The aim of the study was to evaluate the quality of generic product of three DPP-4 inhibitor

The aim of the study was to evaluate the quality of generic product of three DPP-4 inhibitor ( Sitagliptin, Vildagliptin and Linagliptin) used to treat type 2 diabetes, manufactured by Bangladeshi pharmaceutical company. The present investigation deals with all (i) degradation studies including acid, base, thermal and photo stability on the drug substances under the ICH prescribed conditions,(ii) isolated and characterized prominent degradation product through LCMS/MS, IR and NMR (iii) describes plausible degradation pathways and (iv)developed and validated a simple, sensitive and selective stability-indicating RP-UHPLC method for quantification of Sitagliptin, Vildagliptin and Linagliptin. 1.1. Background of the study Medicines are perhaps as old as mankind and the perceptions how their quality has to be ensured has changed progressively over the time.1 The regulation of modern medicines started only after breach progress in the 19th century life sciences which laid a solid groundwork for the modern drug research and development and after the second World War started to grow. Unexpected proceedings have catalysed the development of medicines regulation more than the evolution of a knowledge base. In 1937 more than 100 people in 15 states of the United States died of due to diethylene glycol poisoning in sulfanilamide elixir, which used the chemical as a solvent without any safety testing. 2,3 However, in countries with poor regulatory environment even recently medicines contaminated with diethylene glycol have killed patients.4 In 2009, 25 children were dying after taking paracetamol syrup due to the presence of poisonous diethylene glycol in Bangladesh 5. Lots of case related to substandard and counterfeit drugs around the world. The substandard drugs are ineffective and often dangerous to the patient because of their faulty formulation and also for low quality ingredients which dont meet the correct scientific specifications. Products with the correct ingredients may be included in counterfeit drugs but fake packaging, with the wrong ingredients, with insufficient active ingredients or without active ingredients 6. The health hazard effects of counterfeit drugs are greater than substandard drugs 7. Counterfeit and substandard drugs are a prime cause of morbidity, mortality and loss of public confidence in drugs and health structures 8. WHO has calculated approximately 10 of the global pharmaceuticals market consists of counterfeit drugs, but this percentages are to be increased to 25 in developing countries, and may exceed up to 50 in certain countries 9. FDA finds that up to 25 of the drugs consumed in poor countries are either substandard or counterfeit 10. India and China are recognized as the most leading countries in the production of counterfeit drugs and bulk active ingredients used for counterfeiting worldwide 11. Several studies depicted that counterfeits of pharmaceuticals sourced in China and India were detected in 33 and 42 countries respectively 12. The prevalence of substandard or counterfeit medicines in Lao PDR, Tanzania, Cambodia and Uganda are 12.244.5, followed by Indonesia, Nigeria, Cameroon 1848 and in Myanmar, Cambodia, Lao PDR, Ghana, Kenya, Tanzania, Uganda, Madagascar, Mali, Mozambique, Zimbabwe 1144 13. Substandard and counterfeit drugs are also intensely noticeable in developed countries along with poor and developing countries. For example, in North America, counterfeit atorvastatin 14, erythropoietin 14, growth hormone 15, filgrastim 14,15, gemcitabine 16,17, and paclitaxel 16,17 have been reported. In 20072008, 149 Americans died due to the uses of adulterated blood thinner, heparin that was legally imported. In 2012, 11 people died and sickened another 100 people in the US because of contaminated steroids. In another case, avastin were found to contain no active ingredients in the vials of the cancer medicine 18. In a study, WHO found that about 28 of antibiotic and 2090 of antimalarial drugs were failed quality specifications 19. Drugs are merely not ordinary consumers products. In most cases, consumers are not in a position to make decisions about when to use drugs, which drugs to use, how to use them and to consider potential benefits against risks because no medicine is completely safe. Professional advice from either prescribers or dispensers is needed in making these type of decisions. Pharmaceutical industries are bound to satisfy certain standards to claim it to be a quality drug. The main criterias for the quality of any drug in dosage form are its safety, potency, efficacy, stability, patient acceptability and regulatory compliance 20. The quality of pharmaceutical products must be reliable and reproducible from batch to batch to ensure the safety and efficacy 21. It is required for drug manufacturers to test their products to ensure the requisite quality both during and after manufacturing at various intervals during the shelf-life of the product 22. WHO supports the prescribing practice of generic medicines to minimize the expense of the health care system, but this should be carried out with sufficient and enough evidence for the replacement of one brand for another 23. Generic drugs are not only minimizes the health care expenses 24 but also the quality of the drugs. Comparison of bioequivalence study between the generic products against the innovator product is one of the main challenges and foremost factors for a generic marketing authorization 25. It is very important to do bioequivalence studies for generic products on account of any significant difference in the rate and extent by which the therapeutic ingredients become available at the site of drug action, administered under identical conditions in an adequately designed study 26. Dissolution testing serves as an indicator to identify bioavailability problems 27. Drug products which are biopharmaceutically as well as chemically equivalent must have the same quality, strength, purity, content uniformity, disintegration and dissolution rates 28. In vitro quality control (QC) of pharmaceutical products is a fixed set of investigation started during production by in-process quality control tests and after production by finished product quality control tests according to official pharmacopoeias and different regulatory agencies. QC tests help to avoid the confusion regarding safety, potency, efficacy and stability of pharmaceuticals 29. 