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Product Information Report: Clofazimine

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This document is made possible by the generous support of the American people through the U.S. Agency for International Development. The contents are the responsibility of USP’s Promoting the Quality of Medicines program and do not necessarily represent the views of USAID or the United States Government.

About PQM

The Promoting the Quality of Medicines (PQM) program is a cooperative agreement between the U.S. Agency for International Development (USAID) and the U.S. Pharmacopeial Convention (USP). The PQM program provides technical assistance to strengthen medicines regulatory authorities and quality assurance systems and supports manufacturing of quality-assured priority essential medicines for malaria, HIV/AIDS, tuberculosis, neglected tropical diseases, and maternal and child health.

Recommended Citation

This report may be reproduced if credit is given to the U.S. Pharmacopeial Convention (USP) Promoting the Quality of Medicines (PQM) Program, Rockville, MD. Please use the following citation:

Promoting the Quality of Medicines (PQM). Product Information Report: Clofazimine. 2017. U.S. Pharmacopeial Convention. Rockville, Maryland.

United States Pharmacopeia 12601 Twinbrook Parkway Rockville, MD 20852 USA Tel: +1-301-816-8166 Fax: +1-301-816-8374 Email: [email protected]

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Table of Contents

Acknowledgments ................................................................................................................... v

Executive Summary ................................................................................................................. 1

Key Manufacturing Challenges ................................................................................................. 4

Active Pharmaceutical Ingredient (API) ..................................................................................... 5

Chemical Structure / Formula .................................................................................................. 5Name ......................................................................................................................................... 5Physical Properties.................................................................................................................... 6Chemical Properties ................................................................................................................. 7Structure Characterization...................................................................................................... 12Analysis of CFZ ....................................................................................................................... 15

Analysis of Impurities / Related Substances / Degradation Products .................................. 17Stability of CFZ ....................................................................................................................... 19Test Specifications for CFZ..................................................................................................... 20

Dosage Form ........................................................................................................................ 21

General Summary ................................................................................................................... 21Regulatory Status.................................................................................................................... 21Formulation Barriers to Entry ................................................................................................. 22Formulation Justification ........................................................................................................ 23Analytical Methods of Dosage Form ..................................................................................... 27Stability of Dosage Form ........................................................................................................ 28Dosage Form Test Specifications ........................................................................................... 28

Bioavailability and Pharmacokinetics ...................................................................................... 30

Mechanism of Action .............................................................................................................. 30

Toxicology Information .......................................................................................................... 37

Animal toxicity ........................................................................................................................ 37Genotoxicity............................................................................................................................ 37

HumanToxicity ........................................................................................................................ 38Non-clinical Toxicology........................................................................................................... 39


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Occupational Exposure Limits Calculations ........................................................................... 39Control Band Assignment ...................................................................................................... 40Industrial Hygiene Sampling and Analytical Methods ........................................................... 41Acceptable Daily Exposure Calculations ............................................................................... 41

Choice of Uncertainty and Modifying Factors ....................................................................... 42Information to Patients ........................................................................................................... 43

Manufacturing of Dosage Form.............................................................................................. 44

Facility Design & HVAC Requirements .................................................................................. 44Manufacturing Process ........................................................................................................... 45Process Controls ..................................................................................................................... 45

Cleaning Validation................................................................................................................ 46

Conclusion................................................................................................................................................................47

References................................................................................................................................................................48

Appendix 1 ........................................................................................................................... 52

Concept Note for Development of Tablet/Capsule Dosage Form for Clofazimine (CFZ)... 52References .............................................................................................................................. 57

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Acknowledgments

This report was prepared in collaboration with JM Pharma (United States), with technical guidance and oversight from Nikhil Shah, PQM Senior Manager, Manufacturing Services. The authors also thank Cheri Vincent, Thomas Chiang, Alison Collins, Lisa Ludeman, and Tobey Busch, from USAID for their guidance. Gratitude is also due to the reviewers and editorial staff who provided valuable comments during the development of this document.

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Executive Summary

Clofazimine (CFZ) is a rhimophenazine dye, originally developed for the treatment of tuberculosis. This

drug has both antimicrobial and anti-inflammatory activity and has been found to have diverse uses in

the treatment of leprosy, discoid lupus erythematosus, and pyoderma gangrenostun[1].CFZ is a broad-

spectrum antimycobacterial agent recommended by the World Health Organization (WHO) as a first-

line treatment for leprosy and second-line treatment for multidrug-resistant tuberculosis[2].

CFZ was first synthesized in1957 by Barry et al., at the Laboratories of the Medical Research Council of

Ireland, Trinity College Dublin. WHO has classified CFZ as an "essential drug," and in 1982

recommended its use in combination with other agents to treat all cases of leprosy[3, 4].

In the 1960s, CFZ was largely abandoned for anti-tuberculosis treatment following early experiments

suggesting poor activity in humans with chronic cavitary tuberculosis (TB) and in animal models, and in

the context of emerging effective combination treatments of TB like, streptomycin, isoniazid (INH),

para-aminosalicylic acid[5]. Although CFZ has shown strong activity against Mycobacterium tuberculosis

(MTB) in vitro, including multidrug-resistant (MDR)strains of this pathogen, CFZ is generally considered

to be ineffective in the treatment of pulmonary TB.

CFZ was recently repurposed for managing MDR-TB cases in response to the results of the so-called

Bangladesh study, which demonstrated that a CFZ-containing regimen can cure MDR cases in 9 to 12

months[6]. Despite the lack of indications for treatment of drug-resistant TB using CFZ, it is

recommended by WHO as a Group 5 medicine, i.e., an agent with unclear efficacy, for use in patients

with extensively drug-resistant (XDR) TB [7]. CFZ is currently the only core second‐line medicine for the

treatment of MDR-TB that is not yet included in the WHO Model List of Essential Medicines as an anti-

TB medicine[8]. Médecins Sans Frontiers (MSF) has been using CFZ in its programs since 1988 and has

been using it for the treatment of MDRTB since 1999.

This Product Information Report (PIR) provides a summary of available literature about the active

pharmaceutical ingredient (API), analytical methods, toxicology, and dosage form for the product. The

PIR also provides guidance based on theoretical considerations of switching the CFZ dosage form from

the currently commercialized soft gel capsules to oral, solid dosage forms such as tablets or capsules.

The basic information provided includes the chemical structure/formula, IUPAC name, physico-chemical

properties, and solubility-related data about the CFZ API.

The CFZ API structure has been characterized using various techniques such as UV visible, Fourier-

transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), x-ray diffraction (XRD), mass


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spectrometry (MS), and scanning electron microscopy (SEM). The API structure has been summarized in

the document. Different routes of synthesis for the CFZAPI are also discussed in the PIR.N-aryl ortho-

phenylenediamine is usually the key starting material for the synthesis of CFZ [3].This PIR also provides a

summary of the available literature on the stability of CFZ in the aqueous state and dry state and gives

the mechanism of the degradation and the degradation products formed.

CFZ is a reddish-brown powder with a melting point of 210–215°C. It is readily soluble in benzene;

soluble in chloroform; poorly soluble in acetone and in ethyl acetate; sparingly soluble in methanol and

in ethanol; and virtually insoluble in water. It is classified as a Biopharmaceutics Classification System

(BCS) class II drug because of its poor aqueous solubility and high permeability.

The qualitative formula for the leading marketed formulation and the FDA-approved Reference Listed

Drug (RLD), Lamprene® 50 mg soft gelatine capsules, is described. The description includes the

proposed functions of the excipients, along with the US FDA Inactive Ingredients Database (IID) limits

for each individual excipient.

CFZ is manufactured as soft gelatin capsules. The capsules contain micronized CFZ suspended in an oily

base. The requirements for manufacturing equipment along with the proposed manufacturing process

have been outlined. Possible scale-up requirements are included in accordance with US FDA’s Scale-Up

and Post approval Changes: Chemistry, Manufacturing, and Controls, In Vitro Dissolution Testing, and

In Vivo Bioequivalence Documentation (https://www.fda.gov/media/70949/download).

The absorption of CFZ when taken orally in an oil-wax suspension is approximately 70%. When it is

taken with food, the Cmax of CFZ increases and the time to achieve peak plasma concentration

decreases[3]. It undergoes extensive tissue distribution. CFZ does not cross the blood-brain barrier but

does cross the placenta and is found in human breast milk. A few metabolites of CFZ have been

identified in man, but their biological activity is not known. The elimination half-life of CFZ is variable

and can be as long as 70 days[7].The portion of the ingested drug recovered from the feces may

represent excretion via the bile. A small amount is also eliminated in the sputum, sebum, and sweat.

Lamprene®, the RLD, has a shelf life of 60 months and should be protected from humidity and heat [9].

The capsule shell consists of gelatin, which is known to be sensitive to humidity. Hence, the preparation

is supplied in a humidity-resistant container.

It may be prudent to control relative humidity (RH) during manufacturing steps such as dispensing and

dry mixing, where the API is directly exposed to the environment. The manufacturing facility should be

maintained with optimum temperature and RH conditions to achieve batch-to-batch uniformity.

