The International Pharmacopoeia Quality standards for TB medicines Caroline Mendy - Technical Officer - Quality Assurance and Safety: Medicines Tutorial workshop, Beijing, March 2010

Quality standards for TB medicines – an issue? "[…] reliable quality of medicines is absolutely essential for effective TB programmes" TB Alliance "[…] low quality drugs damage patient recovery and increase the likelihood of drug-resistant strains of TB Stop-TB partnership Several WHO studies carried out or on-going to monitor the quality of marketed TB medicines

Quality standards for TB medicines Monographs available in Ph.Int. Monographs, requirements and identity tests Assay and related substances method development Dissolution / disintegration specifications Isoniazid and Ethambutol hydrochloride tablets Focus: TB FDC tablets

Ph.Int. TB monographs Priorities for inclusion of new specifications for TB medicines: WHO Essential medicines lists WHO specific disease programmes (e.g. Stop-TB, Prequalification) Specifications for first-line and second-line TB drugs available (APIs and FPP)

Ph.Int. TB monographs Amikacin (revision) - Oct 2009 Isoniazid Amikacin sulfate (revision) - Oct 2009 Amikacin injection - Oct 2009 kanamycin monosulfate - Oct 2009 kanamycin acid sulfate - Oct 2009 Kanamycin injection - Oct 2009 Isoniazid Isoniazid tablets Pyrazinamide Pyrazinamide tablets Rifampicin Streptomycin sulfate Streptomycin sulfate powder for injection Thioacetazone Cycloserine Cycloserine capsules Ethambutol HCl (revision)

Ph.Int. TB monographs – to come… Capreomycin - draft in progress Capreomycin injection - draft in progress Ethionamide tablets* Protionamide tablets* Terizidone* Ofloxacin tablets* Levofloxacin tablets* p-aminosalicylic acid granules/tablets* *on 2010 work programme

Ph.Int. monographs: TB FDC dosage forms 4FDC: Rifampicin / Isoniazid / Pyrazinamide / Ethambutol HCl tablets 3FDC: Rifampicin / Isoniazid / Pyrazinamide tablets Rifampicin / Isoniazid / Pyrazinamide dispersible tablets (new) Rifampicin / Isoniazid / Ethambutol HCl tablets (new) 2FDC: Rifampicin / Isoniazid tablets Rifampicin / Isoniazid dispersible tablets (new) Isoniazid / Ethambutol HCl tablets Rifampicin tablets Rifampicin capsules Basic approach for monograph development: top down 9 monographs !

Ph.Int. monographs: TB FDC dosage forms Strategy Start with the most complex combination (4FDC tablets) Work down to most simple situation using same procedures 4FDC Isoniazid Rifampicin 3FDC 2FDC Pyrazinamide Ethambutol

TB 4FDC tablets Ph.Int. monograph: Information section √ ? √

TB 4FDC tablets Monograph: Requirements read General monograph for Tablets √ √ ?

TB 4FDC tablets Monograph: Identity tests Requirement: Specific for each API in the 4FDC tablet identification should be conclusive on presence / absence of the APIs More than one test may apply for conclusive identification tests X and Y There may be a choice between tests either test X or test Y may be applied read instruction in monograph carefully

TB 4FDC tablets Monograph: Identity tests The TB 4FDC monograph states the following: Either tests A and B or test C may be applied Tests A and B are the 2 HPLC assay tests retention time compared to ICRS (reference substance) Test C is a TLC test position (Rf), appearance & intensity of spot compared to ICRS note 3 parameters in one test Choice with respect to visualisation of spots UV or visible (after exposure to iodine vapour) Test C first

TB 4FDC tablets Monograph: Identity test (copy from monograph) HF254

TB 4FDC tablets Monograph: Identity test (results) Eth INH PY Rif Tab Method C.2 In daylight, after exposure to I2 Method C.1 In UV (254 nm), after exposure to I2 In UV (254 nm), before exposure to I2

Structures of the APIs in TB 4FDC tablet Isoniazid Yes Yes Rifampicin UV absorbing groups? Yes No Pyrazinamide Ethambutol

TB 4FDC tablets Monograph: Related substances General recommendation for FDCs Related substances (degradation products) should be tested in the FDC products for APIs which are unstable Very stable APIs may not be required What is the situation in the case of the TB 4FDC tablets? Consult literature for stability Do stress testing under various conditions (part of validation) Temperature, pH, oxidation, light, … API-API interactions

Information from literature & structure Rifampicin hydrolysis (to 25-desacetyl) light sensitive oxidation (to N-oxide) quinone oxidation (to quinone) hydrolysis (to 3-formyl rifamycin)

Information from literature & structure 3-Formyl rifamycin + H2N ― R o R Isoniazid

Information from literature & structure “isonicotinoyl hydrazone” N ― R o R Isoniazid

Information from literature & structure Rifampicin - summary Rifampicin known degradation products Oxidation Rifampicin quinone Rifampicin N-Oxide Hydrolysis 3-Formyl rifamycin 25-desacetyl rifampicin Reaction with Isoniazid isonicotinoyl hydrazone 3-(isonicotinoylhydrazinomethyl)rifamycin light sensitive

