV-2 and MERS-CoV, at the binding interface with its ACE2 receptor. Demonstrating also a low toxicity towards normal lung cells.

The therapeutic doses of GA that are used are variable and were generally determined based on the concentrations of GA in plasma and the time of its elimination from the body through different clinical trials. It is considered that the acceptable doses are: By intravenous route of 20 to 50 mg/kg of weight every 24 hours of Neo-Minophage C (SNMC), and ingested of 1 to 10 mg. diaries. Another recommended dose is 200-800 mg in infusion equivalent to 5-15 g of dry extract in 150 ml of water three times a day after food; for up to 4-6 weeks, and the root is used as a flavoring in a daily dose equivalent to 100 mg of the acid. It is proposed that the maximum safe dose is 1,500 mg/kg of weight with an accumulation time in the body of 2 to 4 months. The maximum reported time of exposure to the compound is 16 years. Another recommended safe dose is 100 mg/day, used chronically and which does not induce pathophysiological changes. In the context of SARS, a dose greater than 300 mg/day has been recommended for oral administration and 240 mg/day for intravenous administration. For a primary prevention indication, they have recommended a dose of 150 mg/day orally, although further studies are recommended to evaluate the effect of this dose. Properties that make Polyoxyethylene-polyoxypropylene-polyoxyethylene the vehicle of choice to formulate Glycircinic Acid and its justification regarding its pharmaceutical use.

Polyoxyethylene-polyoxypropylene-polyoxyethylene is a gel that can be used as a carrier of active molecules by various routes of administration such as the oral, respiratory tract and intranasal (through nebulizations), ocular, vaginal, rectal, topical, intramuscular and even as skin. artificial has also been reported. Surface active block copolymers such as polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE) are widely used as solubilizing and wetting agents. Some of these possess special properties that make them particularly useful for use in topical formulations; these include its low toxicity and its ability to form transparent gels in an aqueous medium. Polyoxyethylene-polyoxypropylene-polyoxyethylene is of particular interest, because concentrated solutions of >20% w/w of the copolymer are transformed from clear, low-viscosity solutions to gel-solids at body temperature. This suggests that when these gels are placed on the skin or injected into a body cavity, the preparation will form a solid artificial barrier and thus a sustained release reservoir for the drug. These characteristics were taken into consideration when choosing Polyoxyethylene-polyoxypropylene-polyoxyethylene as the vehicle for the new formulation of glycirincinic acid. However, for a technician with average knowledge in the matter, it is not obvious how to obtain the formulation of the present invention, since it is not a matter of the simple union of two already known elements, but rather that work had to be done to obtain this product in the new formulation with thermoreversible gel which represents an unexpected and non-obvious element.

DETAILED DESCRIPTION OF THE INVENTION. Glycyrrhizinic acid formulated in Polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE) gel. Preparation of the gel-syrup.

Gels were prepared using the method described by Schmolka, which generally consists of the following:

A solution of Polyoxyethylene-polyoxypropylene-polyoxyethylene was prepared in the concentration range of 20-30% (w/w) and it is when the phenomenon of reverse thermal gelation is observed, which means that at low temperatures it is a liquid and as it the temperature increases the viscosity of the solution as well, generating a gel and specifically the 25% range is preferred without being limiting. The corresponding amount of Polyoxyethylene-polyoxypropylene-polyoxyethylene was added slowly in cold water with gentle mechanical stirring. The Polyoxyethylene-polyoxypropylene-polyoxyethylene solution was left overnight at a low temperature, in a range between 2°C to 6°C but preferably at 4°C for complete hydration of the polymer. Once the polymer was perfectly dissolved, the glycyrrhizinic acid (AG) was previously dissolved in Propylene Glycol (PG) and a preservative was added to this same solution, which could be Propyl-paraben at 0.1% or the same Propylene Glycol but at a concentration of 30-50% among others preferring the Methyl-Paraben at 0.1%. Once the complete dissolution of the AG and the preservative in the Propylene Glycol was guaranteed, it was incorporated into the required volume of gel to prepare the corresponding formulation. Finally, the pH is adjusted in a range of 4-6, which is a more physiological pH at the gastric level, with organic acids such as acetic acid or citric acid, among others that are not toxic or irritating to humans. The formulations were prepared under sterile conditions, for example, poured into glass vials previously sterilized in an autoclave and packaged in a laminar flow hood. It is important to mention that distilled water of Mili-Q quality (Millipore * Corp., Bedford, USA) was used to prepare all the required solutions, in addition to being passed through 0.2p pore size filters to avoid contamination by microorganisms.