2. Drug selection Diabetes is one of the major public health problem, one of four priority non-communicable diseases (NCDs) targeted for action over the world. It is a serious, chronic disease that occurs either when enough insulin is not produced from pancreas, or when the body cannot effectively use the insulin it produces. Both the number of cases and the prevalence of diabetes have been increasing over the past few decades. Globally, it was reported that 422 million adults were living with diabetes in 2014, compared to 108 million in 1980. Since 1980, the global prevalence of diabetes has nearly doubled rising from 4.7 to 8.5 in the adult population.30 This reflects an increase in associated risk factors like being overweight or obese. Diabetes prevalence has been rising faster in low- and middle-income countries than in high-incomecountries over the past decade. It was reported that diabetes caused 1.5 million deaths in 2012. An additional 2.2 million deaths were caused by higher-than-optimal blood glucose , by increasing the risks of cardiovascular and other diseases. 43 of these 3.7 million deaths occur before the 70 years of age. The percentage of deaths caused by high blood glucose or diabetes that occurs prior to age70 is higher in low- and middle-income countries than in high-incomecountries. Because sophisticated laboratory tests are usually required to differentiate between type 1 and type 2 diabetes, separate global estimates of diabetes prevalence for type 1 and type 2 do not exist. The majority of people suffering from diabetes are affected by type 2 diabetes. Earlier this used to occur nearly entirely among adults, but now occurs in children too. Type 2 diabetes mellitus (T2DM) is characterized by both progressive beta cell dysfunction and insulin resistance. To treat dysregulated glucose metabolism focuses on expanding the insulin response to hyperglycemia, improving insulin sensitivity or altering glucose removal through the gut or urine. Dipeptidyl-peptidase-4 (DPP-4) inhibitors or gliptins that block the inactivation of glucagon-like peptide-1 (GLP-1), which stimulates glucose-dependent insulin secretion and inhibits glucagon secretion. Morever, satiety is improved, gastric emptying is slowed, and food intake is reduced 31. With use of GLP-1 receptor agonists these effects are more prominent. There are five DPP-4 inhibitors, including alogliptin, linagliptin, saxagliptin, and sitagliptin in the United States and Europe and vildagliptin which is only available in Europe (Table 1). This class of therapy is administered once per day orally with the exception of vildagliptin which is administered twice per day. DPP-4 inhibitors can be taken without regard to food.DPP-4 inhibitors are not recommended for use as initial monotherapy for Type-2 Diabetes Mellitus treatment 32. These are most frequently prescribed in combination with lifestyle alteration and metformin, sulfonylureas, thiazolidinediones, and/or basal insulin, but selected patients intolerant to metformin have been effectively treated with DPP-4 inhibitor monotherapy. There are a number of combination products available, together with gliptinmetformin and gliptinsodium glucose transporter-2 inhibitor products.33 Table 1. Comparison of available DPP-4 inhibitors used in T2DM. SlDrugApprovalBrand nameDosageDose change in renal dysfunctionDose change in hepatic dysfunctionAvailable in combination1SitagliptinFDA approved Oct 2006Januvia, (Merck)25mgYes No Metformin Simvastatin50mg100mg2VildagliptinEU approved 2008Galvus (Novartis)50mgYesNot recommended for useMetformin 3SaxagliptinFDA approved July 2009Onglyza (AstraZeneca)2.5mgYesNoMetformin5mg 4LinagliptinFDA approved May 2011Tradjenta (Boehringer Ingelheim)5mgNoNoMetforminEmpagliflozin 5AlogliptinFDA approved 2013Nesina (Takeda Pharma Ltd.)6.25mgYesNoMetformin Pioglitazone12.5mg25mg There is very minor risk of hypoglycemia which can be negligible when DPP-4 inhibitors are used as monotherapy or in combination with metformin 34. Hypoglycemia risk is augmented when gliptins are used in combination with sulfonylureas or insulin. Interestingly, in a study of vildagliptin added to insulin therapy, in the setting of superior glycemic enhancement drastically lower rates of hypoglycemia were experienced in patients treated with vildagliptin compared to those receiving placebo 35. Weight gain is usually neutral across the DPP-4 inhibitor class 34. There appears to be neutral effect on lipids, with a general trend toward better triglyceride levels. Systolic blood pressure decrease is very modest and comparable within the class36. The gliptins are considered as safe in renal dysfunction however, alogliptin, saxagliptin, and sitagliptin have requirement of dose adjustment for renal impairment 32,37. Linagliptin and saxagliptin do not need dose adjustment for liver dysfunction. Alogliptin and sitagliptin also do not need dose adjustment for mild or moderate liver dysfunction but should be administered with caution in severe liver impairment. Day by day the requirement of DPP-4 inhibitors was increased for the treatment of T2DM. Thats why three DPP-4 inhibitors, Sitagliptin, Vildagliptin and Linagliptin were chosen for the study. 2.1. Drug Profile 2.1.1. Sitagliptin38 Sitagliptin is a medication which is prescribed for the treatment of type 2 diabetes. It is combined with exercise and diet to improve blood glucose levels in individuals suffering from type 2 diabetes. IdentificationDescriptionSitagliptin, an anti-diabetic drug which is a new oral hypoglycemic, the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. This enzyme-inhibiting drug is to be administered either alone or in combination with metformin or a thiazolidinedione for control of type 2 diabetes mellitus. The mechanism of drug is to competitively inhibit a protein/enzyme, dipeptidyl peptidase 4 (DPP-4), that results in an increased level of active incretins (GLP-1 and GIP), reduced amount of release of glucagon (diminishes its release) and increased release of insulin level.Chemical FormulaC16H15F6N5OStructureMolecular WeightAverage 407.3136 PharmacodynamicsSitagliptin is an orally-active member of new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. The advantage of this medicine is expected to be its lesser side-effects of hypoglycemia in the control of blood glucose values. The drug works to abolish the effects of a protein/enzyme (by the inhibition of this protein/enzyme) on the pancreas at the amount of release of glucagon (diminishes its release) and at the amount of insulin (increases its synthesis and release) until blood glucose levels are restored toward normal, in which case the protein/enzyme-enzyme inhibitor becomes less efficient and the amounts of insulin released abolishes thus diminishing the overshoot of hypoglycemia seen in other oral hypoglycemic drugs.Mechanism of actionSitagliptin is a extremely selective DPP-4 inhibitor, which is believed to work by slowing the inactivation of incretin hormones in patients with type 2 diabetes, thus increasing the concentration and prolonging the action of these hormones. Incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are released through intestine all through the day, and levels are increased in accordance to a meal. These hormones are quickly inactivated by the enzyme, DPP-4. The incretins are part of an endogenous system implicated in the physiologic regulation of glucose