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

2. 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

3. 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

4. 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)

5. 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)

6. 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

7. 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 !

8. 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

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

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

11. 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

12. 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

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

14. 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

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

16. 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

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

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

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

20. 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

21. 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)

22. 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

23. 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)

24. 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.

25. 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++

26. 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

27. 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”

28. 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)

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

30. 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

31. 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

32. 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

33. 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)

34. 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 nm
Method principle
Copper(II) acetate in mobile phase

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. 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 (at 37°C)
No relevance to Ph.Int. solubility definitions, e.g. diazepam
Ph.Int.
Very slightly soluble (1 gram in 1000 to ml of water at 20°C)
BCS
High solubility (5 mg in ≤ 250 ml at 37°C)

42. 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)

43. 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

44. 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

45. 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)
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

46. 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

47. More information… The International Pharmacopoeia
Information extracted from
The International Pharmacopoeia
WHO Prequalification: Training Material and workshops
International Pharmacopoeia – Development of monographs for tuberculosis fixed-dose combination tablets – Professor. T. Dekker

48. Thank you!