A METHOD FOR REDUCING NITROSAMINE DRUG SUBSTANCE-RELATED IMPURITIES (NDSRIs) IN A PHARMACEUTICAL COMPOSITION

Abstract

The present invention relates to a method for reducing nitrosamine drug substance-related impurities (NDSRIs) in a pharmaceutical composition by reducing the amount of nitrates and nitrites present in hydroxyethyl cellulose (HEC), a commonly used pharmaceutical excipient. The method involves purification of HEC using a water miscible, polar solvent in which HEC is insoluble. Purified HEC, when added to a pharmaceutical formulation significantly reduces the amount of Nitrosamine Drug Substance-Related Impurities (NDSRIs) in the formulation.

Description

[0001] TITLE OF THE INVENTION

[0002] A METHOD FOR REDUCING NITROSAMINE DRUG SUBSTANCE- RELATED IMPURITIES (NDSRIs) IN A PHARMACEUTICAL COMPOSITION FIELD OF THE INVENTION

[0003] The present invention relates to a purified excipient used in the formulation of pharmaceutical compositions, with reduced potential for forming nitrosamine impurities. The invention provides a method for reducing nitrosamine drug substance-related impurities (NDSRIs) in a pharmaceutical composition by reducing the amount of nitrates and nitrites present in the pharmaceutical excipient.

[0004] BACKGROUND OF THE INVENTION

[0005] Nitrosamines describe a class of compounds having the chemical structure of a nitroso group bonded to an amine (R'N(-R2)-N=O). The compounds are formed by nitrosating reaction between amines and nitrous acid or nitrite salts under acidic conditions. These compounds are potent genotoxic agents in several animal species and some are classified as possible human carcinogens by the International Agency for Research on Cancer (IARC). They are referred to as ‘cohort of concern’ compounds in the ICH guidance for industry M7 (Rl) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. (M7(R2) Mutagenic Impurities. ICH, Ed. 2023.

[0006] A lot of uncertainty surrounds the presence and acceptable intake limit of nitrosamines in drug formulations, particularly due to non-availability of safety data of the plethora of possible impurities. (The Role of Excipients in Determining N-Nitrosamine Risks for Drug Products IPEC, February 2024). This has led to regulatory challenges, and in some instances, market withdrawal of the drugs, causing disruption in supply chain and even drug shortage. (Sandoz, Inc. Issues Nationwide Recall of 13 Lots of Orphenadrine Citrate lOOmg Extended-Release Tablets Due to Presence of a Nitrosamine Impurity. March 22 2022; Pfizer Voluntary Nationwide Recall of Lots of ACCURETIC™ (Quinapril HCl / Hydrochlorothiazide), Quinapril and Hydrochlorothiazide Tablets,and Quinapril HCl / Hydrochlorothiazide Tablets Due to N-Nitroso-Quinapril Content. March 222022).

[0007] Nitrosamine impurities are classified structurally into two classes: (i) small-molecule nitrosamine impurities that do not share any structural similarity to the active pharmaceutical ingredient (API), and (ii) Nitrosamine Drug Substance-Related Impurities (NDSRIs) that share structural similarity to the API in the drug product, and are generally unique to each API. Since NDSRIs are unique to each API and possess a complex chemical structure, till now there is no safety data available to establish their acceptable limit for intake. The only guidance available till now to determine the acceptable intake is provided by FDA using structural features of NDSRIs to generate a predicted carcinogenic potency and categorizing accordingly on a scale of one to five. (Recommended Acceptable Intake Limits for Nitrosamine Drug Sub stance -Related Impurities (NDSRIs), FDA, August 2023).

[0008] NDSRIs usually find their way to the drug formulations either during manufacturing, packaging and / or storage. Several documents have suggested various methods for reduction of NDSRIs in drug formulations. Nanda et al describes the role of antioxidants such as ascorbic acid, sodium ascorbate, a-tocopherol, caffeic acid, ferulic acid, and amino acids such as glycine, lysine, histidine, as possible inhibitors of nitrosamine formation in oral solid dosage forms. (Nanda, et.al. (2021) Inhibition of N-Nitrosamine Formation in Drug Products: A Model Study. Journal of Pharmaceutical Sciences; 110(12), 3773-3775.)

[0009] Similarly, WO2024127365A1 describes a pharmaceutical composition that provides effective control of the level of nitrosamine drug substance-related impurities (NDSRIs) in the drug product during shelf life of the product by incorporating nitrite quenchers selected from ascorbic acid, amino acids such as cysteine, histidine, glycine, arginine. While these may provide effective control of NDSRI in active ingredients that are amines, such use cannot be generalised over all APIs, and more importantly, will still need extensive experimentation before an excipient is chosen for a particular API.Another approach to reduce NSDRIs in drug formulations, as recommended by FDA, is to incorporate of sodium carbonate that can modify the pH of the product, as nitrosamines are formed in acidic conditions. (Nitrosamine Risk Assessments & FDAs ’ Alternate Methods to Reduce Impurity Levels, Freyr, February 17, 2022). However, such use of alkaline agents, or adjustment of pH to alkaline pH, may not be feasible for all APIs and all dosage forms. For example, injectable dosage forms, as well as base-labile APIs will need pH closer to physiological pH, and alkaline pH would be in fact be detrimental. Therefore, while the use of such alkaline agents could possibly solve the issue with respect to level of NDSRIs, it would create other problems.

[0010] As discussed before, NDSRI can be introduced into drug formulations during manufacturing. Three main factors influence the formation of NDSRIs in drug formulations: a nitrosating agent, a secondary or tertiary amine (vulnerable amines), and appropriate conditions (for example elevated temperatures, acidic conditions, liquid phase) for the reaction. Specifically, NDSRIs can form by nitrosation of APIs and API fragments that contain secondary amine or dimethyl tertiary amine centres, such as by reaction with residual nitrites in excipients used to formulate the drug product. (The Role of Excipients in Determining N-Nitrosamine Risks for Drug Products IPEC, February 2024).

[0011] While the prior art is directed towards reducing nitrosamine content in drug formulations, and while some art points to presence of nitrites / nitrates in excipients as the root cause of NDSRI formation, there is nothing in the aforementioned documents that addresses the concern of residual nitrites and nitrates in excipients.