homeostasis. GLP-1 and GIP increase insulin synthesis when blood glucose concentrations are normal or elevated and release from pancreatic beta cells by intracellular signaling pathways involves cyclic AMP. GLP-1 also lessens glucagon secretion from pancreatic alpha cells, leading to reduced hepatic glucose production. By increasing active incretin levels, sitagliptin increases insulin release level and decreases glucagon levels in the circulation in a glucose-dependent manner. These type of changes lead to a reduction in hemoglobin A1c (HbA1c)levels, and a lower fasting and postprandial glucose concentration. Sitagliptin shows selectivity for DPP-4 and does not inhibit DPP-8 or DPP-9 activity in vitro at concentrations approximating those from therapeutic doses.AbsorptionRapidly absorbed following oral administration, associated with an absolute bioavailability of 87.Volume of distribution198 L healthy subjectsProtein bindingPlasma protein binding of the fraction of Sitagliptin is low (38).MetabolismSitagliptin does not generally undergo extensive metabolism. According to in vitro studies, CYP3A4 (oxidation) with contribution from CYP2C8 was the primary enzyme responsible for the limited metabolism of Sitagliptin.Route of eliminationApproximately 79 of sitagliptin is excreted unchanged condition through the urine with metabolism being a minor pathway of elimination. After administration of an oral 14Csitagliptin dose to healthy individuals, approximately 100 of the administered radioactivity was eliminated through feces (13) or urine (87) within one week of dosing. Primarily elimination of sitagliptin occurs via renal excretion and also involves active tubular secretion.Half life12.4 hoursClearancerenal cl350 mL/min Healthy subjects receiving 100mg oral doseAffected organismsHumans and other mammals 2.1.2. Vildagliptin 39 IdentificationDescriptionFormerly, Vidagliptin was identified as LAF237, which is a new oral anti-hyperglycemic agent (anti-diabetic drug) of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. Vildagliptin works by inhibiting the inactivation of GLP-1 and GIP by DPP-4, in order to enhance the secretion of insulin by GLP-1 and GIP in the beta cells and repress glucaon release by the alpha cells of the islets of Langerhans in the pancreas. At present, it is in clinical trials in the U.S. and has been reported to lessen hyperglycemia in type 2 diabetes mellitus. The drug is still not approved for use in the US, but it was approved in Feb 2008 by European Medicines Agency for use within the EU and is listed on the Australian PBS with some certain restrictions.Chemical FormulaC17H25N3O2StructureMolecular WeightAverage 303.3993Monoisotopic 303.194677059IUPAC Name (2S)-1-2-(3-hydroxyadamantan-1-yl)aminoacetylpyrrolidine-2-carbonitrileCalculated Predicted Partition Coefficient cLogP1.12Calculated Predicted Aqueous Solubility cLogS 2.2Solubility (in water) 1.75 mg/mL (sparingly soluble)Predicted Topological Polar Surface Area (TPSA) 76.36 2PharmacologyIndicationUsed to reduce hyperglycemia in type 2 diabetes mellitus. Structured Indications HYPERLINK https//www.drugbank.ca/indications/DBCOND0029752 Type 2 Diabetes Mellitus PharmacodynamicsVildagliptin is a member of the class of orally active antidiabetic drugs (DPP-IV inhibitors) that is known to have multifunctional advantages as they are not limited into simple blood-glucose control. One of these advantages is a strong protective effect on beta cells of pancreas, which get worse in diabetes. Vildagliptin is considered to be safe, very well tolerated, and efficacious. Gut incretin hormones are released after meal. GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) are the most important incretin hormones. These hormones are secreted in the human small intestine. These are responsible for insulin release due to increased glucose amount levels. GLP-1s dependence on glucose concentration is considered to be beneficial because of a lower risk of hypoglycemia. GLP-1 also inhibits glucagon secretion and increases pancreatic beta cell mass through stimulating proliferation and neogenesis. Nevertheless, the clinical utility of GLP-1 is limited by its short elimination half-life (2 minutes). Proteolytic enzyme DPP-IV degrades GLP-1 in a rapid way.Inhibition of the DPP-IV enzyme is considered as a novel therapeutic approach in the treatment of diabetes that enhance GLP-1 activity. GLP-1s ability is enhanced after administration of vidagliptin to produce insulin in response to increased concentrations of blood glucose that inhibit the release of amount of glucagon following meals, slow down the rate of nutrient absorption into the bloodstream, and also slow down the rate of gastric emptying, and reduce food intake.Mechanism of actionVildagliptin inhibits dipeptidyl peptidase-4 (DPP-4). As a result GLP-1 is invactivated by DPP-4, which allows GLP-1 to enhance the secretion of insulin in the pancreatic beta cells. Dipeptidyl peptidase-4s degrades GIP and GLP-1 in blood glucose regulation.AbsorptionRapidly absorbed after oral administration associated with an oral bioavailability of greater than 90.MetabolismCytochrome p450 3A4Protein binding9.3Half lifeThe elimination half-life is approximately 90 minutes.Affected organismsHumans and other mammals 2.1.3. Linagliptin 40 IdentificationPhysical StateWhite to off white powderDescriptionLinagliptin is a DPP-4 inhibitor developed by Boehringer Ingelheim. It is used in the treatment of type II diabetes Wikipedia. There are two pharmacological characteristics that set linagliptin apart from other DPP-4 inhibitors. They are- it has a non-linear pharmacokinetic profile and is not primarily eliminated by the renal system.Linagliptin was approved by FDA on May 2, 2011.Chemical FormulaC25H28N8O2StructureMolecular WeightAverage 472.5422Monoisotopic 472.23352218IUPAC Name8-(3R)-3-aminopiperidin-1-yl-7-(but-2-yn-1-yl)-3-methyl-1-(4-methylquinazolin-2-yl)methyl-2,3,6,7-tetrahydro-1H-purine-2,6-dionePharmacologyIndicationLinagliptin is used for the management of type 2 diabetes mellitus.Structured IndicationsPharmacodynamicsLinagliptin is a more potent inhibitor of DPP-4 than other member of the same class of drugs with an IC50 of 1 nM. It was found during comparison, sitagliptin, saxagliptin, and vildagliptin have an IC50 of 19, 50, and 62 nM respectively. Activity of DPP-4 by 72.7 and 86.1 from baseline is reduced by a dose of 2.5 mf and 5 mg respectively in healthy male individuals. A dose of 5 and 10 mg is effective as they inhibit 90 of DPP-4 for diabetic patients. Linagliptin is a selective inhibitor DPP-4.In-vitro it is also indicated by the lack of DPP-8 or DPP-9 inhibition at therapeutic exposures.Mechanism of actionLinagliptin is a competitive and reversible dipeptidyl peptidase (DPP)-4 enzyme inhibitor. Mechanism of action of linagliptin is due to slow the breakdown of insulinotropic hormone glucagon-like peptide (GLP)-1 for better glycemic control in diabetic patients. GLP