Information from literature & structure Rifampicin Special storage conditions for the API? International Pharmacopoeia: Rifampicin should be kept in a tightly closed container (exclude exposure to air - moisture and oxygen) (container of low permeability) protected from light and stored at a temperature not exceeding 15°C, or in an atmosphere of nitrogen at a temperature not exceeding 30°C (absence of air oxygen)

Rifampicin impurities (TLC) Reference: Int.Ph. related substances test for rifampicin Silica gel R1 CHCl3/methanol : 85/15 Daylight detection a Rifampicin test substance: 20 mg/ml b Quinone: 0.8 mg/ml (4.0%) c N-oxide: 0.3 mg/ml (1.5%) d 3-Formyl rifamycin: 0.1 mg/ml (0.5%) e Rifampicin: 0.2 mg/ml (1.0%) a b c d e

Information from literature Isoniazid Small molecule Quite stable Can hydrolyze under stress conditions to inter alia isonicotinic acid & hydrazine Primary amine - react with aldehydes, for example reducing sugars / lactose 3-Formyl rifamycin (rifampicin degradation product)

Information from literature Pyrazinamide Small molecule Quite stable Amide group can hydrolyse under strong conditions to pyrazinoic acid & ammonia Test D in Ph.Int. monograph for Pyrazinamide API: Boil 20 mg with 5 ml of sodium hydroxide (~200 g/l) TS; ammonia, perceptible by its odour, is evolved.

Information from literature Ethambutol hydrochloride Small molecule Quite stable (2-aminobutanol – synthesis impurity) Hygroscopic (solubility in water: 50% m/m) In FDC tablets ethambutol can absorb water absorbed water helps degradation of rifampicin Forms metal complexes Very useful in FDC assay test S,S-isomer pharmaceutical :N—C │ Cu++

Information from literature What is important for monograph development? Rifampicin related substances to be tested for Rifampicin quinone Rifampicin N-Oxide 3-Formyl rifamycin isonicotinoyl hydrazone = product with isoniazid 25-desacetyl rifampicin Protect from light (mainly due to rifampicin) Product (packaging) and sample / test solutions Protect from moisture “Manufacture” ► protective packaging + set moisture limit QC testing ► protect tablet powder from moisture uptake

TB 4FDC tablets Examples of water uptake Products B A TB 4FDC tablets Examples of water uptake FPPs (packed products) Unpacked tablets (control) After 5 days at 40°C/75% RH After 5 days at 40°C/75% RH + Light S. Singh, Int. J. Tuberc. Lung. Dis., 7, 298 (2003) Quality of the products not known “bleeding”

TB 4FDC tablets Example of poor FPP Have you seen a TB 4FDC tablet like this when opening the primary packaging? Hopefully not Example: TB 4-FDC tablets one year from expiry Photo from: S. Singh & B. Mohan, Int. J. Tuberc. Lung. Dis., 7, 298 (2003)

TB 4FDC tablets Monograph: Requirements read General monograph for Tablets √ √ ? √

TB 4FDC tablets Assay and related substances The objectives To develop assay methods for all four APIs in the TB 4FDC tablet HPLC as only option To develop methods for determination of degradation products (related substances) Only for rifampicin

Assay TB 4FDC tablets Assay and related substances The approach to HPLC methods Minimise the number of HPLC methods Not 4 HPLC methods for assay of the 4 APIs The column(s) should be easily obtainable The use of an isocratic profile instead of a gradient profile the test can be carried out by any laboratory The assay method(s) should be stability-indicating It would be advantageous if the related substances could be determined with the method(s) used for assay

Assay TB 4FDC tablets Assay and related substances Strategy followed Screening of existing methods Literature Manufacturers Selecting promising candidates Desk and experimental evaluation Optimisation of selected method(s) Validation

Assay TB 4FDC tablets Assay and related substances The outcome Two HPLC methods have been developed One method for simultaneous assay of one or more of Isoniazid Pyrazinamide Ethambutol hydrochloride Another method for rifampicin Assay Related substances (degradation products)

Assay TB 4FDC tablets Isoniazid/Pyrazinamide/Ethambutol HPLC Ethambutol properties were key to assay method Problem Ethambutol lacks useful absorption in UV/visible region USP method - detection at 200 nm Answer Ethambutol compexes with Cu2+ Maximum absorption at 270-280 nm Method principle Copper(II) acetate in mobile phase

Assay TB 4FDC tablets Isoniazid/Pyrazinamide/Ethambutol HPLC buffer pH 5.0 Cu2+ in mobile phase Pyrazinamid Detection at 270 nm resolution ≥ 2 Isoniazid Ethambutol 10 min

Assay TB 4FDC tablets Isoniazid/Pyrazinamide/Ethambutol HPLC Validation All parameters were validated ICH Q2(R1) Specificity of particular interest No “innovator” product Placebo constructed from all known excipients in 4FDC tablets information from package inserts Stress testing water, alkali, acid, hydrogen peroxide at 40°C on one product DAD (Diode Array Detection) for peak purity