The animal toxicity data for CFZ are: oral LD50 (rat): 8400 mg/kg; oral LD50 (mouse): 5000 mg/kg; oral

LD50 (rabbit): 1500 mg/kg; and oral LD50 (guinea pig): 4400 mg/kg.

https://www.fda.gov/media/70949/download

Executive Summary

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The Acceptable Daily Exposure (ADE) value of CFZ has been reported as 30 µg/day. The Occupational

Exposure Limit (OEL) of CFZ is 7 µg/m3. It is assigned as a Category 3 substance in Affygility Solutions’

5-band control banding system. CFZ contact with skin and eye should be avoided. Proper exhaust

ventilation should be ensured to keep the airborne concentrations below the permissible exposure

limits. This OEL is designed to represent an 8 hours/day, 40 hours/week airborne concentration that

nearly all workers may be repeatedly exposed to, day after day, without adverse health effects based on

currently available information.

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Key Manufacturing Challenges

CFZ is a reddish-brown powder, which makes cleaning of the manufacturing equipment very difficult.

Additionally, dedicated manufacturing equipment and processing area for CFZ are recommended

because of the drug’s low OEL value. Use of adequate personal protective equipment (PPE) is also

advised.

The API for CFZ is relatively inexpensive but it currently seems difficult to procure, especially due to

increased global demand with the rollout of the shorter MDR-TB regimen in many countries.

CFZ is a BCS class II drug, which is virtually insoluble in water. Due to this limited solubility, the API

needs to be micronized and suspended in an oil-wax vehicle. The resultant slurry is to be filled in soft

gelatin capsules. API particle size distribution (PSD) is critical for product performance. Hence the API

PSD and polymorphic form are critical attributes in the quality of the dosage form.

CFZ is reported to be photodegradable and degrades up to 23% under UV. It undergoes oxidative

degradation and degrades up to 78% in 10% hydrogen peroxide solution. Protection from light

exposure should be provided during dispensing, compounding, and filling operations. A formulation

with use of antioxidants should be considered.

Cross-linking of gelatin in soft gelatin capsules can severely affect the drug’s performance by reducing

dissolution or causing incomplete dissolution. Appropriate strategies such as a switch to an oral solid

dosage (OSD) form could be considered to mitigate this risk.

Only 50-mg or100-mg soft gel capsules are available. These capsules cannot be opened or dissolved in

water. Smaller children are therefore dosed every second or third day depending on body weight and

the available formulation. This could be acceptable due to the drug’s long elimination half-life.

However, there is a need for child-friendly formulations so that this drug can be used for MDR-TB

treatment trials.

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Active Pharmaceutical Ingredient (API)

The API of CFZ has a melting point of approximately 217°C. CFZ is readily soluble in benzene, soluble

in chloroform, poorly soluble in acetone and in ethyl acetate, sparingly soluble in methanol and in

ethanol, and virtually insoluble in water. Its molecular weight is 473.4[10].

Chemical Structure / Formula

Molecular Formula for C27H22Cl2N4

Name[11]

IUPAC Name N,5-bis(4-chlorophenyl)-3-(propan-2-ylimino)-3,5-dihydrophenazin-2-amine

Others[12, 13]

Traditional Name

2-Phenazinamine, N,5-bis(4-chlorophenyl)-3,5-dihydro-3-[(1-methylethyl) imino]-

3-(p-Chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-(isopropylimino)phenazine

2-Phenazinamine, 3,5-dihydro-N,5-bis(4-chlorophenyl)-3-((1-methylethyl) imino)-

2-Phenazinamine, N,5-bis(4-chlorophenyl)-3,5-dihydro-3-((1-methylethyl) imino)-

Phenazine, 2,10-dihydro-3-(p-chloroanilino)-10-(p-chlorophenyl)-2-(isopropylimino)-


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Phenazine, 3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-(isopropylimino)-

(E)-N,5-bis(4-chlorophenyl)-3-(isopropylimino)-3,5-dihydrophenazin-2-amine

N,5-Bis(4-chlorophenyl)-3,5-dihydro-3-(isopropylimino) phenazin-2-amine

N,5-bis(4-chlorophenyl)-3-[(propan-2-yl) imino]-3,5-dihydrophenazin-2-amine

(3Z)-N,5-bis(4-chlorophenyl)-3-[(1-methylethyl) imino]-3,5-dihydrophenazin-2-amine

MESH Synonyms[14]

Clofazimin, Clofazimina, Clofazimine, Clofaziminum, Riminophenazine, Lamprene®, B 633, Chlofazimine,

UNII-D959AE5USF

Physical Properties

Particle Size CFZ is virtually insoluble in water (0.225mg/L). The selection of the particle size of the API would be a

critical issue for the development of a generic dosage form bioequivalent to the reference product. The

RLD contains micronized CFZ suspended in an oil-wax base filled in soft gelatin capsules[10].

Powder X-ray Diffraction (PXRD) Study CFZ showed multiple characteristic diffraction peaks in PXRD analysis, confirming its crystalline nature

(Figure 1) [3]. The PXRD pattern of CFZ was obtained on a Siemens D-500 X-ray diffractometer, using a

CuX-ray tube, at 40 kV and 40 mA.

Figure 1. X-ray diffractogram for crystalline CFZ

Active Pharmaceutical Ingredient

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Melting Point 201-215°C[3]

Log P 7.66 (Octanol/Water partition coefficient) [15, 16]

pKa 8.51 [16]

Water Solubility 0.225mg/L [16]

Refractive Index 1.63 [16, 17]

Density 1.3 g/cm3[17]

Solubility CFZ is soluble in dilute acetic acid, dimethyl formamide, soluble in 15 parts of chloroform, 700 parts of

ethanol, 1000parts of ether, and practically insoluble in water[12].

Chemical Properties

Stereochemistry [17]

Stereochemistry Achiral

Optical Activity Unspecified

E/Z Centres 1

Defined Stereo Centres 0

Undefined Stereo Centres 0

Charge 0

Hydrogen Bond Donor Count 1

Hydrogen Bond Acceptor Count 4


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Refractivity 142.55 m3·mol-1

Polarizability 51.52 Å3

Number of Rings 5

Synthesis of CFZ A number of literature references describe the process for synthesis of CFZ. The original synthetic

routes to riminophenazines (Barry et al.,1956a; 1956b; 1957 and 1958) have been modified (O'Sullivan,

1984) to give reproducible high yields[3]. The modifications have been summarized by Hooper, 1987[18]

(Figure 2)as follows: N-aryl ortho-phenylenediamine (1) undergoes region specific oxidative dimerization

to yield the parent iminophenazine (2),which reacts further with alkylamines to give substituted

iminophenazines (3). Alternatively, oxidation with benzoquinone in the presence of a carbonyl

compound gives an imidazolophenazine (4), which may be reduced with cleavage of the imino

substituent (5) followed by subsequent aerial oxidation to the parent iminophenazine (2). Amore

selective reduction results in an alternative cleavage of the imidazoline ring (6), which after oxidation

gives a substituted iminophenazine (7). The type of catalyst used in the reduction of these compounds is

crucial and allows full control of the reactions.

Figure 2. Synthetic Routes to Riminophenazines

Reagents: i- FeCl3, H+; ii- NH3; iii- R1NH2, alkylamines; iv- benzoquinone/carbonyl compound R2COR3; v- PtO2/H or Pt/C (10%)/H2;

vi- air; vii- Pd/C (10%)/H2


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In a method for synthesis reported by Gang Zhang et al [19],the synthesis was started from 1,5-difluoro-

2,4-dinitrobenzene (3), which is more reactive towards amine substitution. The two fluoro groups of

compounds (1) were subsequently replaced by N-(4-chlorophenyl) benzenediamine (2) and ammonia to

give the key intermediate (4). The dinitro groups of (4) were reduced either by catalytic hydrogenation

over Pd-C or by zinc powder reduction and the intermediate (5), without isolation, was exposed to air

and cyclized to form the desired riminophenazine core (6) with an amino group pending in the 2-

position. Compound (7) was obtained by displacement of the imino moiety with isopropyl amine in a

sealed bomb. When the palladium catalyzed N-arylation, which was applied to the 2-N arylation of (7)

with 2-bromopyrimidine under the usual reaction conditions, none of the desired product was formed.

After the screening of condition variables, such as Pd catalyst, ligand, base, and solvent, a combination

suitable for the N-arylation of (7) with2-bromo-pyrimidine was found. The target compound (9a) was

thus obtained in 95.8% yield (Figure 3).

Figure 3. Synthesis of CFZ


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Impurity Profiles of CFZ As per the literature[20-24], the following is the list of impurities (Figure 4) with their structures.

Figure 4. Impurities of CFZ

CFZ – Impurity A

N,5-bis(4-chlorophenyl)-3-imino-3,5-dihydrophenazin-2-amine CFZ – Impurity B

5-(4-chlorophenyl)-3-[(1-methylethyl) imino]-N-phenyl-3,5-dihydrophenazin-2-amine

CFZ – Iminophenazine Impurity 5-(4-chlorophenyl)-3,5-dihydro-3-iminophenazin-2-amine

CFZ – Impurity D

Degradation Products A study on the degradation of CFZ was carried out using conditions recommended by ICH to

demonstrate the stability-indicating ability and specificity of the developed method [25, 26]. Standard

samples of known concentration were exposed to different stress conditions. All samples were then

diluted and neutralized, if required, before injection. Control samples were also prepared for analysis.

For acid degradation studies, solutions were prepared using 0.1 and1 N HCl. For base degradation

studies, solutions were prepared using 0.1 N NaOH. For oxidation studies, solutions were prepared

using 10%H2O2. The samples were protected from light and stored at room temperature. Samples were

withdrawn at 0, 1, 2, and 3 h time points and suitably diluted before injection. For photo-degradation

studies, samples were exposed to UV light with an illumination of 7500 lx m with UV radiation at 320–

400 nm in a UV light chamber. Samples were withdrawn over a 6-h time period and diluted before

analysis.

