Evaluating Impurities in Drugs II

58 Pharmaceutical Technology MARCH 2012 PharmTech .com The health implications of drug impurities can be significant because of potential teratogenic, mutagenic, or carcinogenic effects. Controlling and monitoring impurities in APIs and finished drug products, therefore, is a crucial issue in drug development and manufacturing, In Part II of this article, the authors examine impurities from chiral molecules, polymorphic contaminants, and genotoxic impurities. Kashyap R. Wadekar, PhD,* is a research scientist (II), Mitali Bhalme, PhD, is an associate research scientist, S. Srinivasa Rao is a research associate, K. Vigneshwar Reddy is a research associate, L. Sampath Kumar is a research chemist, E. Balasubrahmanyam is a research chemist, and Ponnaiah Ravi, PhD, is senior vice-president of R&D, all with Neuland Laboratories, 204 Meridian Plaza, 6-3- 854/1, Ameerpet, Hyderabad, India, tel. 91 40 30211600, kashyapwadekar@neulandlabs.com. *To whom all correspondence should be addressed. Submitted: Sept. 19, 2011; Accepted Nov. 28, 2011. A D A M G A u L T / O J O I M A G e S /G e T T y I M A G e S Impurities T he public and the pharmaceutical industry are placing greater attention on impurities in drug as evidenced by the attention given to pharmaceutical impurities in books, journal articles, and national and international guidelines (1–10). The health implications of impurities can be significant because of their potential teratogenic, muta- genic, or carcinogenic effects. Controlling and monitoring impurities in APIs and finished drug products, therefore, is a crucial issue in drug development and manufacturing. Part I of this article, which appeared in the February 2012 issue of Pharmaeceutical Technology, discussed the various types and sources of impurities with specific case studies (11). This article, Part II, discusses chiral, polymorphic, and genotoxic impurities (12, 13). Part III, to be published in the April 2012 issue of Pharmaceutical Technology, will examine various degradation routes of APIs, impurities arising from API–excipient interaction during formulation, metabolite impurities, various analytical methodologies to measure impurity levels, and measures to control impurities. Chiral impurities Impurities can be present in the enantiomers of chiral com- pounds. Differences in pharmacological and toxicological profiles have been observed with chiral impurities in vivo (14, 15). The significance of stereochemical purity may be illustrated by formoterol, a selective β2-adrenoceptor ago- nist (16). This compound contains two chiral centers. Initial investigations indicated that the β2-agonist activity resided in the stereoisomer with the (R, R) absolute configura- tion with a rank order of potency (R, R) > (R, S) > (S, S) > (S, R). Subsequent investigation reported much greater dif- ference with the eudismic ratio R, R/S, S increasing from 50 to 850 when the impurity of the eutomer in the diastereomer decreased from approximately 1.5 % to < 0.1% (17). Simi- lar examples of stereochemical isomers can be found in the stereospecific drugs of the (S)-enantiomer of α-methyldopa, picenadol, (R)-sopromidine, (+)-(S)-apomorphine, and ser- traline (18–24). Another example is asenapine maleate, an antipsychotic belonging to the dibenzo-oxepino pyrroles class. Based on its receptor pharmacology, the efficacy is thought to be mediated by its antagonist activity on dopamine (D)-2 and Evaluating Impurities in Drugs Part II of III Kashyap R. Wadekar, Mitali Bhalme, S. Srinivasa Rao, K. Vigneshwar Reddy, L. Sampath Kumar, E. Balasubrahmanyam, and Ponnaiah Ravi Pharmaceutical Technology MARCH 2012 59 serotonin (5-HT)–2A receptors (25). Asenapine shows geometric isomerism and is a racemate of (+) and (-) enan- tiomers. It shows comparable binding affinities, meaning trans-asenapine showed higher affinity at D4 receptors than (+)/cis-asenapine (26). Differences in pharmacological and toxicological pro- files have been observed with chiral impurities in vivo, suggesting that chiral impurities should be monitored carefully. Although development of chiral drugs as single stereoisomers is a preferred approach, consideration must be given to unwanted stereoisomers, which may be present as impurities or degradants in the drug substance or drug product or generated through metabolism in biological systems. Chiral impurities in pharmaceutical samples may