[0012] Wu et al discloses the presence of reactive impurities in various excipients, including nitrate and nitrite impurities that can react with an API. While various general mitigation strategies to reduce reactive impurities have been discussed, nothing in this document provides a strategy for reducing the level of nitrate and nitrite impurities, specifically those that form NDSRIs, in excipients. (Wu Y, Levons J, Narang AS, Raghavan K, Rao VM. Reactive impurities in excipients: profiling, identification and mitigation of drug-excipient incompatibility. AAPS PharmSciTech. 2011Dec;12(4):1248-63. doi: 10.1208 / sl2249-011-9677-z. Epub 2011 Sep 27. PMID: 21948318; PMCID: PMC 3225520.)

[0013] Hydroxyethyl cellulose (HEC) is a thickening and gelling agent that is derived from cellulose. It is a non-ionic, water-soluble polymer that is used in many household products, cosmetics, and pharmaceuticals. HEC belongs to the group of cellulose ethers in which hydroxyl groups have been substituted with one or more of the three hydroxyl groups present in the cellulose ring, as shown in the structure below.

[0014]

[0015] HEC is generally considered safe for use in various applications. It is non-irritating to the skin and eyes at typical concentrations. HEC does not typically cause allergic reactions. It is commonly used as a carrier, suspending agent, viscosity generating agent, coating agent, binder in various pharmaceutical, cosmetic and nutraceutical compositions, in amounts ranging from about 0.1% to about 10% or more, depending upon the purpose. Accordingly, any nitrate and / or nitrite impurities present in HEC can contribute to formation of NDSRI in a pharmaceutical, cosmetic or nutraceutical composition. Such NDSRI, being genotoxic, is highly undesirable, and must be absent or controlled to very low levels.

[0016] A January 2024 draft monograph proposed for inclusion in the Indian Pharmacopoeia suggests that the nitrate levels in HEC should not be more than 3.0 per cent, on the dried basis, if hydroxy ethyl cellulose has a viscosity of 100 mPa.s or less; and should not be more than 0.2 per cent, on the dried basis, if hydroxy ethyl cellulose has a viscosity of more than 100 mPa.s. Such levels of nitrate in the HEC could be a high source for NDSRI formation.HEC is typically obtained by treating cellulose with sodium hydroxide and reacting with ethylene oxide. Bartscherer et cd and Krenzer et al disclose use of isopropyl alcohol (IPA) and carbon dioxide for removal of alkali salt impurities during the manufacturing of HEC, but there is nothing in these references about control of impurities other than the salt impurities. (Bartscherer, KA, de Pablo, J, Bonnin, M C, & Prausnitz, J M (1990). Purification of aqueous cellulose ethers, https: / / doi.org / 10.2172 / 6084196; Purification of Aqueous Hydroxyethylcellulose. Extraction of Sodium Acetate with Isopropanol and High-Pressure Carbon Dioxide Krenzer, M. E.; Pablo, J. J. de; 1989)

[0017] While GB704567A discloses the use of a mixture of isopropanol with water as a displacing medium to remove aqueous washing medium (water) from the hydroxy ethyl cellulose, followed by acetylation to prevent degradation of hydroxyethyl cellulose, there is nothing therein that suggests the removal or reduction of nitrate / nitrite impurities. Similarly, CN109535265 A discloses the use of isopropanol with water to prepare anionic hydroxyethyl cellulose, but does not teach or suggest how to remove or reduce impurities. US3347847A discloses a process for purification of water-soluble hydroxyethyl cellulose using a mixture of acetone and methanol, followed by neutralisation with an acid or a base. JPS6232101 A discloses a method for removing sodium and salts from hydroxy ethyl cellulose using a mixture of acidic esters and solvents including acetone. However, nothing in this document teaches or suggests the removal or reduction of nitrate / nitrite impurities in HEC. Additionally, the neutralisation steps require addition of acid or a base, increasing the complexity of the process. At the time some of these references were published, the presence and risk of nitrosamine impurities was unknown, and therefore, these references could not have taught anything on purification methods to remove the nitrosamine impurities, nor define their limits and / or specifications.

[0018] Thus, none of these documents teach about reducing the level of nitrate and / or nitrite impurities in HEC. Accordingly, there exists a need for addressing the presence of potential NSDRI-forming impurities in HEC, and further devising a strategy to reduce the same.OBJECTIVE OF THE INVENTION

[0019] An object of the present invention is to reduce the amount of nitrates and nitrites present in hydroxyethyl cellulose (HEC) so that, when used in a pharmaceutical composition, its potential to form Nitrosamine Drug Substance-Related Impurities (NDSRIs) is prevented or reduced.

[0020] Another object of the present invention is to reduce the levels of Nitrosamine Drug Substance-Related Impurities in the final pharmaceutical composition.

[0021] Yet another object of the present invention is to provide a process for reducing the nitrate and nitrite impurities that may be present in HEC, so that the potential of HEC to form NDSRIs in a drug product composition is highly reduced or prevented.

[0022] Still another object of the present invention is to provide a pharmaceutical composition comprising purified HEC as per the present invention, wherein the amount of nitrosamine drug substance-related impurities present in the composition is less than lOppm, when stored at a temperature of 40°C, 25% relative humidity for a period of at least 3 months.

[0023] SUMMARY OF THE INVENTION

[0024] In an aspect of the present invention, there is provided a method of reducing nitrosamine drug substance-related impurities (NDSRIs) in a pharmaceutical composition comprising an active ingredient and hydroxy ethyl cellulose (HEC), by reducing the amount of nitrates and nitrites present in hydroxyethyl cellulose wherein the method involves purification of HEC using a water miscible, polar solvent in which HEC is insoluble such as acetone, ethanol, methanol, tertiary butanol, 2- chloro ethanol and isopropyl alcohol (IPA) in a ratio of HEC: water miscible / polar solvent of about 1:1 to about 1:20. In one embodiment, the purification of HEC comprises the steps of: (i) mixing HEC with a liquid medium selected from either water to form an aqueous HEC solution, wherein the aqueous HEC solution is further mixed with a water miscible, polar solvent in a ratio of about 1: 1 to about 1:20 to obtain a homogenous dispersion with a polymer mass; or a water-miscible, polar solvent in a ratio of about 1:1 to about 1:20 to form apolymer dispersion, wherein the polymer dispersion is further mixed with water in a ratio of 1:1 to 20:1 to obtain a polymer mass; (ii) separating the polymer mass from the dispersion obtained in step (i); (iii) optionally redispersing the polymer mass obtained in step (ii) one or more times in the water miscible, polar solvent in a ratio of 1:2 followed by separation; (iv) drying of the polymer mass at a temperature in the range of about 35°C to about 50°C, to obtain purified hydroxy ethyl cellulose polymer mass.