The drug was found to be highly prone to degradation in basic stress conditions followed by acidic and

oxidative stress conditions, while it was found to be comparatively less susceptible to the photolytic

stress condition. In basic stress conditions, treatment with 0.1 N NaOH for 3 h showed nearly 99%


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degradation of CFZ into two degradation products, indicating its high susceptibility to the basic

environment. In acidic stress conditions, the samples treated with 0.1 N HCl for 3 h had negligible

degradation, whereas treatment with 1 N HCl for 3 h resulted in 78.23% degradation of CFZ into four

degradation products. In oxidative stress conditions, treatment with 10% H2O2for 3 h resulted in 35.56%

degradation of CFZ into a single degradation product. Under photolytic stress conditions, the drug

showed 22.70% degradation at the end of 6 h with a maximum number of degradation products

indicating its photosensitive nature. Details of the degradation products and percentage total

degradation are presented in Table 1.

Table 1. Degradation Studies of CFZ Under Different Stress Conditions

Stress Condition Treatment #of Products

Retention Time (RT) of Observed Degradants (min)

% Total Degradation

Standard control sample No treatment — — —

Acid degradation 1N HCl, 3h 4 1.071, 1.334, 1.656, 2.020 78.23

Base degradation 0.1N NaOH, 3h 2 1.094, 1.337 98.99

Oxidation 10% H2O2, 3h 1 1.334 35.56

Photolytic degradation UV exposure, 6h 5 1.042, 1.168, 1.600, 2.378, 3.381

22.70

Environmental Compatibility CFZ is slightly hazardous in water and therefore should be diluted before it reaches ground water or a

sewage system. It should not be disposed of together with household garbage [27].

Avoid generating dust, particularly clouds of dust in a confined or unventilated space, as dust may form

an explosive mixture with air. Any source of ignition such as a flame or spark will cause fire or

explosion[28]. Dust clouds generated by the fine grinding of the solid are a particular hazard because

accumulations of fine dust may burn rapidly and fiercely if ignited.

The combustion products of CFZ include carbon monoxide (CO), carbon dioxide (CO2), hydrogen

chloride, phosgene, nitrogen oxides (NOx), and other pyrolysis products typical of burning organic

material. Avoid contamination with oxidizing agents such as nitrates, oxidizing acids, chlorine bleaches,

and pool chlorine as ignition may result. Use process enclosures, local exhaust ventilation, or other

engineering controls to keep airborne levels below recommended exposure limits[29]. Facilities storing or

utilizing this material should be equipped with an eyewash and a safety shower.


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Safety Data Sheet It is recommended that waste be removed regularly and spills be cleaned up immediately. Avoid

breathing the dust and avoid contact with skin and eyes. For handling CFZ, wear protective clothing,

gloves, safety glasses, and a dust respirator when risk of exposure occurs. To avoid generating dust,

dampen surfaces with water before sweeping and place waste in suitable containers for disposal[28].

Empty containers may contain residual dust, which has the potential to accumulate after settling. Such

dusts may explode in the presence of an ignition source. It is suggested not to cut, drill, grind, or weld

such containers[28]. Material may be irritating to the mucous membranes and upper respiratory tract and

may be harmful by inhalation, ingestion, or skin absorption[29]. It may cause eye, skin, or respiratory

system irritation.

As mentioned earlier also, the toxicity data of CFZ as per the safety data sheet [28]are: oral LD50 (rat):

8400 mg/kg; oral LD50 (mouse): 5000 mg/kg; oral LD50 (rabbit): 1500 mg/kg; and oral LD50 (guinea pig):

4400 mg/kg.

Structure Characterization

UV Spectrum CFZabsorbs in the UV range because of the presence of specific chromophores in the structure that

absorb at a particular wavelength. The UV spectrum of solution of CFZ in 0.01M methanolic

hydrochloric acid is shown in Figure 5[3]. The spectrum was obtained using a Hewlett Packard845 2A

diode array UV visible spectrophotometer and 1-cm quartz cells. The spectrum exhibits two maxima, at

284nm and486nm, with absorbance values of about 1.30 and 0.64, respectively, at a concentration of

0.01% w/v.

Figure 5. UV Absorption Spectrum of CFZ


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FTIR Spectrum The infrared absorbance spectrum of CFZ is shown in Figure 6. The spectrum was recorded with a

Nicolet 5ZDX FT-IR spectrophotometer from a compressed potassium bromide disc[3]. The spectrum

was obtained by scanning at 400 to 2,000 cm-1 at a resolution of 2cm-1.

Figure 6. FTIR Spectrum of CFZ

Mass Spectrum The mass spectrum of CFZ (Figure 7) was obtained using a Finnigan Quadrupole mass spectrometer, by

electron impact at 70 electron volts. The molecular ion (M-H) at m/z 473 was observed[3].

Figure 7. Mass Spectrum of CFZ


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Nuclear Magnetic Resonance Spectrum Both the 1Hand 13C-NMR spectrums of CFZ were obtained in Chloroform-d (CDCI3) using tetramethyl

silane as an internal standard[3]. The 1H and 13C spectrums of CFZ (Figures8 and 10, respectively) were

obtained using a Jeol GX- 270 MHz instrument. A 2D Correlation Spectroscopy (COSY) spectrum was

also obtained (Figure 9).

Figure 8. 1H NMR Spectrum of CFZ

Figure 9. 2D COSY Profile of CFZ


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Figure 10. 13C NMR Spectrum of CFZ

Analysis of CFZ

A number of methods for analysis of CFZ have been described in pharmacopoeias and the literature. A

few of these are summarized below.

Pharmacopoeial Methods

United States Pharmacopeia (USP)

The liquid chromatograph is equipped with a UV detector set at 280nm and a 4.6mm × 25cm column

with5μm packing L7 (Octylsilane chemically bonded to totally porous silica particles, 1.5µm to 10 µm in

diameter or a monolithic silica rod, C8 column) and mobile phase containing a mixture of acetonitrile

and buffer (65:35). The flow rate is kept at about 1.0 mL per minute. Then 20 μL of prepared standard

and test samples, at a concentration of 0.05 mg/mL in mobile phase, are injected into the system and

responses of major peaks are measured. The tailing factor should not be more than 1.5 and the relative

standard deviation for replicate injections should not be more than 0.75%[11].

British Pharmacopoeia (BP) and European Pharmacopoeia (EP)

The assay of CFZ is determined potentiometrically [20, 21]. Dissolve 0.400 gm of CFZ in 5 mL of methylene

chloride; add 20 mL of acetone and 5 mL of anhydrous acetic acid. Titrate the above solution with 0.1M

perchloric acid and determine the end point potentiometrically. Each mL of 0.1M perchloric acid is

equivalent to 47.34 mg of CFZ.

Indian Pharmacopoeia (IP)

The assay of CFZ is determined potentiometrically [22]. Dissolve 0.500 gm of CFZ in 20 mL of chloroform;

add 50 mL of acetone. Titrate the above solution with 0.1M perchloric acid in dioxan and determine the

end point potentiometrically. Each mL of 0.1M perchloric acid is equivalent to 0.04734 gm of CFZ.


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International Pharmacopoeia (Int Ph)

The assay of CFZ is determined by non-aqueous titration. Dissolve 0.500 gm of CFZ in 20 mL of

chloroform; add 50 mL of acetone. Titrate the above solution with 0.1M perchloric acid. Each mL of

0.1M perchloric acid is equivalent to 47.34 mg of CFZ.

Other Reported Methods As per the literature review by Tulshidas Patil et al[25], CFZ had been qualitatively and quantitatively

analyzed using several techniques such as titrimetry, colorimetry, fluorimetry, UV-Vis spectroscopy,

paper chromatography, thin-layer chromatography, and HPLC. The methods indicated were employed

for analysis of CFZ in bulk, dosage form, and biological samples.

Method 1

Quantitative reverse phase (RP) HPLC was performed using a 250 mm × 4.6 mm internal diameter,5 μm

particle, and Inertsil C8-3 column. The mobile phase was a 650:350 mixture of acetonitrile and buffer

(containing 2.25 g of sodium dodecyl sulphate, 0.85 g of tetra butyl ammonium hydrogen sulphate, and

0.885 g of di-sodium hydrogen phosphate in500mL of water; adjust the pH of buffer to 3.0 with dilute

ortho-phosphoric acid)[30]. The flow rate was isocratic at 1 mL/minute. The UV detector was set at 280

nm. The column oven temperature was maintained at 30°C.The injection volume of CFZ samples was

20µl. The run time of analysis was around 25minutes, and the retention time of CFZ was around 14.8

minutes.

The method was validated for parameters like accuracy, linearity, and precision, as per ICH guidelines.

The values of relative standard deviation and percent recovery were found to be satisfactory, indicating

that the proposed method is precise and accurate and hence can be used for the routine analysis of

CFZ in bulk chemical and pharmaceutical formulation dosage form.

The reported regression analysis data and summary of validation parameters for the RP-HPLC method

parameters are shown in Table 2.