occur as side-products of the synthetic process as a result of an inversion of chiral centers due to chemical degradation of the drug substance or both. Similarly, inversion of the chiral center may occur in vivo as a result of metabolism, chemical degradation, or both. Guidelines on the development of chiral compounds are published by regulatory authorities around the world, but they can be general and leave room for interpretation. The issues involved in chiral drug development are com- plex, and a coordinated approach among the many R&D groups is necessary. A multidisciplinary approach serves as a guide to the development of chiral compounds by co- ordinating research efforts in the various phases of devel- opment (22–36). Polymorphic impurities Polymorphism, the ability of a compound to exist in more than one crystalline form, affects the physical, chemical, and biological properties of a compound in question (37). These properties may influence several issues in pharma- ceutical systems, such as processing characteristics, drug stability, and bioavailability. Demonstrating an under- standing of the polymorphs in a given drug is an area of regulatory scrutiny in new drug applications (38). The International Conference on Harmonization’s Q6A guideline, Specification: Test Procedure and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, outlines when and how polymorphic forms should be monitored and controlled (39). For sta- bility concerns, the most stable form is normally used in the formulation. The metastable polymorphic form, how- ever, may be inadvertently generated due to temperature, mechanical treatment, and moisture during processing or storage of the drug product (40). Contamination of polymorphic impurities can adversely influence the stability and performance of the final drug product. Moreover, FDA requires development of validated methods for analysis of the proportion of crystalline forms throughout the drug’s retest period and shelf life (41). For example, olanzapine crystallizes in more than 25 crystalline forms, of which Form II has been designated From Blending... ...to Powder Segregation From Sampling... ...to Tableting & Cleaning Validation Sift-N-Blend™ Patent Pending SimpleBlend™ Pilot Scale Blenders Unit Dose & Bulk Powder, Liquid & Semi-Solid Samplers MaxiBlend® Lab Blender Table Top Rotary & Manual Tablet Presses & Optional Instrumentation Remote Swabbing & Microbiological Sampling Tool & Cleaning Validation Coupons GlobePharma, Inc. 2 B & C Janine Place, New Brunswick, NJ 08901 www.globepharma.com E:sanni@globepharma.com T: (732)296-9700 F: (732)296-9898 Innovative SolutionsProv ding 60 Pharmaceutical Technology MARCH 2012 PharmTech .com the most stable form and is used in the dosage form (42, 43). Olanzapine discolors in the presence of air (44). Polymorphic Forms I and II show very minor differences in their diffracto- grams. Evaluating olanzapine Form I for the presence of Form II, therefore, becomes very important. Salmeterol xinafoate is known to exist in two crystalline polymorphic forms, with Form I being stable and Form II being the metastable poly- morph under ambient conditions (45). These polymorphs have been char- acterized using differential scanning calorimetry, X-ray powder diffrac- tometry, thermogravimetric analysis, and inverse gas chromatography (46). Commercial salmeterol xinafoate is a micronized form with the same crystal structure as that of Form I. The com- mercial drug, however, can contain traces of the Form II polymorph that is formed during the micronization process. Exceptional case impurities When a new process is developed, such as to overcome patent issues, it gener- ally begins with new key starting ma- terials, intermediates, reagents, or sol- vents that may react differently to give byproducts or process impurities. For example, in the synthesis of linezolid and pemetrexed disodium, several pro- cess impurities can be formed due to different process approaches. Pharmaceutical companies can develop new processes based on raw materials, solvents, reagents, process conditions (i.e., temperature), and new polymorphs. Using new materi- als or processes, they may encounter several impurities that may not have been not present in the basic or ini- tial synthesis of an API. After publi- cation of monographs in the United States Pharmacopeia , European Pharmacopoeia, British Pharmaco- poeia, Indian Pharmacopoeia, and