[0025] In an aspect of the present invention, the separation of polymer mass in step (ii) and step (iii) is either done by sieving, centrifugation, filtration or any other suitable separation technique conventionally known in the art.

[0026] In one aspect of the present invention, the purification of HEC comprises the steps of: (i) mixing HEC with water to form an aqueous solution; (ii) mixing the aqueous HEC solution of step (i) with the water miscible, polar solvent in a ratio of 1:4 to obtain a homogenous dispersion with a polymer mass; (iii) separating the polymer mass from the dispersion obtained in step (ii); (iv) dispersing the polymer mass obtained in step (iii) in the solvent in a ratio of 1:2; (v) separating the polymer mass of step (iv), followed by dispersing the polymer mass in the solvent at a ratio of 1:2; and (vi) drying of the polymer mass of step (v) at a temperature of about 35°C to about 50°C, wherein the water miscible, polar solvent is isopropyl alcohol.

[0027] In an alternate aspect of the present invention, the purification of HEC comprises the steps of: (i) mixing HEC with water miscible, polar solvent in a ratio of about 1:5 to about 1:20 to form polymer dispersion; (ii) mixing the polymer dispersion of step (i) with the water, in the ratio of 1: 1 to 20: 1 to obtain a polymer mass; (iii) separating the polymer mass from the dispersion obtained in step (ii); (iv) drying of the polymer mass obtained in step (iii) at a temperature of about 35°C to about 50°C, wherein the water miscible, polar solvent is acetone.

[0028] In an aspect of the present invention, HEC consists of one or more impurities selected from the group comprising nitrous acid, nitrite ions, nitrite salts, nitric acid, nitrates, nitrous acid ions, nitrite esters, peroxynitrite salts, nitrosonium ions, nitro compounds,anhydrous nitrous acid, dinitrogen tetroxide, nitrosyl halides, nitrosyl thiocyanates, nitrosophenols, nitrosothiols, nitric oxide, nitrogen dioxide and nitrile chloride.

[0029] In an aspect of the present invention, the amount of nitrate present in HEC after purification with the water miscible, polar solvent is less than 70ppm.

[0030] In an aspect of the present invention, the amount of nitrite present in HEC after purification with the water miscible, polar solvent is less than 30ppm.

[0031] In another aspect of the present invention, there is provided a pharmaceutical composition comprising purified HEC and an active pharmaceutical ingredient containing one or more amine groups, wherein the amount of nitrosamine drug substance-related impurities present in the composition is less than lOppm, when stored at a temperature of 40°C and 75% relative humidity for a period of 3 months.

[0032] The teachings of the present invention help reduce the levels of Nitrosamine Drug Substance-Related Impurities (NDSRIs) in the final pharmaceutical composition.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0035] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0036] Moreover, the present disclosure also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0037] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.As used herein, “a,” “an,” or “the” can mean one or more than one.

[0038] Furthermore, the term “about,” as used herein, when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±10% of the specified amount. The term composition(s), product(s), preparation(s) and formulation(s) have been used interchangeably.

[0039] The term “comprising”, which is synonymous with “including”, “containing”, or “characterized by” herein is defined as being inclusive or open-ended and does not exclude additional, unrecited elements or method steps, unless the context clearly requires otherwise.

[0040] Also, as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0041] As used herein the term "active pharmaceutical ingredient", abbreviated as API and also known as “drug substance”, refers to the substance (or combination of substances) that is the biologically active component of a drug product. The present invention envisages API as well as salts thereof. The salt maybe any pharmaceutically acceptable salt of the API. The term “drug product” as used herein refers to the finished dosage form or pharmaceutical composition containing the API (or drug substance) and the excipients.

[0042] In an embodiment of the present invention, the API used in a drug product consists of an amine moiety in its skeletal structure, such as primary, secondary, tertiary or quaternary amines, wherein the drug is mixed with, or is in contact with HEC as an excipient.In one embodiment of the present invention, the active pharmaceutical ingredient may be selected from the group comprising acetazolamide, brinzolamide, dorzolamide, methazolamide, sitagliptin, linagliptin, alogliptin, saxagliptin, valsartan, olmesartan, telmisartan, azilsartan, candesartan, orphenadrine, quinapril, Timolol Maleate, Moxifloxacin Hydrochloride, Olopatadine Hydrochloride, Azelastine Hydrochloride, Brimonidine Tartrate, Tobramycin, Dexamethasone, Ciprofloxacin and pharmaceutically acceptable salts and combinations thereof, and wherein purified HEC is used as an excipient.

[0043] The term “pharmaceutical composition” implies a composition comprising an active pharmaceutical ingredient or salt thereof, along with HEC as one of the excipients. The pharmaceutical composition may comprise other additives that are known in the art and may be prepared according to any process conventionally known in the art. The term “pharmaceutical composition”, “composition”, “formulation”, “pharmaceutical formulation”, “drug product” has been used interchangeably.

[0044] As discussed previously, nitrosamine impurities in pharmaceutical formulations arise from reaction between nitrosable compounds such as amines (like those present in API) with nitrosating agents such as nitrites and nitrates under acidic conditions.

[0045] The general chemical structure of nitrosamine impurities is R2N-N=O, wherein R is usually an alkyl group. Nitrosamine impurities are classified structurally into two classes: (i) small-molecule nitrosamine impurities that do not share any structural similarity to the API such as N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosomethylphenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoisophenylethylamine (NIPEA), N-nitrosodibutylamine (NDBA), and N-nitroso-N-methyl-4- aminobutyric acid (NMBA) and (ii) Nitrosamine Drug Substance-Related Impurities (NDSRIs) that share structural similarity to the API in the drug product, and are generally unique to each API.