Table 2. RP-HPLC Method Parameters

Linearity 25 to 75 ppm

USP tailing factor 1.09

Slope 133871

Correlation coefficient 0.999

Intercept (c) 42759

Ruggedness Rugged


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Method 2

Another RP HPLC method reported use of a quaternary pump system equipped with SPD-M20A

Prominence® photo diode array (PDA) detector, SIL-20 AC HT Prominence® autosampler, LC-20 AD

Prominence® liquid chromatograph system, DGU-20A5R degassing unit, and CTO-10AS VP column

oven[25]. It utilized LabSolutions® software for monitoring and processing the output signal. The

optimum chromatographic separation was accomplished using 75:25% v/v ratio of methanol and

ammonium acetate buffer (0.01 mol/L) as the mobile phase at flow rate 1.0 mL/min and UV detection

at284 nm. The method was validated for parameters like accuracy, linearity, and precision, as per ICH

guidelines. The values of relative standard deviation and percent recovery were found to be

satisfactory, indicating that the proposed method is precise and accurate and hence can be used for the

routine analysis of CFZ in the API and pharmaceutical formulation dosage form.

The developed HPLC method was found to be highly sensitive and specific with linearity ranging

between 2 and 10 μg/mL and a correlation coefficient (R2) of 0.9995. The method showed high accuracy

with percent recovery between 99.68% and 100.44%. The detection limit and quantitation limit were

0.0066 μg/mL and 0.0199 μg/mL, respectively.

Analysis of Impurities / Related Substances / Degradation Products

United States Pharmacopeia (USP) The mobile phase and chromatographic system are similar to that of the USP API monograph assay.

Standard Preparation

Prepare 0.5 µg/mL of CFZ USP and 1.5 µg/mL CFZ related compound B in mobile phase.

Test Preparation

Prepare 0.5 µg/mL of CFZ test sample in mobile phase.

Procedure

Separately inject equal volumes (about 20 μL) of the Standard Preparation and the Test Preparation into

the chromatograph, record the chromatograms, and measure the responses for the peaks. The relative

retention times are about 0.81 for CFZ related compound B and 1.0 for CFZ. Calculate the percentages

of CFZ related compound B in the portion of CFZ taken by the formula:

(rU/rS) × (CS/CU) × 100


18

Where:

• rU = peak response of CFZ related compound B from test sample solution

• rS = peak response of CFZ related compound B from standard solution

• CS = concentration of CFZ related compound B in the standard solution

• CU = concentration of CFZ in the test solution

Calculate the percentage of any individual unspecified impurity in the portion of CFZ taken by the

formula:

(rU/rS) × (CS/CU) × 100

Where:

• rU = peak response of any individual unspecified impurity from test sample solution

• rS = peak response of CFZ from standard solution

• CS = concentration of CFZ in the standard solution

• CU = concentration of CFZ in the test solution

The CFZ related compound B should not be more than 1.0%, and any other impurity should not be

more than 0.1%. The total impurities should not be more than 2.0%. Disregard any impurity peaks less

than 0.05%.

Other Pharmacopoeias BP, EP, and IP have adopted similar methods for the analysis of impurities.

Test Solution

Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 100 mL with the

mobile phase.

Reference Solution (a)

Dilute 1.0 mL of test solution to 100mL with the mobile phase. Dilute 1.0 mL of this solution to 10 mL

with the mobile phase.

Reference Solution (b)

Dissolve 5 mg of CFZ in mobile phase and dilute it to 10 mL using mobile phase.

Chromatographic System

Stainless steel column, 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 µm); mixture

containing buffer (acetone, 35:65) adjusted to pH 3.0 as mobile phase at flow rate 1.0 mL/min with

injection volume of 20 µL recorded at 280 nm.


19

CFZ impurity A should not be more that 0.1%, CFZ impurity B should not be more that 0.3%, and any

other impurity should not be more than 0.1%. The total impurities should not be more than 0.5%.

Disregard any impurity peaks less than 0.05%.

The summary of the Pharmacopoeial test limits appears in Table 3.

Table 3. Pharmacopoeial Test Limits for CFZ

Test Limit

USP BP EP IP

CFZ – impurity A - NMT 0.1% NMT 0.1% NMT 0.1%

CFZ – impurity B NMT 1.0% NMT 0.3% NMT 0.3% NMT 0.3%

CFZ – other NMT 0.1% NMT 0.1% NMT 0.1% NMT 0.1%

Impurities all together NMT 2.0% NMT 0.5% NMT 0.5% NMT 0.5%

Assay capsules w/w 90%–110% 95%–105% * 95%–105%

NMT =not more than * Capsules are not official

Reference Standards Availability Reference standards of CFZ are available with USP, BP, EP, and IP. The reference standards of CFZ and

related substances are also available commercially[31, 32].

Stability of CFZ

Dry Powder Stability As per reported literature, CFZ should be stored below 25°C[10]. It is also reported to be

photodegradable [9] and therefore should be protected from light. Monograph of CFZ in USP 41

monograph recommends preserving CFZ in tight, light-resistant containers at room temperature.

The capsule dosage formLamprene® is commercially supplied as a soft gelatin capsule containing CFZ

100mg and 50 mg in HDPE bottle of one hundred (100) capsules, and the labeling recommends that

the product be stored below 25°C. The dosage form should be protected from moisture because the

capsule shell may be adversely affected.


20

Test Specifications for CFZ As per information gathered from USP, EP, and other sources, Table4 provides recommended test

specifications for the CFZ API.

Table 4. Test Specifications for CFZ API

Test Description

Description Reddish brown crystalline fine powder, almost odorless

Identification by HNMR Should be consistent with structure

Loss on drying Should be less than 0.5% w/w

Residue on ignition Should be less than 0.1% w/w

Heavy metals Should be less than 10 ppm

Sulphated ash Should be less than 0.1% w/w

Assay 98.0% -102% (on dry basis)

Related substances As reported in Table 3

21

Dosage Form

General Summary

CFZ is currently the only core second‐line medicine for the treatment of MDR tuberculosis. It is used in

the conventional regimen and also in the standardized shorter regimen lasting 9-12 months. CFZ is

mainly indicated in combination with other anti-leprosy drugs for the treatment of lepromatous leprosy,

including dapsone-resistant lepromatous leprosy, and lepromatous leprosy complicated by erythema

nodosum leprosum[33].

Active drug master files (DMF) for CFZ are listed in Table5[34].

Table 5. Active DMF for CFZ API (as of June 2019)

DMF # Submit Date Holder Subject

33157 06 Sep 2018 Olon Spa, Italy CFZ

16668 26 Jun 2003 Sandoz, Germany CFZ

31256 21 Dec 2016 Zhejiang Huahai Pharmaceutical Co Ltd, China CFZ

33157 06 Sep 2018 Olon Spa, Italy CFZ

All are type II DMFs that include the drug substance and its allied compounds.

Regulatory Status

US FDA approved the New Drug Application (NDA) for the RLD Lamprene® Capsules,50 mg and 100

mg, in 1986 as a prescription drug. Lamprene® is supplied as50-mg brown, spherical soft gelatin

capsules in bottles of 100[10].The soft gelatin capsule containing CFZ 50 mg is spherical without any

imprint[35].

Lamprene® is also supplied as a 100-mg soft gelatin capsule in bottles of 100[10]. The soft gelatin capsule

containing 100mg CFZ is oblong in shape and is imprinted with ‘GEIGY' in white on one side and 'GM'

in white on the other side[35].


22

Lamprene® 100-mg capsules

The irregular color of the capsules is due to the active ingredient being present as a microcrystalline

suspension in an oil-wax base. The capsule fill vehicle is dark brown while the suspended particles are

reddish brown. Sedimentation of the suspended material may lead to an irregular (possibly mottled)

appearance.

Macleods Pharma had submitted an application for CFZ 50-mg and 100-mg tablets to WHO Pre-

Qualification (PQ), as per List of Tuberculosis Pharmaceutical Products Classified According to the

Global Fund Quality Assurance Policy[36]. The application has been reviewed by the Expert Review Panel

(ERP), but more details are not available in the public domain.

Formulation Barriers to Entry The currently available dosage form of CFZ is a soft gelatin capsule containing micronized CFZ

suspended in an oily base[9]. Its shelf life is 60 months. The product should be protected from humidity

and heat. The capsule shell consists of gelatin, which is known to be sensitive to humidity. Hence, the

preparation is supplied in a humidity-resistant container, which should be closed again immediately

after use. The capsules may occasionally stick together, but they remain usable.

Only 50-mg or100-mg soft gel capsules are available, and the capsules cannot be opened or dissolved

in water. Smaller children are therefore dosed every second or third day depending on body weight and

the available formulation.

Despite being inexpensive, CFZ is difficult to procure due to the increased global need for treating

MDR-TB in many countries.

Cleaning of equipment during manufacturing is challenging due to reddish-brown color of CFZ API.

Dedicated manufacturing equipment and processing area are recommended due to low OEL value of -

CFZ. It is also recommended to use adequate personal protective equipment (PPE).

Dosage Form

23

Since CFZ is classified as a BCS class 2 drug[15], it is not a candidate for a biowaiver. However, qualitative

and quantitative (Q1/Q2) matching of the generic product with the RLD product may allow high

assurance of achieving bioequivalence.

The formulation is supplied as a soft gelatin capsule. The main component of the capsule shell, gelatin,

may undergo cross-linking. Cross-linking is the formation of strong chemical linkages beyond simple

hydrogen and ionic bonding between gelatin chains. The process is irreversible and results in insolubility

of gelatin. This can lead to delays in the opening of capsules in dissolution media. Appropriate

strategies, i.e., a switch to OSD form, could be considered to mitigate this risk (See Appendix 1).