Formulation I and II (table I) were prepared at a concentration of 10 and 20 mg/ml of Glycircinic Acid, respectively. These formulations presented the phenomenon of reverse thermal gelation, that is, they are fluid at low temperatures and solidify, forming a gel as the temperature increases, that is, at body temperature.

Formulations III and IV were prepared at a concentration of 100 and 200 mg/ml of the ammonium salt of glycerincinic acid, respectively. It is important to mention that these formulations no longer have the ability to gel due to the high content of PG in them, so the consistency is more fluid, however it is necessary to add that proportion of PG in order to solubilize the AG and even thus it is necessary to heat between 60°C and 120°C with an average temperature of 65-75°C with constant stirring to guarantee the complete dissolution of the AG. In all formulations, 0.1% Methyl-Paraben was used as a preservative.

Table 1. Composition of the formulated gels.

Solubility tests. Prior to the preparation of the gels, it was necessary to determine the solubility of AG in aqueous solution -because the gels are prepared in water- finding a low solubility value: 1.1 mg/ml

Which means that the amount that can be dissolved in water is very small. The AG turned out to be very soluble at pH greater than 9.0, hence it was necessary to test the solubility of the AG in different polar and nonpolar organic solvents: Methyl alcohol, ethyl alcohol, isopropyl alcohol, ether, acetone, dichloromethane, chloroform, hexane. , etc.

Finding that it was insoluble in all of them, this fact is important because it also laid the foundations to implement the analytical method by HTPLC (High Performance Thin Layer Chromatography) for the quantification of glycerinic acid. Finally, it was necessary to test the solubility in other substances that are commonly used in the pharmaceutical industry as vehicles.

The substances that were tested as co-solvents to solubilize glycyrrhizinic acid were:

Glycerin, Transcutol and Propylene glycol.

In Glycerin and Transcutol, glycyrrhizinic acid was insoluble, however in Propylene Glycol the acid dissolved adequately: 89.6 mg/ml This being one of the main reasons why Polyoxyethylene-polyoxypropylene-polyoxyethylene gel was chosen as part of the vehicle to formulate the AG in gel. In addition, it has been shown that PG is a penetration promoter -a chemical substance that increases the partitioning and diffusion- of active ingredients for pharmaceutical or cosmetic use towards and through the skin's permeability barrier. This is a property that can be used to favor the passage of AG through the gastric mucosa at a given moment.

analytical method. It is also important to mention that the analytical method for the quantification of the AG is already practically available and this will be HTPLC in reverse phase plates, showing the following analytical response by the AG. The range of concentrations in which the analytical response is linear is already available and this will be used for the quantification of AG in in vitro tests consisting of permeation tests through pig vagina mucosa.

That is why the formulation of the present invention proves to be viable and inventive since there is no precedent that has a formulation of glycyrrhizinic acid as an active ingredient in a thermoreversible gel and propylene glycol like the one we have described and for therapeutic uses that we have given

Preparation of dextromethorphan and guaifenesin syrup.

En un vaso de precipitados de 400 mi verter 80 mi de agua destilada y calentar entre 70-80°C y adicionar la sacarosa con agitación constante hasta que se disuelva teniendo precaución que la solución no se sobrecaliente. Dextromethorphan hydrobromide is a drug widely used for its properties as an antitussive, because it has a pronounced and specific inhibitory action of the antitussive center, without narcotic or analgesic effects. Does not interfere with expectoration and coughing to maintain airway cleared of secretion. Its structure C18H25NOHBr H20, with a molecular mass of 271.41 g/mol, is a white, crystalline, odorless powder, practically insoluble in water and freely soluble in chloroform.