[0046] The nitrosating agent may include nitrites, nitrates, nitrous acid, nitrite ions, nitrite salts, nitric acid, nitrous acid ions, nitrite esters, peroxynitrite salts, nitrosonium ions, nitro compounds, anhydrous nitrous acid, dinitrogen tetroxide, nitrosyl halides, nitrosylthiocyanates, nitrosophenols, nitrosothiols, nitric oxide, nitrogen dioxide, and nitrile chloride. When such nitrosating agents are present as impurities in excipients such as HEC, they become available for formation of NDSRI of either kind, and especially as conditions change during manufacturing and storage. Under acidic reaction conditions nitrosating agents from the excipient may form nitrous acid, which can react with an amine group present in the API to form a nitrosamine.

[0047] The representative reaction to form nitrosamines is as depicted below:

[0048]

[0049] Source: Control of Nitrosamine Impurities in Human Drugs Guidance for Industry, FDA, Sept 2024 The representative reaction of an API containing a secondary amine functional group in its structure with nitrite under acidic condition to form NDSRI is shown below:

[0050]

[0051] Source: Control of Nitrosamine Impurities in Human Drugs Guidance for Industry, FDA, Sept 2024 A common approach recommended by FDA is to either reduce the impurities in APIs and / or in final pharmaceutical compositions comprising these APIs, using methods as seen in the prior art, or to avoid use of the raw materials and excipients containing nitrosating agents or amines. While removing amine groups is not an option since many APIs have amine in their structure which is necessary for activity, it is nearly impossible to remove the source of nitrosating agents, as this may result in more complex and expensive synthetic routes for formulation of pharmaceutical compositions. Therefore, there exists a need for an improved method to reduce orprevent the formation of nitrosamine impurities in pharmaceutical compositions. While most efforts have focussed on treating the API and / or the raw materials and key intermediates, there are no reported efforts being taken to improve the quality and purity of excipients.

[0052] The inventors also tested formulations containing the treated HEC as per the present invention, and various antioxidants like ascorbic acid, citric acid, sodium sulfite and sodium thiosulfate. It was found that ascorbic acid effectively reduced or controlled the NDSRI impurity levels, however, it caused discoloration of the formulation. On the other hand, trials involving citric acid and sodium thiosulfate did not demonstrate significant improvements in reducing the NDSRI impurity levels. In contrast, the addition of sodium sulfite to the formulation was found to considerably control the NDSRI impurity levels.

[0053] The inventors of the present invention have identified that, since several APIs have amine groups that react with nitrate and / or nitrite moieties present in HEC, rather than targeting the API or the final pharmaceutical composition, it is possible to reduce the amount of nitrosamino impurity in the final pharmaceutical composition by reducing the amount of nitrate and / or nitrite in HEC.

[0054] Accordingly, the present invention provides a method of reducing nitrosamine drug substance-related impurities (NDSRIs) in a pharmaceutical composition comprising an active ingredient and hydroxy ethyl cellulose (HEC), by reducing the amount of nitrates and nitrites present in hydroxyethyl cellulose (HEC), an excipient used extensively in the manufacturing of various pharmaceutical compositions. The method involves purification of HEC using a water miscible, polar solvent in which HEC is insoluble, such as acetone, ethanol, methanol, tertiary butanol, 2- chloroethanol and isopropyl alcohol (IPA).

[0055] In a preferred embodiment, the method primarily involves mixing an aqueous solution of HEC with a water miscible, polar solvent in a specific ratio to obtain a homogenous dispersion with a polymer mass. The homogenous dispersion is then sieved to separate the polymer mass, followed by dispersing of the mass in the solvent in a specified ratio.The polymer mass is once again sieved and dispersed in the solvent in a specific ratio followed by drying at high temperature. When the aqueous dispersion of HEC polymer is prepared by mixing the polymer in water, it facilitates the interaction of polar water molecules with charged ions of nitrates and nitrites, resulting in their dissolution. When the polymer mass is separated by addition of the solvent, the nitrates and nitrites remain in the dispersion after polymer mass separation, and thus get removed.

[0056] In a preferred embodiment, the method primarily involves mixing HEC with the solvent and water in a specific ratio to obtain a homogenous dispersion with a polymer mass. The polymer mass is then separated using suitable separation technique and dried.

[0057] Specifically, the method of the present invention comprises the steps of

[0058] (i) mixing HEC with a liquid medium selected from:

[0059] (a) water to form an aqueous HEC solution, wherein the aqueous HEC solution is further mixed with a water miscible, polar solvent in a ratio of about 1:1 to about 1:20 to obtain a homogenous dispersion with a polymer mass; or (b) a water-miscible, polar solvent in a ratio of about 1:1 to about 1:20, to form a polymer dispersion, wherein the polymer dispersion is further mixed with water in a ratio of 1: 1 to 20: 1 to obtain a polymer mass;

[0060] (ii) separating the polymer mass obtained in step (i);

[0061] (iii) optionally, redispersing the polymer mass obtained in step (ii) one or more times in the water-miscible, polar solvent in a ratio of 1:2, followed by separation;

[0062] (iv) drying the polymer mass of step (ii) or (iii) at a temperature in the range of about 35°C to about 50°C to obtain the purified hydroxyethyl cellulose polymer mass.In the above method the separation of polymer mass in steps (ii) and (iii) is either done by sieving, centrifugation, filtration or any other suitable separation techniques conventionally known in the art.

[0063] The term ‘water miscible, polar solvent’ and ‘solvent’ is used interchangeably. In the context of the present disclosure polar solvents are those having positive and negative electrical charges asymmetrically distributed throughout their structure. These solvents are miscible with other polar solvents like water due to interaction between opposite charges.

[0064] In a preferred embodiment of the present invention the water miscible, polar solvent is isopropyl alcohol.