Formulation Justification

Reverse Engineering A qualitative formula for the leading marketed formulation was retrieved from the US FDA documents

of the RLDLamprene® NDA. Lamprene®for oral administration contains50mg and 100 mg of CFZ

suspended in an oil-wax base filled in soft gelatin capsules. The other inactive ingredients in capsules

include beeswax, butylated hydroxytoluene, citric acid, ethyl vanillin, gelatin, glycerin, iron oxide,

lecithin, p-methoxy acetophenone, parabens, plant oils, and propylene glycol[10, 35].

Excipients A list of excipients with their proposed functions in the RLD Lamprene®capsule is provided in Table 6.

The US FDA’s Inactive Ingredient Database (IID)can be accessed for additional information on the

individual inactive ingredients. The IID provides the dosage forms for which the excipient is approved

and the maximum concentration approved for that dosage form. Quantitative limits for excipients used

in Lamprene®were confirmed using the IID[37].

Table 6. List of Inactive Ingredients with Proposed Function and IID Limits

Ingredients Function Reference

(Page of Reference) IID Limit for Soft

Gel capsule[37]

Beeswax Controlled-release agent, stabilizing agent, stiffening agent

[38] pp 779 60 g/kg*[39]

Butylated hydroxytoluene

Antioxidant [38] pp 75 0.25 mg


24

Citric acid Acidifying agent; antioxidant, buffering agent, chelating agent, flavor enhancer, preservative

[38] pp 181 1.0 mg

Ethyl vanillin Flavoring agent [38] pp 261 0.64 mg

Gelatin Component of capsule shell [38] pp 278 733 mg

Glycerin Plasticizer in gelatin shell [38] pp 283 204.2 mg

Iron oxide Colorant [38] pp 340 NL# (tablets – 0.79mg)

Lecithin Emulsifying agent, solubilizing agent [38] pp 385 325 mg

para-Methoxy acetophenone

Flavoring agent NL NL

Parabens Antimicrobial preservatives [38] pp 441 0.68 mg

Plant oils Vehicle for capsule fill NL NL

Propylene glycol Vehicle for capsule fill [38] pp 592 17.7 mg

NL =Not listed in IID *Not listed in IID; proposed limit is from reference[38]and [39]

# Value for tablets is given, since no information was available for capsule

Formulation Challenges CFZ should not be directly exposed to an acidic or basic environment. Selection of neutral excipients or

other antioxidants to improve the stability of the CFZ formulation is suggested. CFZ is a reddish-brown

powder, which makes cleaning and cleaning validation (necessary to demonstrate the control of cross-

contamination) very difficult. The impact of cross-contamination risk is increased by the low

Occupational Exposure Limit (OEL) value of CFZ also. As such, the product needs to be manufactured

using dedicated equipment and rooms. This restraint can present a significant financial, technical, and

operational challenge for manufacturers. Similar challenges exist with rifampicin, which requires

manufacturers to have segregated manufacturing operations. CFZ is also reported to be

photodegradable and therefore it should be protected from light.

As the gelatin capsule is considered to be sensitive to moisture, it is recommended that during

manufacturing, the capsules should be protected from humidity. The manufacturing facility should be

maintained with optimum temperature and RH conditions to achieve batch-to-batch uniformity.

Dosage Form

25

CFZ dosage forms are available only as 50-mg or100-mg soft gel capsules that cannot be opened or

dissolved in water, thus making dosing for children very difficult. Smaller children are therefore dosed

every second or third day depending on their body weight and the available formulation dosage, and

this may result in inaccurate dosing.

There is a need for development of a child-friendly formulation for administration of regular and

accurate dosages to treat children with MDR-TB. Therefore, an opportunity and/or challenge to

innovate exists for the potential manufacturers to develop an age-appropriate dosage form, as

compared with the currently available soft gelatin capsules. One of the strategies that should be

considered is to attempt developing an OSD form, such as tablets, in place of the currently

commercialized soft gel capsule dosage form. This approach would also mitigate the risk of the cross-

linking problem of the soft gel capsule shell.

A theoretical concept is explored and provided in Appendix 1.The concept is based on the physico-

chemical properties of CFZ and the traditional as well as state-of-the-art innovative manufacturing

technologies available. Recommendations are also made for CFZ OSD form selection. This will help

the manufacturer to develop engineering study data with the help of the roadmap provided in this

document.

Manufacturing Process CFZ capsules are preferably prepared by suspending the API in an oil-wax base filled in soft gelatin

capsules. Major unit operations in soft gel capsule manufacture should include micronization of API,

preparation of gelatin gel mass for encapsulation, fill material preparation, encapsulation, drying of

filled capsules, cleaning/sorting of capsules, and packaging [40]. A schematic flow diagram of soft gel

capsule manufacturing process is shown in Figure 11.

Figure 11. Schematic Flow Diagram of Soft Gelatin Capsule Manufacture


26

As indicated above, based on reported photo-degradation of the CFZ API, it is recommended that the

API and CFZ finished pharmaceutical product (FPP) should be protected from light during

manufacturing and during raw material weighing, mixing, filling, and packaging. Use of a sodium vapor

light source is recommended to achieve adequate photo-protection.

The first step may be the milling of raw material. However, this is an optional step based on the particle

size of supplied API raw material. Airjet mill or Quadro mill can be used for milling. Milled powder is

then subjected to sieving to achieve uniform particle size. The micronized CFZ is mixed with an oil-wax

base with other excipients to form fill material paste for soft gelatin capsules. The process of filling the

fill material into soft gelatin capsules is shown in Figure 12. The material to be encapsulated flows by

gravity. The gelatin sheets are fed on rolls containing a small orifice lined up with the die pocket of the

die roll. Two plasticized gelatin ribbons are continuously and simultaneously fed with the paste fill

between the rollers of the rotary die mechanism where the capsules are simultaneously filled, shaped,

hermetically sealed, and cut from the gelatin ribbon. The sealing of the capsule is achieved by

mechanical pressure on the die rolls and the heating (37-40°C) of the ribbons by the wedge[41].

Figure 12. Filling of Soft Gelatin Capsules[41]

Precautions for safe handling include avoiding contact with skin and eyes. Additionally, formation of

dust and aerosols should be avoided. Adequate general or local exhaust ventilation should be provided

to keep airborne concentrations below the permissible exposure limits. Normal measures should be

taken for fire prevention protection. For nuisance exposures, use type P95 (US) or type P1 (EU EN 143)

particle respirator. For higher-level protection use type OV/AG/P99 (US) or type ABEK-P2 (EU EN 143)

Dosage Form

27

respirator cartridges. Use respirators and components tested and approved under appropriate

government standards such as National Institute for Occupational Safety and Health (US) or European

Committee for Standardization (EU).

Analytical Methods of Dosage Form

Pharmacopoeial Methods

USP

Dissolution

The medium used for dissolution is 500 mL of water. The USP 2 apparatus is used at 50 rpm for 15

minutes. The analysis is performed by placing the soft gelatin capsule in each vessel. The capsule is

allowed to sink before starting the paddle rotation. The capsules are observed for the time taken to

rupture the capsule shell.All the capsules should rupture in less than 15 minutes.

Assay

The mobile phase and chromatographic system are similar to that of the API. For capsule analysis,

obtain the contents of 20 soft gelatin capsules and mix together. Transfer the contents equivalent to

500 mg of CFZ into a 250-mL conical flask, add 50 mL of mobile phase in increments, and shake well.

Transfer the contents completely to a 1000-mL volumetric flask and make up the volume with mobile

phase. Stir the resultant mixture at high speed to make the stock sample solution homogenous. Filter

about 20 mL of the stock sample solution. Transfer 1 mL of the filtered solution into a 10-mL volumetric

flask and dilute to volume using mobile phase. Inject the standard and test preparations and calculate

the percentage of labeled amount of CFZ in the portion of capsules taken by the formula:

(rU/rS) × (CS/CU) × 100

Where:

• ru = peak response of CFZ from test sample solution

• rs = peak response of CFZ from standard solution

• Cs = concentration of CFZ in the standard solution (mg/mL)

• Cu = nominal concentration of CFZ in the test solution (mg/mL)

Indian Pharmacopoeia

Assay

Weigh accurately a quantity of the mixed contents of 20 capsulescontaining about 0.15 g of CFZ and

dissolve in sufficient choloroform to produce 100 mL. Filter the solution through a chloroform-washed

plug of cotton wool. Dilute 5 mL of the clear filtrate to 100 mL with choloform. To the 5 mLfiltrate, add


28

5 mL of 0.1M methanolic hydrochloric acid and sufficient choroform to produce 50 mL. Measure the

absorbance of the resulting solution at about 491 nm using a blank containing 5 mL of 0.1M methanolic

hydrochloric acid diluted to 50 mL with chloroform. Calculate the content of CFZ,considering 650 as the

specific absorance at 491 nm.

Other Methods In one particular method, as reported under other method for API analysis[6], for the 50-mg strength 10

capsules were placed in a beakerwith sufficient quantity of extraction diluent and were stirred with a

magnetic stirrer at about 2000 rpm for 30 minutes. CFZ was extracted from the capsule contents. For

the 100-mgstrength, 5 capsules were placed in a beaker along with sufficient quantity of diluents, and

CFZ was extracted usinga similar procedure as with the 50-mg capsules. The final concentrations of

thesolutions were set to approximately 50 ppm for CFZ. An assay method was developed and validated

for determinationof the CFZ in capsules. The method was precise, accurate, stability indicating, and

robust.