and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones. These hormones increase the production and release of insulin from beta cells of pancreas and decrease the release of glucagon from pancreatic alpha cells. This leads to a overall reduction in glucose production in the liver and increase an of insulin in a glucose-dependent manner.AbsorptionPeak Plasma Concentration, Cmax, 5 mg, healthy subjects 8.32 nmol/L Time to attain peak plasma concentration, Tmax, 5 mg, healthy subjects 1.75 hours Area under the curve, AUC(0-24 hours), 5 mg, healthy subjects 119 nmol h/L Bioavailability (Rate or extent of absorption, healthy subjects 30. After administration of a dose of 5 mg once daily, steady state is attained by the third dose. In spite of reduction of Cmax by high fat meal, it increases AUC, this interaction with food is clinically insignificant. Linagliptin can be taken with or without food.Volume of distributionVd 1110 LProtein bindingApproximately it is bound to plasma protein at 70-80 , the extent to which is concentration dependent. As linagliptin has tendency to bind to plasma protein, it has a long terminal half-life and a non-linear pharmacokinetic profile. In this context linagliptin is unique as other DPP-4 inhibitors have linear pharmacokinetic profiles.MetabolismLinagliptin is not extensively metabolized. 90 of dose of linagliptin is excreted unchanged during metabolism. So very small portion of drug is metabolized. The main metabolite is CD 1790 which is pharmacologically inactive. Glucuronidation forms some of its other minor metabolites.Route of eliminationLinagliptin is eliminated via the feces/enteroheptic system (80) and urine (5). Other DPP-4 inhibitors are primarily eliminated by the renal system.Half lifeTerminal/Elimination half life 131 hours. As linagliptin has longer elimination half-life, once daily dosing is appropriate to sustain inhibition of DPP-4 activity. Effective half-life for accumulation of drug is 12 hours when multiple oral doses of 5 mg are given.ClearanceRenal clearance, steady state 70 mL/min 3. Stability Stability is the ability of a drug substance or a drug product to remain within established or recognized specifications to make sure its identity, strength, quality and purity all through the retest period or expiration dating period, as appropriate. 41 In a rational design and evaluation of dosage forms for drugs, the stability of the active components is the most major criterion to determine their suitability. Several forms of instability can occur. First, there may be chemical degradation of the drug, leading to significant lowering of the amount of the therapeutic agent in the particular dosage form. In case of drugs with narrow therapeutic indices it is even of greater significance, where the patient needs to be carefully titrated as a result serum levels are not so high which are potentially toxic or so low that they are ineffective. Second, although the degradation of the active drug may not be that extensive, a toxic degradant may be formed in the decomposition process. An example of a product of degradation that is significantly more toxic is conversion of tetracycline to epianhydrotetracycline. Third, instability of a drug product can lead to a decrease in its bioavailability, rather than to loss of drug or the formation of toxic degradation products. This reduction in bioavailability can result in a substantial lowering in the therapeutic efficacy of the dosage form. This phenomenon, for example, can be caused by physical and chemical changes in the excipients in the dosage form, independent of whatever changes the active drug may have undergone. Fourth, there may be substantial changes in the physical appearance of the dosage forms Since most drugs are organic molecules, it is important to recognize that many pharmaceutical pathways are, in principle, similar to reactions described for organic compound. The major difference that has to be considered is that most pharmaceutical reactions occur due to or are governed by water, oxygen, or light, rather than other active ingredients. Thus, the most common routes of decomposition are hydrolysis, oxidation, photolysis, racemization, and decarboxylation . 42. 3.1. Stability testing The aim of stability testing is to endow with evidence or verification on how the quality of a drug substance or drug product varies with time due to diversity of environmental factors such as temperature, humidity, and light, and to establish a retest period for the drug substance or a shelf life for the drug product and recommended storage conditions. 43 3.2. Stress testing or Degradation studies Stress testing of the drug substance can recognize the possible degradation products and degradation pathways. Stability testing is necessary to analyze the inherent stability of the molecule and validate the stability representing power of the analytical procedures used. The nature or methodology of the stress testing will vary on each drug substance and the category of drug product involved. Stress testing is typically to be carried out on a single batch of the drug substance. It should include the effect of temperatures (in 10C increments (e.g., 50C, 60C, etc.) above that for accelerated testing), humidity (e.g., 75 RH or greater) where appropriate, oxidation, and photolysis on the drug substance. The stress testing is to be supposed to also evaluate the susceptibility of the drug substance to hydrolysis. It is done within a wide range of pH values either in solution or suspension. Photo stability testing should be done with high importance as it is considered to be an essential part of stress testing. The standard conditions or criteria for photo stability testing are described in ICH Q1B. In stability study, degradation products are evaluated under various stress conditions is useful to establish degradation pathways and developing and validating appropriate analytical procedures. However, if any degradation product has been demonstrated that they not formed under accelerated or long term storage conditions then it is not necessary to evaluate that certain product specifically. Results from these studies will outline a vital part of the information provided to regulatory authorities 44. 3.2.1. Reasons for conducting forced degradation studies Forced degradation studies are carried out for the following reasons Development and validation of stability indicating methodology Determination of the intrinsic stability of a drug molecule, and structure elucidation of degradation products Determination of degradation pathways of drug substances and products Discernment of drug vs. non drug related degradation products in the formulations 3.2.2. FDA recommended degradation studies for a drug substance The following are FDA recommended degradation studies for a drug substance (FDA 1998) Stressing the drug substance in solution and suspension at acidic and alkaline pH and under high oxygen environment Stressing the solid drug at temperature and temperature/ humidity conditions in excess to accelerated conditions Stressing the drug photolytically in the solid state and solution Demonstration of the stability indicating methods with forced degraded / spiked samples Isolation and/or full characterization of degradation products (by NMR, MS, UV etc) Determination of the mechanism and kinetics of formation of the degradation products if possible. Thus, for degradation study of a drug substance, it should be exposed to acid /base, oxidative, exposure to light, thermal and humidity. 