1. 1 TG Dekker – WHO, MalaysiaFeb 2005 Active Pharmaceutical Ingredients (APIs) Workshop on GMP and Quality Assurance of Multisource Tuberculosis Medicines Kuala Lumpur – Malaysia 21-25 February 2005 Theo Dekker, D.Sc., consultant to WHO Research Institute for Industrial Pharmacy North-West University, Potchefstroom, South Africa iiftgd@puk.ac.za

2. 2 TG Dekker – WHO, MalaysiaFeb 2005 Abbreviations APIActive pharmaceutical ingredient BPBritish Pharmacopoeia CEPEU certificate of suitability EOIExpression of interest FDCFixed dose combination FPPFinished pharmaceutical product GMPGood manufacturing practices ICHInternational Conference on Harmonization Int.Ph.International Pharmacopoeia Ph.Eur.European Pharmacopoeia SmPCSummary of product characteristics TBTuberculosis USPUnited States Pharmacopeia

3. 3 TG Dekker – WHO, MalaysiaFeb 2005 Presentation approach 1.Collect and interpret all available information on the APIs (pre-dossier studies):  The possible manufacturer(s)  Literature, all aspects  Monographs in pharmacopoeia 2.Dossier requirements  Nomenclature  Properties  Manufacturing and site  Specifications  Container closure  Stability testing / re-test period

4. 4 TG Dekker – WHO, MalaysiaFeb 2005 Part 1. Available information on API  Collecting and interpretation of all available information of the API through a systematic approach, should always be done upfront. Some outcomes: 1.Sound choice of API source (manufacturer) 2.Sound scientific understanding of the API, with respect to properties, stability, specifications, etc. 3.Assists in FPP pharmaceutical development 4.Assists in dossier compilation 5.Reduction of time / cost  Forms part of Product Profile Report

5. 5 TG Dekker – WHO, MalaysiaFeb 2005 Info on potential API manufacturer(s) The decision on the API manufacturer should be finalised before starting or early during FPP development studies  Changes afterwards may be major of nature 1.Is the manufacturer reliable / reputable? 2.Is the open part of DMF available and according to all requirements? 3.Is a valid CEP available? 4.GMP inspection of API site by FPP manufacturer

6. 6 TG Dekker – WHO, MalaysiaFeb 2005 DMFs accepted & CEPs by Jan 2005 Number of manufacturers APIDMFCEP Ethambutol 2HCl21 Isoniazid31 Pyrazinamide33 Rifampicin5 Streptomycin1 Total145

7. 7 TG Dekker – WHO, MalaysiaFeb 2005 Literature information Know your API before development, through:  Standard works / series / books – such as:  (Analytical) Profiles of Drug Substances and Excipients [ed: (Florey) Brittain) – 30 volumes]  The Merck Index (for structures, properties)  Pharmaceutical Codex (12 th edition)  Journals through search facilities such as  International Pharmaceutical Abstracts, Chemical Abstracts, Analytical Abstracts & internet  Pharmacopoeial monographs (current)  Analysis of structure & stereochemistry

8. 8 TG Dekker – WHO, MalaysiaFeb 2005

9. 9 TG Dekker – WHO, MalaysiaFeb 2005 Examples of existing API information in standard works  In the table on the next pages it is indicated in which standard works the APIs, appearing in the 5 th invitation for Expression of Interest (TB), is included. Abbreviations:  Apr = Analytical Profiles of Drug Substances and Excipients (contains chapters on APIs)  BP = British Pharmacopoeia  Cod = Pharmaceutical Codex (12 th ed, 1994)  EP = Ph.Eur.  Int = International Pharmacopoeia  US = USP  MI = Merck Index (13 th ed, 2001)

10. 10 TG Dekker – WHO, MalaysiaFeb 2005 Table of API occurrence APIAPrCodBPEPUSIntMI Rifampicin (rifampin) ✓✓✓✓✓✓✓ Ethambutol 2HCl ✓✓✓✓✓✓✓ Pyrazinamide ✓✓✓✓✓✓ Isoniazid ✓✓✓✓✓✓✓ Streptomycin sulfate ✓✓✓✓✓✓ Amikacin ✓✓✓✓✓✓✓ Kanamycin ✓✓✓✓✓ Capreomycin ✓✓✓

11. 11 TG Dekker – WHO, MalaysiaFeb 2005 Table of API occurrence (con.) Apology: JP was not available at time of preparation APIAPrCodBPEPUSIntMI Cycloserine ✓✓✓ Ethionamide ✓✓✓✓✓ Ofloxacin ✓✓✓✓ Protionamide ✓✓ p-Aminosalicylic acid ✓✓✓ Moxifloxacin ✓