Stability-Indicating Method A stability-indicating RP-HPLC method for a quantitative determination of CFZ in the API and dosage

form is summarized under the section Degradation Products above [25]. A quality-by-design (QbD)-

based, simple, rapid, economical, and stability-indicatingHPLC method wassuccessfully developedfor

CFZ. The forced degradation studies of CFZ were carried ot under a variety of stressed conditions. The

developed method was able to detect andquantify CFZ in its bulk chemical form and its formulation

dosage form. The optimized methodwas further validated for linearity, limit of detection (LOD), limit of

qunatitation (LOQ), specificity,accuracy, precision, robustness, and system suitabilitytesting parameters.

Stability of Dosage Form

It is recommended that Lamprene®capsules be stored at temperatures below 25°C and protected from

light. USP recommends preserving CFZ in tight, light-resistant containers at room temperature. The

shelf life of the RLD Lamprene® capsule product is 60 months.

Dosage Form Test Specifications

Test specifications for CFZ capsulesas per various pharmacopoeias are summarized in Table 7.

Dosage Form

29

Table 7. Test Specifications for CFZ Capsules

Test Description

USP BP/IP

Identification Should pass Should pass

Dissolution test Capsule shell ruptures in NMT 15 minutes

Not required

Uniformity of dosage form

Should meet the requirements (90-110% w/w of the label claim)

Should meet the requirements (95-105% w/w of the label claim)

Related substances As reported in API specifications As reported in API specifications

Assay 90-110% w/w of the label claim 95-105% w/w of the label claim

30

Bioavailability and Pharmacokinetics

Clofazimine absorption following oral administration isincomplete and varies significantly from patient

to patient.Following oral administration as coarse crystals, only about 20% isabsorbed. However, if the

drug is given orally as a microcrystallinesuspension in an oil-wax base, an absorption rate of 70%can

beachieved[9].

Mechanism of Action

CFZ exerts a slow bactericidal effect on Mycobacterium leprae (Hansen’s bacillus). CFZ inhibits

mycobacterial growth and binds preferentially to mycobacterial DNA. CFZ also exerts anti-inflammatory

effects in treating erythema nodosum leprosum. However, its precise mechanisms of action are

unknown[10].

In Mycobacterium tuberculosis, CFZ appears to act as a pro-drug, which is reduced by NADH

dehydrogenase (NDH-2) to release reactive oxygen species upon reoxidation by oxygen. CFZ

presumably competes with menaquinone, a key cofactor in the mycobacterial electron transfer chain,

for its reduction by NDH-2[42].

The mechanism of action for the antimycobacterial activity of CFZ can be postulated through its

membrane-directed activity including the bacterial respiratory chain and ion transporters. Intracellular

redox cycling, involving oxidation of reduced CFZ, leads to the generation of antimicrobial reactive

oxygen species (ROS), superoxide, and hydrogen peroxide (H2O2). In addition, interaction of CFZ with

membrane phospholipids results in the generation of antimicrobial lysophospholipids, which promote

membrane dysfunction, resulting in interference with K+ uptake[43]. Both mechanisms result in

interference with cellular energy metabolism by disrupting ATP production (Figure13). Anti-

inflammatoryactivity of CFZ is primarily through inhibition of T lymphocyte activation and proliferation.

CFZ may indirectly interfere with the proliferation of T cells by promoting the release of ROS and E-

series prostaglandins (PGs), especially prostaglandin E2 (PGE2), from neutrophils and monocytes.


31

Figure 13. Mechanism of Action of CFZ

Measurement of the minimum inhibitory concentration (MIC) of CFZ against leprosy bacilli in vitro is not

yet feasible.

CFZ augments PGE2 production in normal neutrophils as well as neutrophils from chronic myelocytic

leukemia and chronicgranulomatousdisease, although the meaning of this in terms of neutrophil action

hasnot beenfully elucidated. CFZ has alsobeen found to partially reverse inhibitionof monocyte function

by a Mycobacterium tuberculosis glycolipid[44].

BCS Class of the Product According to the USFDA definitions, APIs as per BCS have been classified into four categories [15]:

• BCS class I: high solubility – high permeability

• BCS class II: low solubility – high permeability

• BCS class III: high solubility – low permeability

• BCS class IV: low solubility – low permeability

CFZ has low solubility and high permeability (Peff- 4.38X10-4 cm/s)[2]and therefore has been placed in

BCS class II.

Pharmacokinetics From the US FDA approved labeling of Lamprene®[10], the pharmacokinetic parameters are summarized

as below:


32

Absorption

CFZ has a variable absorption rate in patients, ranging from 45% to 62% with 9% to 74% of an

administered dose appearing in feces. About 20% of a dose is absorbed from the gastrointestinal tract

when CFZ is administered as coarse crystals, but 45% to 70% of a dose may be absorbed when the drug

is administered as a micronized suspension in an oil-wax base after oral administration of Lamprene®.

Simultaneous ingestion of food increases the bioavailability in terms of area under the curve by 60% and

tends to increase the rate of absorption. The average serum concentrations of CFZ in patients treated

with Lamprene®100 mg and 300 mg daily were 0.7 µg/mL and 1 µg/mL, respectively.

Time to reach peak plasma concentration (median Tmax) of CFZ decreases from 12 hours to 8 hours

under fed conditions relative to the fasted state.

Distribution

CFZ is highly lipophilic and tends to be deposited predominantly in fatty tissue and in cells of the

reticulo-endothelial system. It is taken up by macrophages throughout the body. In autopsies

performed on leprosy patients who had received Lamprene®, CFZ crystals were found predominantly in

the mesenteric lymph nodes, adrenals, subcutaneous fat, liver, bile, gall bladder, spleen, small intestine,

muscles, bones, and skin.

CFZ is bound to alpha-and beta-lipoproteins in serum, particularly the beta-lipoproteins, and the

binding was saturable at plasma concentrations of approximately 10 µg/mL. Binding to gamma-globulin

and albumin was negligible.

Metabolism

Three CFZ metabolites were found in urine following repeated oral doses of Lamprene®. Information on

the metabolism of CFZ is limited. Figure 14 shows the details of metabolites of CFZ [3].

Figure 14. Metabolites of CFZ


33

Elimination

After ingestion of a single 300-mg dose of Lamprene®, elimination of unchanged CFZ and its

metabolites in a 24-hour urine collection was negligible. CFZ is retained in the human body for a long

time, and elimination of CFZ is slow. In healthy subjects, after a single administration of 200mg CFZ,

mean plasma elimination half-life is reported as 10.6 (± 4.0) days, but it has also been reported to be as

little as 70 hours. Part of the ingested drug recovered from the feces may represent excretion via the

bile. Fecal elimination of CFZ exhibits considerable inter-individual variation, and 35% to 74% of a single

oral dose may be excreted unchanged in feces over the first 72 hours after the dose. A small amount is

also eliminated in the sputum, sebum, and sweat. The elimination half-life of CFZ following repeated

oral doses of 50 or 100 mg Lamprene® in patients was highly variable, with estimates ranging from 6.5

to 160 days. The overall mean half-life of CFZ in these patients was approximately 25 days.

Dissolution Profile of Reference Product A dissolution test for the capsules is available in the monograph for CFZ capsules in USP 41[11]. No

dissolution profile data are available for the reference product. According to the USP monograph, the

capsule shell should rupture in less than 15 minutes in the dissolution medium under the prescribed

conditions.

Food Effect on Pharmacokinetics A study was performed on the effect of food on the bioavailability and pharmacokinetics of single oral

doses of CFZ[45]. Following administration with food, the area under the curve (AUC)for plasma

concentration versus time and the peak plasma concentration (C), were 62% and 30% higher,

respectively, compared to results obtained in the fasted state (Figure 15). The gastrointestinal

absorption of CFZ in terms of the median time (Tmax) to reach Cmax was 8hours with food and 12 hours

without food.

Figure 15. Mean Plasma Concentration of CFZ after Single Dose of CFZ

Where: ◼ = Fasted volunteers; ⚫ = Fed volunteers


34

Bioequivalence Study Protocol Guidance As per the WHO guidance document dated 18 November 2016 entitled “Notes on the Design of

Bioequivalence Study: Clofazimine” [46],the following guidance about study design should be taken into

account:

Notes on the design of bioequivalence studies with products invited for submission to the WHO

Prequalification Team: medicines (PQTm) are issued to aid manufacturers with the development of their

product dossier. Deviations from the approach suggested below may be considered acceptable if

justified by sound scientific evidence. Below, additional specific guidance is provided on those WHO-

invited immediate-release products that contain CFZ.

Guidance for the Design of Bioequivalence Studies

Taking into account the pharmacokinetic properties of CFZ, the following guidance with regard to the

study design should be considered:

Design

Due to the long half-life of CFZ, a parallel design is recommended. However, a cross-over design might

be considered.

Dose

A 100-mg dose of CFZ (the highest capsule strength) should be used in the bioequivalence study since

the pharmacokinetics is reported to be non-linear.

Fasting vs. Fed State

The bioequivalence study should be conducted in the fed state as CFZ may exhibit a higher absorption

in the presence of food and it is recommended that CFZ be taken with meals.

Subjects

Healthy adult subjects should be enrolled. It is not necessary to include patients in the bioequivalence

study.