3.2.3. Regulatory considerations In accordance to the International Conference on Harmonization (ICH) guidelines 45, impurities in pharmaceuticals can be defined as components that remain with the active pharmaceutical ingredients, or arise during the manufacturing process and/or storage of the drug substance. The performance of the pharmaceutical products may be influenced by the presence of these impurities, even in small amounts. The ICH and FDA have published guidelines for the identification and qualification of impurities in new drug substances and drug products 45-47. According to the guidelines, impurities can be characterized as organic or inorganic impurities and residual solvents. Organic impurities may include impurities in starting synthesis materials, synthesis by-products, degradation products and intermediates. For degradation products, the ICH Guidance Q3B (R2) 46 provides recommendations for reporting, control, identification and qualification in drug products. The critical values for reporting, identifying and qualifying impurities vary For a given degradation product, its acceptance criteria (allowable level) should be established no higher than its qualified level and along with safety considerations 47. Sometimes the qualification thresholds are exceeded and adequate data are not available to qualify the degradation product. In this case, additional studies should be conducted on the drug product containing the degradant or isolated degradation products. The guidelines of FDA and ICH provide a feasible way to control drug degradation products. However, degradation products that exceed qualification thresholds or that are potentially toxic compounds are not under this guidance as they do not provide a rationale for them. 3.2.4. Purposes of forced degradation studies Forced degradation studies are carried out to achieve the following purposes To establish degradation pathways of drug substances and drug products. To differentiate degradation products that are related to drug products from those that are generated from non-drug product in a formulation. To elucidate the structure of degradation products. To determine the intrinsic stability of a drug substance in formulation. To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis or photolysis of the drug substance and drug product 48, 49. To establish stability indicating nature of a developed method. To understand the chemical properties of drug molecules. To generate more stable formulations. To produce a degradation profile similar to that of what would be observed in a formal stability study under ICH conditions. To solve stability-related problems 50. 3.2.5.Forced degradation testing time Before stress testing, it is very important to know the appropriate time or when to perform forced degradation studies for the development of new drug substance and new drug product. FDA guidance states that stress testing should be performed in phase III of regulatory submission process. Stress studies should be done in different pH solutions, in the presence of oxygen and light, and at elevated temperatures and humidity levels to determine the stability of the drug substance. Generally, force degradation studies are carried out on a single batch. The results should be summarized and submitted in an annual report 51 Starting stress testing would be very effective if it is done during early in preclinical phase or phase I of clinical trials. These tests are conducted on drug substance to attain adequate time for identifying degradation products and structure elucidation and also to optimize the stress conditions. An early stress study also provides appropriate recommendations form a king improvements in the manufacturing process and suitable selection of stability-indicating analytical procedures 52,53. 3.2.6. Degradation Limits Many discussions among pharmaceutical scientists have already been held about the question of how much degradation is sufficient. Degradation of drug substances between 5 and 20 are considered as acceptable and reasonable for validation of chromatographic assays 54,55. Some pharmaceutical scientists suggest that 10 degradation can be favorable to use in analytical validation for small pharmaceutical molecules. So acceptable stability limits of 90 of label claim is frequent 56. Others suggestion is like that the drug substance spiked with a combination of known degradation products can be used to challenge the methods engaged to monitor in stability of drug product 49. No such limits for physiochemical changes, loss of activity or degradation during shelf life have been established for individual types or groups of biological products 57. It is not always mandatory that forced degradation study would result in a degradation product. The study can be concluded if no degradation is observed after drug substance or drug product has been exposed to stress conditions than those conditions mentioned in an accelerated stability protocol 58. Over-stressing a sample is not recommended as this may cause the formation of a secondary degradation product that is not to be seen in formal shelf-life stability studies. On the other hand, under-stressing may not generate sufficient degradation products which can fail the whole stress study 59. Protocols for generation of product related degradation may be different for each drug substance and drug product because of their differences in matrices and concentrations. It is recommended that maximum of 14 days for stress testing in solution (a maximum of 24h for oxidative tests) to provide stressed samples for methods development 60. 