[0065] In one embodiment of the present invention, the method comprises the steps of:

[0066] (i) mixing HEC with water to form an aqueous solution;

[0067] (ii) mixing the aqueous HEC solution of step (i) with a water miscible, polar solvent at a ratio of 1:4 to obtain a homogenous dispersion with a polymer mass;

[0068] (iii) separating the polymer mass from the dispersion obtained in step (ii)

[0069] (iv) dispersing the polymer mass obtained in step (iii) in the water miscible, polar solvent at a ratio of 1:2;

[0070] (v) further separating the polymer mass in step (iv) and dispersing the polymer mass in the water miscible, polar solvent at a ratio of 1:2;

[0071] (vi) drying the polymer mass of step (v) at a temperature of about 35°C to about 50°C. In a preferred embodiment of the present disclosure, the method comprises the steps of:

[0072] (i) mixing HEC with water at a ratio of about 1: 30 to about 1: 50 to form an aqueous solution;

[0073] (ii) mixing the aqueous HEC solution of step (i) with isopropyl alcohol at a ratio of 1:4 to obtain a homogenous dispersion with aa polymer mass;(iii) separating the polymer mass from the dispersion obtained in step (ii)

[0074] (iv) dispersing the polymer mass obtained in step (iii) in isopropyl alcohol at a ratio of 1:2;

[0075] (v) further separating the polymer mass in step (iv), followed by dispersing the polymer mass in isopropyl alcohol at a ratio of 1:2;

[0076] (vi) drying of the polymer mass of step (v) at a temperature of about 35°C to about 50°C.

[0077] In the above method, an optimum ratio of 1:4, 1:2 and 1:2 is maintained in steps (ii), (iv) and (v), respectively, for complete polymer (HEC) recovery. When the aqueous solution of HEC is prepared by mixing it in water, it facilitates the interaction of polar water molecules with charged ions of nitrates and nitrites, resulting in their dissolution. When the polymer mass is separated by addition of solvent, i.e. isopropyl alcohol, the nitrates and nitrites remain in the hydroalcoholic dispersion after polymer mass separation, and thus get removed. The solvent is usually recovered from the homogenous dispersion left behind after separating the polymer mass, and the remaining dispersion is disposed. Residual moisture in the polymer mass can cause hydrolysis of the polymer during drying, and that would impact the final viscosity of the formulation. Separation and dispersion of the polymer mass is therefore carried out twice prior to drying, to remove the residual moisture trapped in the mass.

[0078] In the above method the separation of polymer mass in steps (iii) and (v) is either done by sieving, centrifugation, filtration or any other suitable separation techniques conventionally known in the art.

[0079] In yet another embodiment of the present invention, the water miscible, polar solvent is acetone.

[0080] In one embodiment of the present invention, the method comprises the steps of:

[0081] (i) mixing HEC with water miscible, polar solvent in a ratio of about 1:5 to about 1:20 to form polymer dispersion;(ii) mixing the polymer dispersion of step (i) with water, in the ratio of 1:1 to 20:1 to obtain a polymer mass;

[0082] (iii) separating the polymer mass obtained in step (ii); and

[0083] (iv) drying the polymer mass obtained in step (iii) at a temperature of about 35°C to about 50°C.

[0084] In yet another preferred embodiment of the present invention, the method comprises the steps of:

[0085] (i) mixing HEC with acetone in a ratio of about 1:5 to about 1:20 to form polymer dispersion;

[0086] (ii) mixing the polymer dispersion of step (i) with water, in the ratio of 1:1 to 20:1 to obtain a polymer mass;

[0087] (iii) separating the polymer mass obtained in step (ii);

[0088] (iv) drying the polymer mass obtained in step (iii) at a temperature of about 35°C to about 50°C.

[0089] In the above method the separation of polymer mass in steps (iii) is either done by sieving, centrifugation, filtration or any other suitable separation techniques conventionally known in the art.

[0090] When hydroxyethyl cellulose (HEC) is initially dispersed in acetone, no solubilization of the polymer occurs. Upon the subsequent addition of water, the polar water molecules facilitate the dissociation and dissolution of ionic impurities, such as nitrates and nitrites, by interacting with their charged species. During this process, the polymer remains in a non-solubilized state, allowing the separation of the polymer mass while the dissolved nitrates and nitrites remain in the hydroalcoholic medium. As a result, these impurities are effectively removed. This approach minimizes polymer loss, thereby improving overall yield. Although the fundamental mechanism of nitrate and nitrite removal is consistent with that observed in aqueous-based purification processes, the use of acetone providesdistinct advantages, including higher polymer recovery and reduced drying time, owing to the lower water content in the system.

[0091] By using the above method of the present invention, the amount of nitrates and nitrites in HEC are reduced, and hence, the amount of NDSRIs formed due to nitrosating reactions between the nitrites and / or nitrates of HEC and the API is controlled, i.e. prevented or reduced.

[0092] In an embodiment of the present invention, the amount of nitrate present in HEC after purification with the water miscible, polar solvent is less than 70ppm. In another embodiment, the amount of nitrate present in HEC after purification is less than 65ppm. In yet another embodiment, the amount of nitrate present in HEC after purification is less than 60ppm.

[0093] In an embodiment of the present invention, the amount of nitrite present in HEC after purification with the water miscible, polar solvent is less than 30ppm. In another embodiment, the amount of nitrate present in HEC after purification is less than 25 ppm.

[0094] The present invention is further illustrated by reference to the following examples which are for illustrative purposes only, and do not limit the scope of the invention in any manner.

[0095] EXAMPLES

[0096] Example 1: Purification Of Excipient using isopropyl alcohol:

[0097] Dorzolamide compositions containing HEC were taken as a working representative. This formulation is commercially available to patients under the brand name Trusopt®, containing dorzolamide hydrochloride as its active ingredient, for use in treating increased ophthalmic pressure. The marketed product composition uses HEC as a polymer for enhancing the viscosity of the formulation. Different ratios of excipient Polymer solution and isopropyl alcohol from 1:1 to 1:20 were used for optimum purification of HEC. Two commercially available grades of HEC, Natrosol™ 250 HX (Molecular weight: 10,00,000 Daltons, Brookfield viscosity at 25°C: 1,500-2,500 mPa·s) and Natrosol™ 250 HHX (Molecular weight: 13,00,000 Daltons, Brookfield viscosity at 25°C: 3,500-5,500 mPa·5) were used for this.

[0098] Concentrated phase of two grades of Hydroxyethyl cellulose was prepared.

[0099] (i)Preparation of Natrosol™ 250 HX: 600 ml of hot water (80°C - 90°C) was taken in a stainless steel manufacturing vessel. Gradually, 20 grams of Natrosol 250 HX was added to the hot water. The resulting mixture was homogenized for 2 hours to get homogeneous dispersion ensuring that no lumps were present.