Sample Size

CFZ AUC and Cmax in the fed state have a moderate intra-subject variability (<30%). These data (i.e., for

the intra-subject variability for AUC and Cmax) may facilitate the calculation of a sufficient sample size for

a cross-over bioequivalence study. However, in the case of a parallel design, inter-subject variability

must be taken into account and these data are not presently available.

Washout

In case of a cross-over design, the long half-life of CFZ must be taken into account, but it is not possible

to define since its half-life may vary from 70 hours up to at least 10 days. Therefore, a washout period of

at least 2 months should be applied to prevent carryover.


35

Blood Sampling

The blood sampling should take into account that CFZ absorption is slow and that Tmax occurs after 6–12

hours. Entero-hepatic recycling seems to occur as well. Therefore, the blood sampling does not need to

be very frequent during the initial few hours, but needs to be sufficiently frequent (e.g., every 30

minutes) during the first 12hours after administration to properly characterize the Cmax of CFZ.

Considering the elimination half-life, it is sufficient to take blood samples up to 72 hours after

administration for the characterization of CFZ pharmacokinetics.

Analytical Considerations

Information currently available in PQTm indicates that it is possible to measure CFZ in human plasma

using liquid chromatography–mass spectrometry (LC-MS)/MS analytical methodology. The bioanalytical

method should be sufficiently sensitive to detect concentrations that are 5% of the Cmax in most profiles

of each formulation (test or comparator).

Parent or Metabolite Data for Assessment of Bioequivalence

The parent drug is considered to best reflect the biopharmaceutical quality of the product. The data for

the parent compound should be used to assess bioequivalence.

Statistical considerations

The data for CFZ should meet the following bioequivalence standards in a single-dose, crossover, or

parallel design study:

• The 90% confidence interval of the relative mean AUC0-72 h of the test to reference product

should be within 80% to 125%

• The 90% confidence interval of the relative mean Cmax of the test to reference product should

be within 80% to125%

Information currently available in PQTm indicates that the comparator product is not a highly variable

drug product for AUC and Cmax in the fed state. However, if a parallel design is selected, it must be

taken into account that the inter-subject variability of the drug product is probably large.

Bioanalytical Methods

A simple, specific, and rapid HPLC assay for the determination of CFZ in human plasma was

developed[47]. The drug and the internal standard (medazepam) were extracted from 0.5 mL plasma with

dichloromethane/diisopropyl ether (1:1, v/v) at pH3.0, after precipitating the proteins with methanol.

The drugs were then quantitated on a reversed-phase C8 HPLC column using a mobile phase consisting

of a mixture of methanol/0.25N sodium acetate buffer at pH 3.0 (74:26, v/v). The flow rate and UV

detector wavelength were set at1 mL/min and 286 nm, respectively. The precision, linearity, and limit of

quantitation of the method were within acceptable limits. The method was considered adequate and


36

could be applied in studies involving blood level monitoring and pharmacokinetics inpatients. The

validation parameters of the method are given in Table 8.

Table 8. Bioanalytical Method Parameters

Linearity 0.003 – 1µg/mL

Correlation coefficient 0.9962

Slope 85.70

Intercept 480.41

Limit of quantitation 0.003µg/mL

Precision (CV)

Intra-day precision 5.6

Inter-day precision 8.5

Percentage recovery 85% (CV- 5.2%)

Interference No interference peaks from plasma components were observed in chromatograms.

Another method for determination of CFZ in plasma has been reported, with a sensitivity limit of about

10 mg/mL [48]. This method involved extraction of CFZ into organic solvents, separation of CFZ from

potential interfering materials by HPLC, and quantitation via the high absorbance of CFZ at 285 nm.

CFZ was extracted from 1-mL aliquots of rat or human plasma by addition of 1 mL of phosphate-citrate

buffer, pH 6.0, and 14 mL of chloroform-methanol (4:1, v/v) in a50-mL culture tube. The tube was closed

with a foil-lined cap and was shaken for 20 min at 80–100 strokes/min on a shaker (Eberbach, Ann

Arbor, MI, USA). After centrifuging for 10 min at 400 g, the aqueous layer was aspirated off and10mL of

the organic layer was transferred to a 16 X 100 mm test tube and evaporated to dryness under a gentle

stream of high-purity nitrogen using a Meyer N-Evap (Organomation Assoc., Shrewsbury, MA, USA).

The residue was reconstituted in 150 µL of mobile phase (0.0425 M phosphoric acid in 81%methanol)

and 0.5 mL of hexane. Following centrifugation to separate the phases, the hexane layer was discarded

and the mobile phase was transferred to an injection vial.

As per the literature review article by Tulshidas Patil et al[6], CFZ had been estimated in biological

samples using HPLC equipped with UV and PDA detectors. It is important to accept the fact that none

of the methods is impeccable. During the last three decades, various HPLC methods have been

developed and validated to estimate CFZ. But very few methods are of composite purpose, i.e., used to

estimate assay and impurities or to estimate drug from both dosage forms as well as biological fluids.

Most of the analytical methods utilize UV detection. Every analytical method has its own stipulations,

benefits, and pitfalls. Experienced formulators and method development scientists should collaborate

to minimize the issues in the estimation of the drug.

37

Toxicology Information

As per literature review, the following is the toxicity data for CFZ.

Animal toxicity

Acute toxicity LD50 orally in mice, rats, and guinea pigs: >4 g/kg; in rabbit: 3.3 g/kg. CFZ toxicity has been decreased

by the use of liposome-encapsulated drug with no reported change in the MIC[12]

An additional reference [50] provides acute animal toxicity information as follows:

• Oral LD50 (rat): 8400 mg/kg

• Oral LD50 (mouse): 5000 mg/kg

• Oral LD50 (rabbit): 1500 mg/kg

• Oral LD50 (guinea pig): 4400 mg/kg.

Reproductive Toxicity At 25 times the normal human dose, CFZ impaired fertility in rats. Fetal toxicity in mice was found at 12

to 25 times the normal human dose and included retardation of fetal skull ossification, increased

incidence of abortions and stillbirths, and impaired neonatal survival. The skin and fatty tissue of

offspring became discolored approximately 3 days after birth, which was attributed to the presence of

Lamprene® in the maternal milk.

Genotoxicity

CFZ was Ames negative but inhibited growth of human fibroblasts at 2.5 mg/mL, showed dose-related

changes in mitotic indexes, and showed elevated incidence of chromosomal aberrations in mice treated

with 40 mg/kg daily for seven days[12].

No long-term carcinogenicity studies in animals have been conducted with Lamprene®. Lamprene® was

not teratogenic in laboratory animals at dose levels equivalent to 8 times (rabbit) and 25 times (rat) the

usual human daily dose.


38

HumanToxicity

It has been found that Lamprene® crosses the human placenta. The skin of infants born to women who

had received the drug during pregnancy was found to be deeply pigmented at birth. No evidence of

teratogenicity was found in these infants. There are no adequate and well-controlled studies in

pregnant women. Based on previous observations, discoloration gradually faded over the first year.

Lamprene® should be used during pregnancy only if the potential benefit justifies the risk to the

fetus[12].. Limited data is available regarding the reversibility of discoloration.

Human Drug-Drug Interactions Dapsone may inhibit the anti-inflammatory activity of Lamprene® but have not been confirmed[10]. CFZ

reduces rifampicin absorption in leprosy patients, increasing the time required to reach peak serum

concentration and prolonging the elimination half-life. Bioavailability was not affected, so this

interaction is unlikely to be clinically significant. In patients receiving high doses of CFZ (300 mg daily)

and isoniazid (300mg daily), elevated concentrations of CFZ were detected in plasma and urine,

although skin concentrations were found to be lower.

Human Potential Toxicity There are reports of death following severe abdominal symptoms. Autopsies have revealed crystalline

deposits of CFZ in various tissues including the intestinal mucosa, liver, spleen, and mesenteric lymph

nodes. Ames test reveals no evidence of carcinogenicity risk but long-term studies are incomplete.

Human Adverse Reactions

Gastrointestinal Toxicity

Gastrointestinal symptoms include: abdominal and epigastric pain, diarrhea, nausea, vomiting,

gastrointestinal intolerance (40 - 50%); discoloration of urine, feces, sputum, sweat; elevated blood

sugar; and elevated erythrocyte sedimentation rate.

Eyes

Eye pigmentation may arise due to CFZ crystal deposits.

Central Nervous System (CNS)

CNS symptoms include headache, dizziness, drowsiness, fatigue, and taste disorder. Some patients

developed depression because of the skin discoloration.


39

Skin

Pigmentation from pink to brownish-black was observed in75 to 100% of the patients within a few

weeks of treatment; ichthyosis and dryness (8-28%); rash antipruritic (1-5%). Reddish black reversible

skin discoloration may take several months or years to disappear after the conclusion of therapy.

Non-clinical Toxicology[49]

Long-term carcinogenicity studies in animals have not been conducted with Lamprene®. Results of

mutagenicity studies were negative. There is some evidence of clastogenic potential in mice.

Impaired female fertility (reduced number of offspring and lower proportion of implantations) was

observed in one study in rats receiving Lamprene® (from 9 weeks before mating until weaning) at 50

mg/kg/day, equivalent to about 2.4 times the maximum recommended clinical dose. No non-clinical

data on male fertility are available.

No specific data are available on the treatment of over dosage with Lamprene®. However, in the event

of an overdose, the stomach should be emptied by inducing vomiting or by gastric lavage, and

supportive symptomatic treatment should be employed. Lamprene® is contraindicated in patients with

known hypersensitivity to CFZ or any of the excipients of Lamprene®.