3.2.7.Approach for degradation conditions selection Forced degradation is conducted to make representative samples for the development of stability-indicating methods for drug substances and drug products. The options of stress conditions are supposed to be consistent with the products breakdown under normal manufacturing process, storage, and use conditions which are specific in each individual case 56. A common procedure of degradation conditions used for drug substance and drug product is shown in Scheme 1. To conduct force degradation studies successfully a minimal list of stress factors recommended to take account like acid and base hydrolysis, thermal degradation, photolysis, oxidation 52,61-63 and may include freezethaw cycles and shear stress conditions 57. There is no specification in regulatory guidelines about the conditions of pH, temperature and specific oxidizing agents to be used. The protocol of photolytic degradation studies is left to the applicants judgment even though Q1B specifies that the light source is supposed to produce combination of visible and ultraviolet (UV,320400 nm)outputs, and that exposure levels should be reasonable 58. The initial trial should have the aim to come upon the conditions that degrade the drug by approximately 10. Some conditions mostly used for forced degradation studies are presented in Table 164. Some scientists have found it practical to begin with extreme conditions such as 80 1C or even higher temperatures and testing at shorter (2,5,8,24h,etc.) multiple time points, so that the rate of degradation can be evaluated 65. The primary degradants and their secondary degradations products can be illustrated by testing at initial stage. Thus improved degradation pathway can be established. In another approach degradation is started by considering the drug substance to be labile and doing degradation at the conditions mentioned in Table1. Then stress would be increased or decreased to obtain sufficient degradation. As compared to harsher environment and less time approach, this tactic is better because some reasons. They are (i) If there is any modification in the mechanism of reaction during a harsh condition , and (ii) If there is any practical difficulty in neutralizing or diluting every sample, when it is associated with a high concentration of reactants, e.g., acid or base, before an injection can be made on the HPLC column. Both these reasons suggest as normal as possible conditions to carry out the decomposition of the drug 66. Studies should be repeated when formulations or methods change because the change may lead to the production of new degradation products. 3.2.8 Diagram of stress study Figure1 Diagram of stress study used for degradation of drug substance and drug product67 3.2.9. Conditions for degradation study 3.2.9.1. Hydrolytic conditions Hydrolysis is one of the most common degradation chemical reactions over a wide range of pH. Hydrolysis is a chemical process that includes decomposition of a chemical compound by reaction with water. Hydrolytic study under acidic and basic condition involves catalysis of ionizable functional groups present in the molecule. Acid or base stress testing involves forced degradation of a drug substance by exposure to acidic or basic conditions which generates primary degradants in desirable range. The selection of the type and concentrations of acid or base depends on the stability of the drug substance. Hydrochloric acid or sulfuric acids(0.11M) for acid hydrolysis and sodium hydroxide or potassium hydroxide(0.11M) for base hydrolysis are suggested as suitable reagents for hydrolysis 68,69. If the compounds for stress testing are poorly soluble in water, then co-solvents can be used to dissolve the min HCl or NaOH. The selection of co-solvent is based on the drug substance structure. Stress testing trial is normally started at room temperature and if there is no degradation, elevated temperature(5070 1C) is applied. Stress testing should not exceed more than 7days. The degraded sample is then neutralized using suitable acid, base or buffer to avoid further decomposition. 3.2.9.2. Oxidation conditions 3.2.9.3. Photolytic conditions The photo stability testing of drug substances must be evaluated to demonstrate that a light exposure does not result in unacceptable change. Photo stability studies are performed to generate primary degradants of drug substance by exposure to UV or fluorescent conditions. Some recommended conditions for photostability testing are described in ICH guidelines 58. Samples of drug substance and solid/liquid drug product should be exposed to a minimum of 1.2million lx hand 200 Wh/m2 light. The most commonly accepted wavelength of light is in the range of 300 800 nm to cause the photolytic degradation 73,74. The maximum illumination recommended is 6 million lxh 72. Light stress conditions can induce photo oxidation by free radical mechanism. Functional groups like carbonyls, nitro aromatic ,N-oxide, alkenes, aryl chlorides, weak CH and OH bonds, sulfides and polyenesare likely to introduce drug photosensitivity 75. 3.2.9.4. Thermal conditions 3.3.1. Sample generation For generating samples for SIM the API is force degraded at conditions more severe than accelerated degradation conditions. It involves degradation of drug at hydrolytic, oxidative, photolytic and thermal conditions as discussed earlier. The forced degradation of API in solid state and solution form is carried out with an aim to generate degradation products which are likely to be formed in realistic storage conditions 80. This sample is then used to develop an SIM. 3.3.2. Method development 4. Quality by Design (Qbd) approach in method development and optimization Joseph M. Juran was first defined the term Quality by Design (QbD) 83 and applied with great success. The underlying concept of QbD is that quality must be designed in to a product through the systematic implementation of a approach to establish a absolute understanding of the product and the processes utilized to develop and manufacture it. To improve quality control strategies are developed and used to verify continuously. Recently the FDA has begun to approve the QbD approach for the pharmaceutical sector.84 There are a lots of allegation of the concept, including the necessities for systematic experimental design strategies, modeling the influence of variables on quality, and ensuring the traceability of information from the stages of design through validation. Now a days analytical chemists have started to apply QbD approaches for chromatographic methods development, prompting a revisit of method development strategies. Modern technology allows to investigate the strategies for chromatographers in the context of Quality by Design. Pharmaceutical industry has paying attention on product Quality, Safety, and Efficacy. By applying scientific tools QbD (Quality by Design) product quality has been improved. These QbD tools will minimize the threat by increasing the quality and productivity. The implementation of ICH quality guidelines Q8 to Q11 are always recommended by regulatory authorities 85-88. Analytical Quality by Design (AQbD).As per ICH, QbD is defined as A systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management. The outcome of AQbD is well establised and suitable for intended purpose with robustness throughout the lifecycle. Different tools of AQbD life cycle are ATP (Analytical Target Profile), CQA(Critical Quality Attributes) 89,90, Risk Assessment, Method Optimization and Development with DoE (design of experiment), MODR (method operable design region), Control Strategy and Risk Assessment, AQbD Method Validation, and Continuous Method Monitoring. Figure 1 represents the AQbD life cycle with