[0100] (ii)Preparation of Natrosol™ 250 HHX: 900 ml of hot water (80°C - 90°C) was taken in a stainless steel manufacturing vessel. Gradually, 20 grams of Natrosol 250 HHXwas added to the hot water. The resulting mixture was homogenized for 2 hours to get homogeneous dispersion ensuring that no lumps were present.

[0101] (iii) Purification of Natrosol™ 250 HX with Isopropyl alcohol

[0102] About 20 grams of Natrosol 250 HX was solubilised in 600 ml water to produce a solution. About 2400 ml of isopropyl alcohol (IPA) (1:4 ratio of solution to IPA) was added to this solution and stirred for 30 minutes at 1600 rpm to form a homogenous mixture. The mixture was then sieved to separate the lumpy polymer mass. About 80 grams of the polymer mass was then dispersed in 160 ml of IPA such that the ratio of polymer mass to IPA was maintained at 1:2. The dispersion was once again sieved to separate the polymer mass. About 70 grams of polymer mass obtained was once again dispersed in 140ml of IPA. The final polymer mass was dried at 40°C for about 48 hours.

[0103] (iv) Purification of Natrosol™ 250 HHX with Isopropyl alcohol

[0104] About 20 grams of Natrosol 250 HHX was solubilised in 900 ml water to produce a solution. About 3600 ml of isopropyl alcohol (IPA) (1:4 ratio of solution to IPA) was added to this solution and stirred for 30 minutes at 1600 rpm to form a homogenous mixture. The mixture was then sieved to separate the lumpy polymer mass. About 80 grams of the polymer mass was then dispersed in 160 ml of IPA such that the ratio of polymer mass to IPA was maintained at 1:2. The dispersion was once again sieved toseparate the polymer mass. About 70 grams of polymer mass obtained was once again dispersed in 140ml of IPA. The final polymer mass was dried at 40°C for about 48 hours.

[0105] Nitrate and Nitrite content was determined potentiometrically using an indicator, a nitrate selective electrode and a silver-silver chloride electrode with 13.2 g / L solution of ammonium sulphate as reference electrode. The comparative results of nitrate and nitrite level before and after treatment of the excipients are provided is Table 1 below:

[0106] TABLE 1:

[0107] Natrosol™ 250 HX Natrosol™ 250 HHX USP Nitrate Nitrite USP Nitrate Nitrite Limit (PPm) (PPm) Limit (PPm) (PPm) (ppm)

[0108] Observed

[0109] value before IPA 441.27 20.75 271.2 35.1 treatment

[0110] Observed value after

[0111] IPA 2000 2000

[0112] 62.3 20.2 58.3 27.8 treatment

[0113]

[0114] It was observed that after treatment of HEC, the nitrate and nitrite levels were significantly reduced in comparison to untreated HEC.

[0115] Example 2: Preparation of Dorzolamide ophthalmic formulation:

[0116] Different batches of Dorzalamide ophthalmic formulation were prepared. The HECs (both grades) were prepared by purification as described in Example 1 above. Post purification, the HECs were sterilized by subjecting to autoclaving at 121°C for about 30 minutes. The drug phase containing Dorzolamide was prepared by dissolving buffering agents, osmogents and preservatives in 60% of batch quantity water. The pH was adjusted using 0.1N sodium hydroxide. The drug phase was sterilized by filtering through 0.2 micron sterilizing grade filter, and was then mixed with the HECs to obtain the final formulation.

[0117] Example 3: Determination of NDSRI levels in final Dorzolamide ophthalmic formulation:The NDSRI levels in final Dorzolamide ophthalmic formulation of Example 2 were determined using LC-MS / MS (UPLC system equipped with UV-VIS detector and MS / MS detector). Standard solution of NDSRI (N- Nitroso dorzolamide) impurity was prepared. The sample solution and placebo solutions were prepared. The placebo, Standard, and sample were injected and peak response was recorded. The Dorzolamide formulations prepared as per Example 2, containing treated excipient and untreated excipient, were tested for NDSRI level in the final formulation. The results are summarised in table 2 below

[0118] TABLE 2:

[0119] Formulation Formulation N-Nitroso Dorzolamide

[0120] No. Details

[0121] 1. Formulation Time Stability NDSRI with

[0122] point conditions

[0123] unpurified (PPm) HEC <25*

[0124] Initial 14.021 1 month 25°C / 40%RH 12.381

[0125] 40°C / 25%RH 14.946 50°C / 75%RH 36.337 3 months 25°C / 40%RH 26.171

[0126] 40°C / 25%RH 33.397 50°C / 75%RH 50.941 2. Formulation Initial 1.0831

[0127] with purified

[0128] HECs 1 month 40°C / 25%RH 2.6583

[0129] 3 months 3.3882 1 month 50°C / 75%RH 3.22

[0130] 3 months 3.9876 3 Reproducible Initial 5.0196

[0131] batch

[0132] (Formulation 2) 1 month 40°C / 25%RH 6.00

[0133] with purified

[0134] HECs 3 months 4.0227

[0135] 3 months 25°C / 40%RH 3.5697 3 months 30°C / 65%RH 3.6326

[0136]

[0137] RH: relative humidity

[0138] NMT: Not more than

[0139] *This is based on recommended Acceptable intake o by USFDA for Dorzolamide

[0140] Example 4: Preparation of Dorzolamide-Timolol ophthalmic formulation:

[0141] Different batches of Dorzolamide-Timolol ophthalmic formulation were prepared using conventional technique. The polymer HEC excipients were prepared and purified as described in Example 1. Post purification, the excipients were sterilized by subjecting to autoclaving at 121°C for about 30 minutes. The drug phase containing combination of Dorzolamide-Timolol active ingredients was prepared by dissolving buffering agents, osmogents and preservatives in 60% of batch quantity water. The pH was adjusted using 0.1N sodium hydroxide. The drug phase was then sterilized by filtering through 0.2 micron sterilizing grade filter, and was then mixed with the polymer excipients to obtain the final formulation.