Japanese Pharmacopoeia, they may not have a control of those impuri- ties that are formed due to different process approaches. After publica- tion of the monograph, companies have to change the analytical method or control these impurities as non- pharmacopeial impurities, including genotoxic impurities, with separate analytical methods, such as high- performance liquid chromatography (HPLC) or gas chromatography (GC). For example, during the synthesis of linezolid, impurities based on a bis- linezolid compound and a bis-benzyl impurity are formed due to the non- infringed patent process (47–49). Some published patents have different poten- tial process impurities, which cannot be separated in a single HPLC method, and which result from synthetic routes different from the synthetic route in the basic patent (47–55). Pemetrexed disodium heptahydrate, the API in Eli Lilly’s Alimta, is a multi- targeted antifolate used to treat meso- thelioma and a second-line treatment for non-small-cell lung cancer. Alimta also is under investigation for multiple other cancers (56). Each non-infringed process patent has different potential impurities (see Figure 1, Process Impu- rities 1, 2, 3, and 4) (57–60). It may not be possible to analyze these impurities in a single HPLC method. Impurities due to the piperazine ring The piperazine moiety is present in the chemical structure of more than 200 drugs. The biotransformation of the piperazine ring involves several well- known metabolic reactions, including N-oxidation, hydroxylation, N-deal- kylation, and ring cleavages to N-sub- stituted as well as N,N ’-disubstituted ethylenediamines. In addition, several unexpected metabolic pathways have been reported for the piperazine ring: N-glucuronidation, N-sulfonation, formation of carbamoyl glucuronide, and glutathione adducts (61). Some compounds containing the piperazine ring indicate that the ring is normally metabolically stable when both nitro- gen atoms are substituted with groups larger than ethyl. The lack of partial degradation of the piperazine ring to form ethylene- diamine in olanzapine (2-methyl-4-(4- methyl-1-piperazinyl)10H-thieno[2,3- We make pharmaceutical feeding processes work PureFeed¨ Pharmaceutical Feeders For pharmaceutical feeding solutions, call: (800) 558-0184 www.accuratefeeders.com C O N T I N U O U S M I X I N G H O T M E L T E X T R U S I O N C O N T I N U O U S G R A N U L A T I O N F E E D I N G E X P E R I E N C E I N : Product Line Features: • Feed rate range from .02 kg/hr to 150 kg/hr. • Easy disassembly and cleaning in minutes. • 316L stainless steel. • Ceramic feed disc delivers pulse-free metering. • Disposable, flexible hopper speeds cleaning and eliminates cross contamination. Federal Equipment Company has the pharmaceutical processing equipment you need for your project and understands that delivery and budget are critical elements in any equipment purchase. 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When one of the nitrogen atoms is substituted by hy- drogen on the piperazine ring, whether its methyl or ethyl, ethylenediamine formation is normally observed. An ex- ample is levofloxacin, S-(-)-9-fluoro-2,3-dihydro-3-methyl- 10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4] benzoxazine-6-carboxylic acid, which is the (S)-isomer of ofloxacin. In levofloxacin, the piperazine nitrogen atom is substituted with methyl due to several photodegradation im- purities (see P 2 to P 10, Figure 3) (64–67). Some process im- purities also are observed (see Figure 3). If the levofloxacin process involved methylenedichloride as a solvent, a chloro methyl impurity may form, and after isolation of the final product, the same impurity may convert to a di-quaternary cyclic piperazine impurity. Additionally, when the ciprofloxacin (1-cyclopropyl-6- fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl) quinoline-3- carboxylic acid) nitrogen atom is substituted by hydrogen on the piperazine ring, several metabolites and process im- purities are formed (see Figure 3) (68–74). When nitrogen is substituted with hydrogen during the reaction, two dimer impurities (F-F dimer ciprofloxacin and F-Cl dimer cipro- floxacin) also are observed (75). Impurities Figure 1: Reaction scheme for different process approaches for pemetrexed sodium impurities, respectively labeled as 1, 2, 3, and 4 (Refs. 57–60). Ph. Eur. is European Pharmacopoeia. Key starting material