Assay / related substances TB 4FDC tablets Rifampicin HPLC Rifampicin related substances to be tested for Rifampicin quinone Rifampicin N-Oxide 3-Formyl rifamycin 3-(isonicotinoylhydrazinomethyl)rifamycin "hydrazone“ obtained from 3-formylrifamycin & isoniazid reaction Sample donated by Dr S. Singh, NIPER, India 25-desacetyl rifampicin Also applicable to: 3FDC, 2FDC tablets and rifampicin tablets and capsules

Assay / related substances TB 4FDC tablets Rifampicin HPLC – Isonicotinoyl hydrazone In situ preparation of 3-(isonicotinoylhydrazinomethyl)rifamycin hydrazone formed from rifampicin and isoniazid reaction solution (5) For solution (5) dissolve 4 mg of rifampicin RS and 2 mg of isoniazid RS in 25.0 ml of acetic acid (~60g/l) TS and keep the solution at room temperature for 30 minutes Rationale of in situ preparation based on molecules structures/interactions

Assay TB 4FDC tablets Rifampicin HPLC Validation All parameters were validated ICH Q2(R1) Specificity of particular interest No “innovator” product Placebo constructed from all known excipients in 4FDC tablets information from package inserts Known impurities & validation Stress testing water, alkali, acid, hydrogen peroxide at 40°C on one product DAD (Diode Array Detection) for peak purity

Dissolution / disintegration specifications BCS The Biopharmaceutics Classification System (BCS) of an API is based on its aqueous solubility and intestinal permeability According to the BCS an API falls in one of four classes: Permeability Solubility Class High 1 Low 2 3 4

Dissolution / disintegration specifications BCS High solubility definition The highest single unit dose of an API is completely soluble in 250 ml or less of aqueous medium over the pH range 1.0 - 6.8 (at 37°C) No relevance to Ph.Int. solubility definitions, e.g. diazepam Ph.Int. Very slightly soluble (1 gram in 1000 to10 000 ml of water at 20°C) BCS High solubility (5 mg in ≤ 250 ml at 37°C)

Dissolution / disintegration specifications BCS Classification of TB APIs WHO Technical Report Series 937, Annex 8 (2006) Solubility Class API Low 2 Rifampicin (300 mg) High 3/1 Isoniazid (300 mg) Pyrazinamide (400 mg) 3 Ethambutol 2HCl (400 mg)

Dissolution / disintegration specifications Isoniazid/Ethambutol tablets Acceptance criteria Either (A) Dissolution or (B) Disintegration may be applied Dissolution All 6 units ≥ 80% of label claim dissolved in 30 min. If one tablet fails, average of 12 tablets ≥ 75%, none <60% Criteria as set at EC meeting of Oct. 2007 Comply with 5.4 Disintegration test for tablets and capsules, operating the apparatus for 10 minutes If the tablets do not comply, carry out test A above More stringent than dissolution test Generally applicable to Class 1 & 3 APIs

Dissolution / disintegration specifications Isoniazid/Ethambutol tablets Dissolution conditions of Ph.Int. for Tablets/capsules containing a BCS highly soluble API Samples analysed by HPLC method for assay A B Apparatus Paddle, 75 rpm Dissolution medium pH 6.8 phosphate buffer Volume of medium 500 ml Degassed? No (undegassed) Temperature 37°C ± 0.5°C Sampling points 10, 15, 20, 30, 45, and 60 C D

Isoniazid/Ethambutol tablets Discussion of results The dissolution profiles of the 2 APIs in a particular product are similar (this is true for all 4 products) Both APIs are highly soluble (BCS definition) The products show different dissolution rates Dissolution rate A > B ≈ C >> D Disintegration (min) 7 11 11 21 Dissolution rate is related to disintegration time Acceptance criteria: Disintegration tighter than dissolution For BCS highly soluble APIs The dissolution rate is controlled mainly by disintegration of the tablets/capsules

Closing remarks Approach for development of TB FDC tablet monographs Top down (4FDC → 3FDC → 2FDC → rifampicin tabs/caps) In this way all monographs can have same test methods For rifampicin and rifampicin related substances One HPLC method Includes detection of isonicotinoyl hydrazone (API-API reaction) For isoniazid, pyrazinamide & ethambutol hydrochloride One method, no related substances Dissolution/disintegration for isoniazid/ethambutol 2HCl tablets Acceptance criteria: Disintegration tighter than dissolution Ph.Int. takes lead with setting alternative criteria for Class 1 & 3 API tablets/capsules

More information… The International Pharmacopoeia Information extracted from The International Pharmacopoeia http://www.who.int/medicines/publications/pharmacopoeia/en/ WHO Prequalification: Training Material and workshops International Pharmacopoeia – Development of monographs for tuberculosis fixed-dose combination tablets – Professor. T. Dekker http://apps.who.int/prequal/

Thank you!