[0142] Example 5: Determination of NDSRI levels in final Dorzolamide-Timolol ophthalmic formulation:

[0143] The NDSRI levels in the Dorzolamide-Timolol ophthalmic formulation of Example 4 above were determined using LC-MS / MS (UPLC system equipped with UV-VIS detector and MS / MS detector). Standard solution of NDSRI (N- Nitroso Timolol) impurity was prepared. The sample solution and placebo solution were prepared. The placebo, Standard, and sample were injected and peak response was recorded. The Dorzolamide-Timolol formulations prepared as per Example 4, containing treated excipient and untreated excipient, were tested for NDSRI level in the final formulation. The results are summarised in table 3 below:

[0144] TABLE 3:

[0145] Formulation Formulation N Nitroso Dorzolamide

[0146] No: Details

[0147] 1. Formulation Time Stability NDSRI with

[0148] point conditions (ppm)<37.5* unpurified

[0149] HEC Initial 46.2283

[0150]

[0151] 3 months 25°C / 40%RH 44.41

[0152] 40°C / NMT25%RH 45.69

[0153] 2. Formulation Initial 2.3599

[0154] with purified 3 months 40°C / NMT25%RH 7.1049

[0155] HECs

[0156] 3 months 25°C / 40%RH 6.0861

[0157] 3 months 30°C / 65%RH 6.4072

[0158]

[0159] RH: Relative humidity

[0160] NMT: Not more than

[0161] *This is based on recommended Acceptable intake by USFDA for Dorzolamide formulation.

[0162] EXAMPLE 6: Purification of Excipient using acetone:

[0163] Different ratios of acetone: water from 1:1 to 20:1 was used for optimum purification of HEC. Two commercially available grades of HEC, Natrosol™ 250 HX (Molecular weight: 10,00,000 Daltons, Brookfield viscosity at 25°C: 1,500-2,500 mPa-s) and Natrosol™ 250 HHX (Molecular weight: 13,00,000 Daltons, Brookfield viscosity at 25°C: 3,500-5,500 mPa·5) were used for this.

[0164] Concentrated dispersion of two grades of Hydroxyethyl cellulose was prepared.

[0165] Preparation of Natrosol™ 250 HX with acetone: 50 grams of Natrosol 250 HX was dispersed in 500 ml of acetone, stirred to get homogeneous lump free dispersion.

[0166] Preparation of Natrosol™ 250 HHX with acetone: 50grams of Natrosol 250 HHX was dispersed in 500ml of acetone, stirred to get homogeneous lump free dispersion.

[0167] (i) Purification of Natrosol™ 250 HX with acetone

[0168] About 50 grams of Natrosol 250 HX was dispersed in 500 ml of acetone. About 150 ml of water was added to this dispersion and stirred to get homogenous mixture. The mixture was then sieved to separate the lumpy polymer mass. The resultant polymer mass was dried at 40°C for about 48 hours.

[0169] (ii) Purification of Natrosol™ 250 HHX with acetone

[0170] About 50grams of Natrosol 250 HHX was dispersed in 500ml of acetone. About 150 ml of water was added to this dispersion and stirred to get homogenous mixture. Themixture was then sieved to separate the lumpy polymer mass. The resultant polymer mass was dried at 40°C for about 48 hours.

[0171] Nitrate and Nitrite content was determined potentiometrically using an indicator, a nitrate selective electrode and a silver-silver chloride electrode with 13.2 g / L solution of ammonium sulphate as reference electrode.

[0172] The comparative results of nitrate and nitrite level before and after treatment of the excipients are provided is Table 4 below:

[0173] TABLE 4

[0174] Natrosol™ 250 HX Natrosol™ 250 HHX USP Nitrate Nitrite Nitrate Nitrite Limit for (ppm) (ppm) (PPm) (PPm) (ppm)

[0175] Observed

[0176] value before treatment 1306.66 13.55 662.99 60.17 with acetone

[0177] Observed value after 26.03 58.44

[0178] 2000

[0179] treatment with acetone 8.38 25.94

[0180]

[0181] It was observed that after treatment of HEC with acetone, the nitrate and nitrite levels were significantly reduced in comparison to untreated HEC.

[0182] Example 7: Preparation of Dorzolamide ophthalmic formulation:

[0183] Different batches of Dorzolamide ophthalmic formulation were prepared. The HECs (both grades) were prepared by purification as described in Example 6 above. Post purification, the HECs were sterilized by subjecting to autoclaving at 121°C for about 30 minutes. The drug phase containing Dorzolamide was prepared by dissolving buffering agents, osmogents and preservatives in 60% of batch quantity water. The pH was adjusted using 0.1N sodium hydroxide. The drug phase was sterilized by filtering through 0.2 micron sterilizing grade filter, and was then mixed with the HECs to obtain the final formulation.

[0184] Example 8: Determination of NDSRI levels in final Dorzolamide ophthalmic formulation:The NDSRI levels in final Dorzolamide ophthalmic formulation of Example 7 were determined using LC-MS / MS (UPLC system equipped with UV-VIS detector and MS / MS detector). Standard solution of NDSRI (N- Nitroso Dorzolamide) impurity was prepared. The sample solution and placebo solutions were prepared. The placebo, Standard, and sample were injected and peak response was recorded.

[0185] The Dorzolamide formulations prepared as per Example 7, containing treated excipient and untreated excipient, were tested for NSDRI level in the final formulation.

[0186] The results are summarised in Table 5 below:

[0187] TABLE 5:

[0188] Formula Formulation Details N-Nitroso Dorzolamide tion No.

[0189] 1. Formulation with unpurified Time point NDSRI (ppm) HEC Initial 14.021

[0190] IM (40°C / 25% 14.946

[0191] RH)

[0192] 2. Formulation with purified HEC Initial 8.41

[0193] IM (40°C / 25% 9.52

[0194]

[0195] RH)

[0196] RH: relative humidity

[0197] Based on the USFDA recommended acceptable intake for dorzolamide of 100ng / day, the maximum permissible limit of NDSRI impurity is 25 ppm. Accordingly, the use of purified HEC was found to keep the NDSRI level way below 25ppm in this composition.