for pemetrexed H 2 N N N N H OH H 2 N H 2 N H 2 N NH 2 NH NH NH NH 1 Process impurity 2 Process impurity 3 Process impurity 4 Process impurity NO 2 H 2 N N N N H NH O NH N H N H O NH O COONa COONa COONa COONa COONa COONa Structure in literature/patents Structure in draft Ph. Eur. monograph Pemetrexed disodium Tautomer impurity OH H 2 N N N H O H 2 N N N H O COOCH 3 COOCH 3 COOAr COOAr COOR COOH COOH COOR N N H N N N N N N N N N N N H H H H O O O O OH O O O OH O A L L f IG u R e S A R e c O u R T e S y O f T H e A u T H O R S C e L e B r a T I N G Y e a r s35 KEEPING YOU CURRENT SINCE 1977 • www.pharmtech.com Meet us in New York! | May 1–3, 2012 Celebrate with us at Jacob Javits Center, NYC 64 Pharmaceutical Technology MARCH 2012 PharmTech .com Genotoxic impurities There was no specific document on control of genotoxic impurities before 2000. ICH guidelines made passing references to compounds of unusual toxicity. Geno- toxic impurities are chemical compounds that may be mutagenic and could potentially damage DNA (76). Non-monoalkylated agents are classified as genotoxic due to the nature of the functional groups they pos- sess and also of related aniline derivatives. Addition- ally, salt-forming steps can introduce genotoxic im- purities. Some examples include formation of methyl chloride as a side reaction of hydrochloric acid in meth- anol or esters of methanesulfonic acid as byproducts from the methanesulfonic acid salt-formation step in alcohol-based solvents (77, 78). EMA issued guidelines on the threshold of toxicological concern (TTC) that recommended limits for exposure to potential genotoxic impurities to be 1.5 mcg per day for commercially approved drugs (79). As per the guidelines, testing will be required for all potential impurities from an API’s synthetic route containing structural elements that are the cause of concern for genotoxicity potential using the well-established Salmonelle Ames test. The Ames test is a screening test that is used to help identify chemicals that affect the structure of DNA. The test exposes Salmo- nella bacteria to chemicals and looks for changes in the way bacteria grow. These changes result from mutations that occur when the structure of DNA is altered in certain places and the micronuclei test for mutagenicity (80, 81). Impurities Figure 2: The piperazine ring and metabolite impurities of olanzanpine. Ph. Eur. is European Pharmacopoeia. USP is US Pharmacopeia. H N N N N S H N N N N S 7-Hydroxy glucuronide metabolite 7-Hydroxy N-Oxide metabolite N-Oxide-2-acid metabolite N-Oxide-2-Hydroxy methyl metabolite N-desmethyl-2-Hydroxy methyl metabolite N-desmethyl-2-acid metabolite 4’- N - Glucuronide metabolite 10- N - Glucuronide metabolite Ring-opening amide impurity process impurity Chloro methyl impurity Ph. Eur. impurity C process impurity Cl- Cl- Cl- 2-acid metabolite 2-Hydroxy methyl metabolite N-Oxide process impurity Ph. Eur. impurity D and metabolite N-Formal impurity process impurity N-Des methyl process impurity and metabolite 7-Hydroxy metabolite O O Gluc-O- HO H N N N N S H N N N N S O H N N N N S H N N N N S N N N S HO OH OH H N N N N S H N N N S OH N N Gluc Gluc O H N N N N S H N N N S OH OH NH H N N N S OH O O NH NH 2 NH 2 NH S O NH 2 NH N N N N N+ Cl N HS S N N+ N+ N H S O Plausible Structure NH 2 N N H S Oxidative ring-opening of piperazine N N NH NH 2 H S Further degradation impurity NH N N NH H S Amide process impurity USP impurity B Ph. Eur. impurity BN N H H S O N N N N H S O N Olanzapine N N N H S H N N N N (?) Olanzapine Tautomer impurity S H N N N NH S N N N N S H N N S Dimer of olanzapine process impurity N N N N O H S N-Acetyl impurity process impurity N N H O HO S Degradation impurity N N N N H S OH N-Hydroxy methyl impurity process impurity H N N S H N N N N S Piperazine dimer impurity process impurity Manufacturing Innovation: Achieving Excellence in Sterile and Emerging Biopharmaceutical Technology April 16-18, 2012 JW MARRIOTT DESERT RIDGE RESORT • PHOENIX, ARIZONA Just Confirmed: Steven Lynn, Office Director (acting), Office of Manufacturing and Product Quality, Office of Compliance, CDER, FDA to speak in the closing plenary session. Featuring: A New Contamination Issue Never Before Experienced: A Novel Bacterial Contamination in Biopharmaceutical Manufacturing, by PDA Chair, Anders Vinther, PhD, Vice President, Quality Biologics, Genentech. 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