each tool. Figure Lifecycle of Quality by design tools(QbD) 4.1. Analytical Target Profile(ATP) General ATP for analytical procedures is as follows (a)Target analytes selection (API and impurities), (b)Technique selection (HPLC, GC, HPTLC, Ion Chromatography, chiral HPLC, etc.), (c)Method requirements selection (assay or impurity profile or residual solvents). (a) Target Analytes Selection. Common ATP for HPLC method during impurities profile Table-1 Common ATPs for impurity profile by HPLC method. SlMethod requirments for impurity profile1Number of analytes (API and impurities)2Separation of all analytes3Mobile Phase (buffer and organic modifier)4Elution method (gradient or isocratic)5Sample concentration6Sample diluent7Sample solution stability8Sample preparation process (dilution process and sonication time, etc.)9Filter or centrifuge10Impurity specification limits11Column type (stationary phase and dimensions)12Detection (UV/RID/ELSD)13Flow rate14Injection volume15Column oven temperature16Runtime17System suitability parameters selection with limits18LOD and LOQ concentrations establishment19Impurities/ Degradants calculation method20Recovery establishment (b) Technique Selection. Every analytical technique has its own principle so based on the analytes characteristic it can be chosen. Analytical test items and analytical techniques are as follows (1)identification by IR FT-IR spectrophotometer, (2)impurity profile (Chromophore) HPLC with UV detector, (3)impurity profile (non-Chromophore) HPLC with RID/ELSD and so forth, (4)assay by HPLC (Chromophore) HPLC with UV detector, (5)assay by HPLC (non-Chromophore) HPLC with RID/ELSD and so forth. (c) Method Requirements Selection.Method requirements can vary from one method to another. The common ATPs for impurity profile by HPLC method are listed in TableHYPERLINK https//www.hindawi.com/journals/jchem/2015/435129/tab2/ t _blank1. 4.2. Critical Quality Attributes (CQA) Critical quality attributes for analytical methods describes method attributes and parameters. Different CQA is required for each analytical technique. For HPLC analysis CQA are Buffer of mobile phase, pH of mobile phase, Diluent, Column selection, Organic modifier and Elution method. For GC analysis CQA are Gas flow, Oven temperature and program, Injection temperature, Sample diluent, and Concentration. For HPTLC method CQA are TLC plate, Mobile phase, Injection concentration and volume, Plate development time, Color development reagent and Detection method. The CQA for analytical method development based on the nature of impurities and DS can such as solubility, pH value, polarity, charged functional groups, boiling point, and solution stability. 4.3. Risk Assessment. Risk Assessment is a science-based process can be performed from initial stage of method development to continuous method monitoring. AQbD come up to involves the risk detection at early stages of progress followed by appropriate improvement plans with control strategies that will be recognized. In general, Ishikawa fishbone diagram can be used for risk identification and assessment. In the FigureHYPERLINK https//www.hindawi.com/journals/jchem/2015/435129/fig5/ t _blank5that shows fishbone risk identification approach for typical analytical test procedure. Figure- Fishbone for Risk identification 4.4. Design of Experiments(DoE) for method optimization and development Once the potential and critical analytical method variables are defined with initial risk assessment, then DoE can be performed to confirm and refine critical method variables based on statistical significance. It can be determined per unit operation or combination of selected multiple method variables and their interactions and responses (critical method attributes). This approach provides an excellent opportunity to screen a number of conditions generated from a limited number of experiments. Then, data evaluations by using statistical tools are very important to identify critical method variables and the appropriate optimal ranges for method variables where a robust region for the critical method attributes could be obtained. As per ICH Q8 guidance process robustness is defined as Ability of a process to tolerate variability of materials and changes of the process and equipment without negative impact on quality. Starting materials properties will affect the drug substance synthetic process robustness, impurity profile, physicochemical properties, process capability, and stability. Process understanding will provide the sufficient knowledge for establishing robustness parameters by evaluating different operating conditions, difference scales, and different equipments. 4.5. Method Operable Design Region (MODR) Method operable design region (MODR) is used for establishment of a multidimensional space based on method factors and settings MODR can provide suitable method performance. It is also used to establish meaningful method controls such as system suitability, RRT, and RRF. Further method verification exercises can be employed to establish ATP conformance and ultimately define the MODR. 4.6. Control Strategy and Risk Assessment Control strategy 91-92 is a planned set of controls, derived from analyte nature and MODR understanding. Method control strategy can be established based on the complete statistical data collected during the DoE and MODR stages as discussed above. Using this statistical experimental data, correlations can be drawn between method and analyte attributes for the ability to meet ATP criteria. Control strategy will resolve the method parameters inconsistency (e.g., reagent grade, instrument brand or type, and column type). Method control strategy does not appear dramatically different under the AQbD approach when compared to the traditional approach. However, method controls are established based on CQA, DoE, and MODR experimental data to ensure a stronger link between the method purpose and performance. 4.7. Analytical Quality by Design (AQbD) Method Validation AQbD 93 method validation approach is the validation of analytical method over a range of different API batches. It uses both DoE and MODR knowledge for designing method validation for all kinds of API manufacturing changes without revalidation. The approach provides the required ICH validation elements as well as information on interactions, measurement uncertainty, control strategy, and continuous improvement. This approach requires fewer resources than the traditional validation approach without compromising quality. 