12. 12 TG Dekker – WHO, MalaysiaFeb 2005 Example: solubility from 2 literature sources 1 Merck Index 13 th ed 2 Pharmaceutical Codex 12 th ed APIWaterCHCl 3 Ethanol RifampicinWater: Slightly 1,2 pH 7.5: 0.3% 2 pH 5.3: 0.4% 2 pH 2.0: 10% 2 Freely 1,2 Slightly 2 Ethambutol 2HCl50% 2 0.1% 2 20% 2 Ethambutol baseSparingly 2 Very 2 Isoniazid14% 1 0.1% 1 2% 1 Pyrazinamide1.5% 1 0.7% 1 0.6% 2

13. 13 TG Dekker – WHO, MalaysiaFeb 2005 Information from the structures  APIs which are organic compounds, have unique chemical structures & stereochemistry  These structures, together with the solid/liquid state conditions, are basically responsible for chemical and physical properties of the APIs  It is thus always appropriate to analyse the structure of the API, especially if limited literature information is available  Few examples to follow

14. 14 TG Dekker – WHO, MalaysiaFeb 2005 Rifampicin structure hydrolysis oxidation hydrolysis

15. 15 TG Dekker – WHO, MalaysiaFeb 2005 3-Formyl rifamycin formation Source: S. Singh et al. Pharm. Pharmacol. Commun., 6, 405-410 (2000)

16. 16 TG Dekker – WHO, MalaysiaFeb 2005 Rifampicin structure and properties Oxidation  Hydroquinone group  Main degradation of API (to rifampicin quinone)  Enhances solubility in alkaline medium  Tertiary amine  Moderately prone towards oxidation (to N-oxide)  Enhances solubility in acid medium  Oxidation enhanced by  Metal ions  Low pH

17. 17 TG Dekker – WHO, MalaysiaFeb 2005 Rifampicin structure and properties (2) Hydrolysis  Hydrazone (imine) group  Hydrolysis to 3-formyl rifamycin  25-acetyl (ester) group  Hydrolysis to 25-desacetyl rifampicin (minor) Light sensitive  Due to conjugation in molecule (unsaturated) Storage of bulk raw material (BP/Ph.Eur.):  Store under nitrogen in an airtight container, protected from light at temperature of ≤ 25ºC

18. 18 TG Dekker – WHO, MalaysiaFeb 2005 Rifampicin impurities (TLC)  Reference: Int.Ph. related substances test for rifampicin Silica gel R1 CHCl 3 /methanol : 85/15 Daylight detection  BP limits for capsules:- a) Rifampicin: 20 mg/ml b) Quinone: 0.8 mg/ml (4.0%) c) N-oxide:0.3 mg/ml (1.5%) d) 3-Formylrifamycin: 0.1 mg/ml (0.5%) e) Rifampicin: 0.2 mg/ml (1.0%)

19. 19 TG Dekker – WHO, MalaysiaFeb 2005 Isoniazid structure Small molecule (quite stable)  Basic amino functions  Primary amine - react with aldehydes/lactose (see presentation: Pharmaceutical R&D Considerations)  Can hydrolyze under stress conditions to inter alia isonicotinic acid & hydrazine  Oxidize in presence of strong oxidants (e.g. permanganate), with metals as catalyst

20. 20 TG Dekker – WHO, MalaysiaFeb 2005 Pyrazinamide structure Small molecule (quite stable)  Basic amino functions (in aromatic ring)  Amide group can hydrolyse under strong conditions to pyrazinoic acid & ammonia  USP ID test C: Boil 20 mg with 5 ml of 5 N sodium hydroxide: the odor of ammonia is perceptible  Forms metal complexes (slight pink bulk API / product?)  Sublimes when heated

21. 21 TG Dekker – WHO, MalaysiaFeb 2005 Pyrazinamide synthesis  Reagents: NaOH, water as solvent, touch of EDTA  Product directly crystallised from the reaction mixture  No residual solvents (only water used)  2-CPZ to be included as a possible synthesis impurity in API specifications  EDTA for metal complexation (prevent colouration)

22. 22 TG Dekker – WHO, MalaysiaFeb 2005 Ethambutol hydrochloride structure Small molecule  Basic amino groups (in free base)  No vulnerable groups for degradation under mild conditions (2-aminobutanol – synthesis impurity)  2 chiral carbon atoms, optically active (test)  Hygroscopic (solubility in water: 50% m/m)  Can dissolve in absorbed water at high relative humidity  Forms metal complexes  USP: Preserve in well-closed containers

23. 23 TG Dekker – WHO, MalaysiaFeb 2005 p-Aminosalicylic acid + CO 2  Carboxylic acid and phenolic group: acidic  Weak basic group (amphoteric)  Saturated solution: pH of 3.0-3.5 (USP)  Sodium salt available (monograph in USP)  Labile: Decarboxylate when heated  Limit test for m-aminophenol in USP (API & tablets)  More stable in alkaline medium than in acid medium  Store in cool place!!