[0198] Example 9: Preparation of Dorzolamide Hydrochloride and Timolol ophthalmic formulation:

[0199] Different batches of Dorzolamide hydrochloride and Timolol ophthalmic formulation were prepared. The polymer HEC excipients were prepared and purified as described in Example 6. Post purification, the excipients were sterilized by subjecting to autoclaving at 121°C for about 30 minutes. The drug phase containing Dorzolamide hydrochloride and Timolol active ingredient was prepared by dissolving bufferingagents, osmogents and preservatives in 60% of batch quantity water. The pH was adjusted using 0.1N sodium hydroxide. The drug phase was then sterilized by filtering through 0.2 micron sterilizing grade filter, and was then mixed with the polymer excipients to obtain the final formulation.

[0200] Example 10: Determination of NDSRI levels in final Dorzolamide Hydrochloride and Timolol ophthalmic formulation:

[0201] The NDSRI levels in the Dorzolamide Hydrochloride and Timolol ophthalmic formulation of Example 9 above were determined using LC-MS / MS (UPLC system equipped with UV-VIS detector and MS / MS detecto

[0202] r). Standard solution of NDSRI (N- Nitroso Dorzolamide) impurity was prepared. The sample solution and placebo solution were prepared. The placebo, Standard, and sample were injected and peak response was recorded.

[0203] The Dorzolamide Hydrochloride and Timolol formulations prepared as per Example 9, containing treated excipient and untreated excipient, were tested for NSDRI level in the final formulation.

[0204] The results are summarised in Table 6 below:

[0205] TABLE 6

[0206] Formula Formulation Details N-Nitroso Dorzolamide tion No.

[0207] 1. Formulation with unpurified Time point NDSRI (ppm) <37.5*

[0208] HEC Initial 46.2283

[0209] IM (40°C / 25% 51.2687

[0210] RH)

[0211] 2. Formulation with purified HEC Initial 7.7979

[0212] IM (40°C / 25% 9.8675

[0213]

[0214] RH)

[0215] RH: Relative humidity

[0216] Based on the USFDA recommended acceptable intake for the combination product of dorzolamide and timolol, the maximum permissible limit of NDSRI impurity is 37.5 ppm. Accordingly, the use of purified HEC was found to keep the NDSRI level way below 37.5 ppm in this composition.

Claims

CLAIMS:

1. A method of reducing nitrosamine drug substance-related impurities (NDSRIs) in a pharmaceutical composition comprising an active ingredient and hydroxyethyl cellulose (HEC), wherein the amount of nitrates and nitrites present in hydroxyethyl cellulose is reduced through purification of HEC using a water miscible, polar solvent in which HEC is insoluble.

2. The method according to claim 1, wherein the water miscible, polar solvent is selected from the group comprising acetone, ethanol, methanol, tertiary butanol, 2- chloro ethanol and isopropyl alcohol (IPA).

3. The method according to claim 1, wherein a ratio of HEC: water miscible, polar solvent is about 1: 1 to about 1:20.

4. The method according to claim 1, wherein the method comprises the steps of: (i) mixing HEC with a liquid medium selected from:(a) water to form an aqueous HEC solution, wherein the aqueous HEC solution is further mixed with a water miscible, polar solvent in a ratio of about 1:1 to about 1:20 to obtain a homogenous dispersion with a polymer mass; or(b) a water-miscible, polar solvent in a ratio of about 1: 1 to about 1:20 to form a polymer dispersion, wherein the polymer dispersion is further mixed with water in a ratio of 1: 1 to 20: 1 to obtain a polymer mass;(ii) separating the polymer mass obtained in step (i);(iii) optionally, redispersing the polymer mass obtained in step (ii) one or more times in the water-miscible, polar solvent in a ratio of 1:2 followed by separation;(iv) drying the polymer mass at a temperature in the range of about 35°C to about 50°C to obtain the purified hydroxy ethyl cellulose polymer mass.

5. The method according to claim 4, wherein the separation of polymer mass in step (ii) and step (iii) is either done by sieving, centrifugation, filtration or any other suitable separation technique.

6. The method according to claim 4, wherein the purification of HEC comprises the steps of:(i) mixing HEC with water to form an aqueous solution;(ii) mixing the aqueous HEC solution of step (i) with the water miscible, polar solvent in a ratio of 1:4 to obtain a homogenous dispersion with a polymer mass;(iii) separating the polymer mass from the dispersion obtained in step (ii);(iv) dispersing the polymer mass obtained in step (iii) in the solvent in a ratio of 1:2;(v) separating the polymer mass of step (iv), followed by dispersing the polymer mass in the solvent at a ratio of 1:2; and(vi) drying of the polymer mass of step (v) at a temperature of about 35°C to about 50°C,wherein the water miscible, polar solvent is isopropyl alcohol.

7. The method according to claim 4, wherein the purification of HEC comprises the steps of:(i) mixing HEC with water miscible, polar solvent in a ratio of 1:5 to about 1:20 to form polymer dispersion;(ii) mixing the polymer dispersion of step (i) with the water, in the ratio of 1: 1 to 20: 1 to obtain a polymer mass;(iii) separating the polymer mass from the dispersion obtained in step (ii);(iv) drying of the polymer mass obtained in step (iii) at a temperature of about 35°C to about 50°C,wherein the water miscible, polar solvent is acetone.

8. The method according to claim 1 to 7, wherein HEC consists of one or more impurities selected from the group comprising nitrous acid, nitrite ions, nitrite salts, nitric acid, nitrates, nitrous acid ions, nitrite esters, peroxynitrite salts, nitrosonium ions, nitro compounds, anhydrous nitrous acid, dinitrogen tetroxide, nitrosyl halides, nitrosyl thiocyanates, nitrosophenols, nitrosothiols, nitric oxide, nitrogen dioxide and nitrile chloride.

9. The method according to claims 1 to 7, wherein the amount of nitrate present in HEC after purification with the water miscible, polar solvent is less than 70ppm.

10. The method according to claims 1 to 7, wherein the amount of nitrite present in HEC after purification with the water miscible, polar solvent is less than 30ppm.

11. The pharmaceutical composition comprising purified HEC according to claim 1 and an active pharmaceutical ingredient containing one or more amine groups, wherein the amount of nitrosamine drug substance-related impurities (NDSRIs) present in the composition is less than lOppm, when stored at a temperature of 40°C and 75% relative humidity for a period of 3 months.

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