4.8. Continuous Method Monitoring (CMM) and Continual Improvement Life cycle management is a control strategy used for implementation of design space in commercial stage. CMM is final step in AQbD life cycle it is a continuous process of sharing knowledge gained during development and implementation of design space. This includes results of risk assessments, assumptions based on prior knowledge, statistical design considerations, and bridge between the design space, MODR, control strategy, CQA, and ATP. Once a method validation is completed, method can be used for routine purpose and continuous method performance can be monitored. This can be performed by using control charts or tracking system suitability data, method related investigations, and so forth. CMM allows the analyst to proactively identify and address any out-of-trend performance. Advantages and Recommendations. AQbD is an approach that moves away from reactive troubleshooting to proactive failure reduction. The type and extent of the risk assessment depends on the stage of the project in the development timeline. AQbD success rate depends on right approach, planning, tools usage, and performance of work in a suitable time. Applying the appropriate risk assessment tools at the right time could lead to prevention of method failures and better understanding on the design space and control strategy 94-106. 5.Method Validation Analytical method validation is the process which is established by doing laboratory studies, that fulfill the efficiency of the method to meet the official requirements for the intended analytical application. Validation is required for any new or modified method to confirm that it is capable of providing precise, reproducible and robust results with a variation of equipment, operators in the same or different laboratories 107. The validation process for analytical methods begins with the planned and systematic approach by the applicant of the validation data to support analytical procedures 108. The obtained results from method validation can be used to evaluate the quality, acceptability, reliability and stability of analytical results. The analytical methods validation is conducted as per ICH guidelines. Validation or revalidation of analytical methods is required 109 Before their introduction into routine use Whenever any conditions are changes Whenever the method is changed 5.1. Typical parameters recommended by FDA, USP, and ICH are as follow.109, 111 Specificity Linearity Range Precision Method precision (Repeatability) Intermediate precision (Reproducibility) Accuracy (Recovery) Solution stability Limit of Detection (LOD) Limit of Quantification (LOQ) Robustness System suitability Forced degradation studies Components required for validation The common compendial requirements for the establishment of analytical methods for finished products are categorized in following ways Category 1 Identification and Qualification of main components or active ingredients in its finished pharmaceutical products. Category 2 Determination of impurities in bulk drug substances or degradation compounds in finished pharmaceutical products. It includes the quantitative assay and limit tests. Category 3 Determination of performance characteristics Category 4 Identification tests. Table-5.1. Parameters to be covered in validation 5.2.Linearity and Range The linearity of an analytical method is its ability to obtain response that is directly proportional to the concentration (amount) of analyte in the sample. A linear relationship should be assessed across the range of the analytical procedure. It is described directly on the drug substance by dilution of a standard stock solution of the drug product, using the proposed method. Linearity is usually articulated as the confidence limit about the slope of the regression line.109-111 For determining the linearity, minimum of five concentrations are recommended according to ICH guideline.112 The range of an analytical method is the difference between the upper levels and lower levels which have been confirmed to be determined with linearity, precision and accuracy using the method.110 5.3.Precision The precision of an analytical procedure expresses the closeness of agreement or the degree of scatter between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels repeatability, intermediate precision and reproducibility.112The precision of an analytical procedure is usually expressed as the standard deviation or relative standard deviation of series of measurements. Precision may be either the degree of reproducibility or of the repeatability of the analytical procedure under normal conditions. Intermediate precision (also known as ruggedness) expresses within laboratories variations, as on different days, or with different analysts or equipment within same laboratory. Precision of an analytical procedure is determined by assaying a sufficient number of aliquots of a homogeneous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation.113 5.4.Accuracy (Recovery)The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. lt is determined by applying the method to samples to which known amounts of analyte have been added. These should be analysed against standard and blank solutions to ensure that no interference exists. The accuracy is then calculated from the test results as a percentage of the analyte recovered by the assay. It may often be expressed as the recovery by the assay of known, added amounts of analyte.111,112 5.6.Solution stability During validation the stability of standards and samples is established under normal conditions, normal storage conditions, and sometimes in the instrument to determine if special storage conditions are necessary, for instance, refrigeration or protection from light.111 5.8.Limit of Quantification (LOQ) The limit of Quantitation (LOQ) or Quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. For analytical procedures such as HPLC that exhibit baseline noise, the LOQ is generally estimated from a determination of S/N ratio (101) and is usually confirmed by injecting standards which give this S/N ratio and have an acceptable percent relative standard deviation as well.112,113 5.9.Robustness is defined as the measure of the ability of an analytical method to remain unaffected by small but deliberate variations in method parameters (e.g. pH, mobile phase composition, temperature and instrumental settings) and provides an indication of its reliability during normal usage. Determination of robustness is a systematic process of varying a parameter and measuring the effect on the method by monitoring system suitability and/or the analysis of samples.111,112 5.10.System Suitability System suitability tests are an integral part of liquid chromatographic methods. They are used to verify that the detection sensitivity, resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. Factors, such as the peak resolution, number of theoretical plates, peak tailing and capacity have been measured to determine the suitability of the used method.109-113 Table-1. Acceptance criteria (limits) of system suitability parameters Solid Solution / Suspension Photolytic Thermal Thermal/ Humidity Acid / Base Hydrolysis Oxidative Photolytic Thermal Thermal/ Humidity Oxidative Photolytic Thermal Thermal/ Humidity Photolytic Thermal Oxidative 5t2/196)
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