24. 24 TG Dekker – WHO, MalaysiaFeb 2005 Cycloserine R-configuration  Optically active H 2 0  Stable in anhydrous solid state, protected from water  Degrades in solution, or when solid is exposed to moisture  Pathway1: Dimerisation through one molecule attacking other  Pathway 2: Hydrolysis to ß-aminoxy-D-alanine  Stability: alkaline medium > neutral >> acid medium  Dissolution medium capsules: buffer pH 6.8 !!! (USP)  Primary amine: react with aldehydes/ketones

25. 25 TG Dekker – WHO, MalaysiaFeb 2005 Ofloxacin Moxifloxacin  Structurally related as encircled (see also ciprofloxacin)  Both APIs contain acid and basic groups  Chirality: both intrinsic chiral (optically active)  Ofloxacin: 1 chiral centre: racemate (±) used  Moxifloxacin: 2 chiral centra: S,S-enatiomer used  Both APIs have enone system (in circle): photosensitive?

26. 26 TG Dekker – WHO, MalaysiaFeb 2005 Amikacin – semi synthetic Kanamycin APIs differ only here 4 x NH 2 groups, 2H 2 SO 4 salt hydrolysis

27. 27 TG Dekker – WHO, MalaysiaFeb 2005 Literature information sources The information on the APIs mentioned in the previous slides are backed by inter alia:  Analytical Profiles of Drug Substances and Excipients (ed: Florey/ Brittain) (see next slide)  The Pharmaceutical Codex: Principles and Practice of Pharmaceutics. 1994. Lund, W., ed. 12 th edition, London: The Pharmaceutical Press  The Merck Index, 13 th edition (2001)

28. 28 TG Dekker – WHO, MalaysiaFeb 2005 Literature support style Literature information used in the dossier should always be accompanied by  Full traceable reference citations, for instance:  Devani, M.B., Shishoo, C.J., Doshi, K.J. & Patel, H.B. Kinetic studies of the interaction between isoniazid and reducing sugars. Journal of Pharmaceutical Sciences, 74, 427-432 (1985)  Hassan, M.M.A., Jado, A.I., & Zubair, M.U. Aminosalicylic acid. In Florey, K., ed. Analytical Profiles of Drug Substances, vol. 10. New York: Academic Press, p. 1-27 (1981)  Photocopies of the relevant pages

29. 29 TG Dekker – WHO, MalaysiaFeb 2005 Part 2. Dossier requirements for Active pharmaceutical ingredient (API) Refer to Section 2 of: Guideline on Submission of Documentation for Prequalification of Multi-source (Generic) Finished Pharmaceutical Products (FPPs) used in the Treatment of HIV/AIDS, Malaria and Tuberculosis (hand-out) As from page 3/33

30. 30 TG Dekker – WHO, MalaysiaFeb 2005 Part 2. Dossier requirements for Active pharmaceutical ingredient (API) 3/33 2.1Nomenclature (INN, Systematic, CAS, etc.) 2.2Properties (structure, stereochemistry, etc) 2.3Site of manufacture 2.4Route of synthesis (impurities, etc) 2.5Specifications (pharmacopoeia?) 2.6Container closure system 2.7Stability testing – re-test period & storage -  Open part of Drug Master File - submit (DMF)  CEP (only limited information required)

31. 31 TG Dekker – WHO, MalaysiaFeb 2005 2.2 Properties of APIs 3/33 Three aspects: 2.2.1 API not described in BP, Int.Ph., JP, Ph.Eur., or USP (non-compendial) 2.2.2 API described in BP, Int.Ph., JP, Ph.Eur., or USP (compendial) 2.2.3 Information from literature (discussed)  All the APIs on 5 th Invitation for EOI fall in category 2.2.2, except moxifloxacin

32. 32 TG Dekker – WHO, MalaysiaFeb 2005 2.2.1 Properties non-compendial APIs  Structure, stereochemistry, MF and RMM  Proof of structure/stereochemistry correctness  Single crystal X-ray structure (sufficient) or  Spectrometric data (IR, 1 H & 13 C NMR, MS, etc.) in form of QA certified copies of the spectra and tabulated of data with - assignments against structure or - correlation against API spectral data from peer reviewed literature, preferable by the innovator (in tabulated form!!). Strongly recommended for non-compendial APIs  Physico-chemical properties as discussed on next slide