In a 400 ml beaker, pour 80 ml of distilled water and heat to between 70-80°C and add the sucrose with constant stirring until it dissolves, taking care not to overheat the solution.

Once dissolved, stop heating and add the glycerin with constant stirring until completely incorporated. Allow the solution to cool until it reaches 40°C.

Separately, in a 50 ml beaker place 2 ml of ethanol and dissolve the Methyl-Paraben, this solution is added to the one previously obtained. In the same way, in a 59 ml beaker, place 2 ml of ethanol and dissolve the Propyl-Paraben, add this solution to the one initially obtained.

In another 50 ml beaker, pour 10 ml of distilled water and dissolve the dextromethorphan hydrobromide while stirring. Add this solution to the one obtained previously.

Add the anhydrous glucose to the rest of the syrup with constant stirring until completely incorporated.

Make up to volume with distilled water and allow the syrup (solution) to cool to room temperature. Refrigerate for 24 hours and then filter.

Claims

CLAIMS Having sufficiently and clearly described my invention, and considering the document PA/a/2006/003643 as a reference and precedent; I consider as a novelty and therefore I claim as my exclusive property, what is contained in the following clauses: 1.- Gel formulation of the ammonium salt of glycyrrhizinic acid as active agent with a thermoreversible gel former, such as polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE) as a vehicle characterized by comprising POE-POP-POE in concentrations of 20-30% (w/w) and the active agent is found in concentrations of 10, 20, 100 and 200 mg/ml. 2. - Gel formulation of the ammonium salt of glycyrrhizinic acid; with the Polyoxyethylene-polyoxypropylene-polyoxyethylene POE-POP-POE as vehicle according to claim 1 where the concentration of 25% (w/w) of the POE-POP-POE thermoreversible gel is preferred and the concentrations of 10,20,100 and 200 mg/ml. of the active agent of ammonium salts of glycyrrhizinic acid. . 3. -Process for making the gel formulation of claims 1 and 2 consisting of the following steps: a) The polyoxyethylene-polyoxypropylene-polyoxyethylene thermoreversible gel former dissolves slowly in water at low temperature in a range between 2° C to 6°C, but preferably at 4°C overnight for the complete hydration of the polymer and it remains at the preferred concentration of 25% (w/w). b) The ammonium salt of glycyrrhizinic acid as active agent is dissolved in propylene glycol. c) To the previous mixture of the ammonium salt of gI icyrrhizinic acid and propylene glycol, 0.1% methylparaben was added as a preservative. d) With the dissolved Polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE), the required volume of gel is incorporated into the mixture of subparagraph c) in the following proportions: 85: 15 / 85: 15 / 35:65 / 35 :65 to obtain the final concentrations of 0, 20, 00, and 200 mg/ml of the ammonium salt of glycyrrhizinic acid. f) Once the final concentration is obtained, the pH is adjusted in a range of 4-6 with organic acids such as acetic acid and citric acid. 4.- Process according to claim 3, where in order to prepare the gel formulation at concentrations of 10 and 100 mg/ml of the ammonium salt of glycyrrhizinic acid, it is necessary to heat the mixture, to guarantee the complete dissolution of the active compound. , with constant stirring in a temperature range from 60°C to 120°C, preferring 65-75°C. 5.- Process according to claim 3 where in the final stage of the process the pH is adjusted, preferring the pH range from 4 to 6 as it is the most physiological in the gastric cavity. 6. -Use of the gel formulation of claims 1, 2 and 3 to prepare a drug for therapeutic use in SARS-CoV-2 infections and pathologies associated with COVID-19. 7. -Use of the gel formulation according to claim 6 wherein the syrup formulation is for oral administration. 8. -Use of the syrup formulation according to claim 6, for the management of SARS-CoV-2 and COVID19 infections previously described where the 15 formulation is used for a period of 7 to 15 days on average and even up to 20-30 days depending on the degree of symptoms or persistence of the viral infection. 9. -Use of the syrup formulation according to claim 6 for the prophylaxis of COVID-19 and other severe clinical manifestations caused by SARS- CoV2. 10.- Use of the formulation claiming as reference and background the document PA/a/2006/003643. . ..