33. 33 TG Dekker – WHO, MalaysiaFeb 2005 2.2.2 Properties compendial APIs  Physicochemical and other relevant properties of the API, such as  Solubility in water, other solvents such as ether, ethanol, acetone, and buffers of different pH  pKa, partition coefficient  Existence/absence of polymorphs and pseudo-polymorphs e.g. solvates (with XRPD, DSC, IR)  Polymorphism: see presentation Pharmaceutical R&D Considerations (rifampicin)  Hygroscopicity: see presentation Pharmaceutical R&D Considerations (ethambutol hydrochloride in 4FDC tablet)  Particle size

34. 34 TG Dekker – WHO, MalaysiaFeb 2005 2.3 Sites of manufacture 3/33 For each facility where all/part of manufacturing occurs:  Name of manufacturer  Street address  Postal address  Phone & fax numbers  E-mail addresses

35. 35 TG Dekker – WHO, MalaysiaFeb 2005 2.4 Route(s) of synthesis 4/33 Three aspects: 2.4.1 API not described in BP, Int.Ph., JP, Ph.Eur., or USP (non-compendial) 2.4.2 API described in BP, Int.Ph., JP, Ph.Eur., or USP (compendial) 2.4.3 Specifications of raw materials and intermediates used in the synthesis

36. 36 TG Dekker – WHO, MalaysiaFeb 2005 2.4.1 Synthesis non-compendial APIs  A flow diagram of the synthesis process, that includes inter alia chemical structures of starting materials and intermediates, reagents, catalysts, conditions, solvents and purification steps  Example: see pyrazinamide synthesis (slide 21)  A full description of each process, including purification and reprocessing (justified)  (Possible) process impurities should be discussed:  Organic compounds  Catalysts and other inorganic impurities  Residual solvents

37. 37 TG Dekker – WHO, MalaysiaFeb 2005 2.4.2 Synthesis compendial APIs  Valid CEP available  Only outline of synthesis necessary  No CEP  Same as for non-compendial APIs

38. 38 TG Dekker – WHO, MalaysiaFeb 2005 2.5 API specifications 4/33 2.5.1 API not described in BP, Int.Ph., JP, Ph.Eur., or USP (non-compendial) 2.5.2 API described in BP, Int.Ph., JP, Ph.Eur., or USP (compendial) General note An API has only one set of specifications, applicable at release and throughout the re- test period (FPPs may have two sets of specifications)

39. 39 TG Dekker – WHO, MalaysiaFeb 2005 2.5.1 Specs: non-compendial APIs ICH Q6A (new APIs and products) – for instance:  Provide justification for proposed specifications  Impurities (synthesis, degradation & residual solvents) to be characterised and limits set  Analytical methods with validation  Preparation and potency determination/specification of primary and secondary (working) standards, with CoAs  Valid CoAs for at least 2 batches

40. 40 TG Dekker – WHO, MalaysiaFeb 2005 2.5.2 Specs: compendial APIs  Additional critical specifications not included in monograph e.g.  particle size & polymorphic form  synthesis related impurities  residual solvents  Valid CoAs for at least 2 batches 2.5.3 Container-closure system for API 4/33  see guideline

41. 41 TG Dekker – WHO, MalaysiaFeb 2005 2.7 Stability testing 6/33 2.7.1 Stress testing (forced degradation) 2.7.2 Stability testing (regulatory)

42. 42 TG Dekker – WHO, MalaysiaFeb 2005 2.7.1 Forced degradation  Literature information and/or CEP in support or to replace studies  Forced degradation studies  To identify possible degradants for stability studies  To verify specificity of stability assay method - Diode array detection for API peak purity!!  Different stress conditions in solution (guideline)  Different stress conditions in solid state (guideline)

43. 43 TG Dekker – WHO, MalaysiaFeb 2005 2.7.2 Stability testing (regulatory)  Only degradants that form under the real-time and accelerated conditions needs to be considered  Stability protocol, particulars of batches (3), tabulated results and discussion of data  Typical schedule given under FPPs  Propose re-test period when stored under defined conditions

44. 44 TG Dekker – WHO, MalaysiaFeb 2005 Some conclusions 1.Get to know your API by  Analysis of literature information  Analysis of the structures / functional groups  Lab studies, e.g. forced degradation, spectral data and physical data  Considering the dossier requirements 2.Decision on API manufacturer should include  DMF quality, GMP inspection, CEP availability 3.API manufacturers are encouraged to apply for CEPs for their APIs