Risk Summary (use in specific populations) There is no data with Lamprene® use in pregnant women to inform associated risk. Retardation of fetal

skull ossification, increased incidences of abortions and stillbirths, and impaired neonatal survival were

observed in mice following prenatal exposure to Lamprene® at 25 mg/kg, equivalent to the 0.6 times

maximum recommended human daily dose (200 mg), based on body surface area comparisons.

Pregnant women should be advised of the potential risk to the fetus.

The main adverse effect of CFZ is skin discoloration or darkening occurring in the majority of patients

receiving the drug. This may be distressing to patients and lead to stigmatization. Reddish black

reversible skin discoloration may take several months or years to disappear after the conclusion of

therapy[9].

Occupational Exposure Limits Calculations

The occupational exposure limit (OEL) of CFZ is 7 µg/m3[50].

Using the uncertainty/safety factor method (now referred to as adjustment factor method) for

determining OELs as presented by Sergent and Kirk[51] while considering the uncertainty factors


40

discussed by Naumann and Weidemann[52], Sargent, et al[53] and as outlined in the new ISPE Risk-Mapp®

Baseline Guide[54], the OEL for CFZ can be calculated as follows:

OEL = PoD(mg/day) / (AF)(SS)()(vol)

OEL = 60(mg/day) / (225)(4)(1)(10 m3) = 0.0066 mg/m3 = 6.7 µg/m3*

* As an Industrial hygiene standard practice, this value will be rounded to 7 µg/m3.

Where:

• AF = Adjustment factor

o 3 for low therapeutics dose to no observed effect level (NOEL)extrapolation,

o 5 for human variability,

o 1 for chronic exposure,

o 1 for possible irreversible effects,

o 3 for missing data (carcinogenicity and mixed genotoxicity), and

o 5 for potential reproductive effects and potential severe effects (QT prolongation)

• SS = steady state based on elimination half-life = 4

• = pharmacokinetic factor based on bioavailability = 1

• Vol = volume of air breathed in a shift = 10 m3

This OEL is based on information currently available and is designed to be an 8-hour a day, 40-hour a

week, airborne concentration, which nearly all workers may be repeatedly exposed to day-after-day

without adverse health effects. It does not take into account hyper-sensitive or otherwise unusually

responsive individuals or persons with hypersensitivity to CFZ, whose symptoms may be exacerbated by

exposure to this drug.

Control Band Assignment

Based on numerical OEL, CFZ is assigned as a Category 3substance in the Affygility Solutions’ 5-band

control banding system[60]. This converts to a Category 3 in a traditional 4-band system.


41

Table 9. Band System for Hazardous Chemicals

Band No.

Target Range of Exposure Concentration

Hazard Group Control

1 >1 to 10 mg/m3 dust >50 to 500 ppm vapor

Skin and eye irritation Use good industrial hygiene practice and general ventilation

2 >0.1 to 1 mg/m3 dust >5 to 50 ppm vapor

Harmful on single exposure Use local exhaust ventilation

3 >0.01 to 0.1 mg/m3 dust >0.5 to 5 ppm vapor

Severely irritating and corrosive

Enclose the process

4 <0.01 mg/m3 dust <0.5 ppm vapour

Very toxic in single exposure, reproductive hazard, sensitizer

Seek expert advice

Industrial Hygiene Sampling and Analytical Methods

Precautions for Safe Handling Based on information captured in Table 9, the following precautions are recommended for safe

handling of CFZ. Avoid contact with skin and eyes. Avoid formation of dust and aerosols. Use adequate

general or local exhaust ventilation to keep airborne concentrations below the permissible exposure

limits. Normal measures for preventive fire protection are recommended.

Acceptable Daily Exposure Calculations

The Acceptable Daily Exposure (ADE) of CFZ is30µg/day[50].

The ADE is the daily dose of a substance, below which no adverse effects are anticipated by any route,

even if exposure occurs over a lifetime. The reported ADE is based on an adult human body. If it has to

be identified in paediatrics, then additional adjustments need to be applied.

Using the uncertainty/modifying factor method (now referred to as adjustment factor method) for

determining Acceptable Daily Exposure (ADE) values as presented in the revised ISPE Risk-Mapp®

Baseline Guide [54, 55]while also considering the methods discussed by Sergant, et al. [53],and the

European Medicines Agency[56], an ADE for CFZ can be calculated as follows:

ADE = (PoD mg/day) / AFC x MF x PK

ADE = 60 mg/day /90 x5 x 4= 0.033 mg/day = 33 µg/ day*

* As an industrial hygiene practice, this value will be rounded to 30 µg/day.


42

Where:

• PoD = Point of Departure

• AFc= Composite Adjustment Factor (AFA X AFH X AFS X AFL X AFD)

• AFA= Interspecies variability

• AFH= Intraspecies variability

• AFS= Study duration

• AFL= Low dose extrapolation

• AFD= Database completeness

• MF = Modifying Factor (severity)

• PK = Pharmacokinetic adjustment(s)

Choice of Uncertainty and Modifying Factors

In calculating the ADE value for CFZ, a composite AFc of 90 was used. The choice was made to account

for the following factors:

1. The low oral daily therapeutic dose was selected as the point of departure and this dose is

based on the human data[50]; therefore, a factor of 1 was applied to AFA.

2. In the absence of specific intraspecies data variability, a conservative default factor of 10 is

applied to AFH to extrapolate from the general human population to sensitive subgroups, such

as pediatric and geriatric patient.

3. The data reviewed was based on sub-chronic studies; therefore, to extrapolate for chronic

exposure, a factor of 1 was applied to AFS.

4. A minimum daily therapeutic dose has been established and an adjustment factor of 10 was

already applied in AFH to protect sensitive subgroups. Therefore, to extrapolate from a low

therapeutic dose to a probable NOEL, an adjustment factor of 3 is applied to AFL.

5. The information database was in complete, missing carcinogenicity data and mixed genotoxicity

data; therefore, an adjustment factor of 3 was applied to AFD.

6. CFZ was associated with potential reproductive effects and potentially severe effects including

QT prolongation; therefore, a modifying factor (MF) of 5 was applied.

7. A composite PK factor of 4 was used to account for a long elimination half-life and variable

human pharmacokinetics.


43

Information to Patients

The Product monograph of Lamprene® provides the following information to patients undergoing

treatment with CFZ:

• Inform patients to take Lamprene® with meals.

• Inform patients to report abdominal pain or other gastrointestinal symptoms, such as nausea or

vomiting, to their healthcare provider.

• Inform patients that Lamprene® frequently causes a red to brownish-black discoloration of the

skin as well as discoloration of the conjunctivae, tears, sweat, sputum, urine, and feces. Advice

patients that skin discoloration may take several months or years to resolve after the conclusion

of therapy with Lamprene®.

• Inform patients that skin discoloration may result in psychological effects and advise them to

report any symptoms of depression or suicidal ideation.

• Advice females of reproductive age to use effective contraception while taking Lamprene® and

for at least 4 months after stopping treatment with Lamprene®. It is also recommended that

they have a pregnancy test prior to starting treatment with Lamprene®.

• Advice males taking Lamprene® to use a condom during intercourse while in treatment and for

at least 4 months after stopping treatment.

• Inform patients of the importance of compliance with the prescribed drug regimen in order to

prevent drug resistance. Irregularity in administration of medication and poor compliance can

lead to delayed and incomplete cure, and could result in infecting other people. Poor

compliance can result in disease progression and ultimately result in the development of

disabilities and deformities. Whenever possible, ensure that non-compliant patients receive

adequate assessment, health education, and supervised treatment.

• Instruct patients on how to recognize signs and symptoms of inflammatory reactions and

relapses during and following completion of treatment.

• Instruct patients on the importance of immediately reporting the earliest manifestations of

inflammatory reactions and relapse signs to their healthcare provider.

44

Manufacturing of Dosage Form

CFZ is a reddish-brown powder that makes cleaning of the manufacturing equipment very difficult.

Therefore, dedicated manufacturing equipment and processing area for CFZ is recommended.

As already discussed in section Formulation barriers to entry, Lamprene®, the Reference Listed Drug

(RLD), has a shelf life of 60 months. The capsule shell consists of gelatin, which is known to be sensitive

to humidity. Hence, the preparation is supplied in a humidity-resistant container. It may be prudent to

control relative humidity (RH) during manufacturing steps such as dispensing and dry mixing, where API

is directly exposed to the environment. The manufacturing facility should be maintained with optimum

temperature and RH conditions for the reasons already detailed out in section. Standard cleaning

protocols and good manufacturing practices should be strictly followed. Lamprene® product

monograph reports use of micronized API suspended in oily-wax base. Use of controlled particle size of

micronized CFZ has to be considered to manufacture a formulation equivalent to the RLD while also

ensuring batch-to-batch uniformity.

Facility Design& HVAC Requirements

Pharmaceutical facilities are closely inspected by the WHO-PQ inspectors, who require manufacturing

companies to conform to cGMP (current Good Manufacturing Practices). According to cGMP

regulations drug manufacturers, processors, and packagers are required to take proactive steps to

ensure that their products are safe, pure, and effective. GMP regulations require a quality approach to

manufacturing, enabling companies to minimize or eliminate instances of contamination, mix ups, and

errors.

The WHO Guidance for HVAC [57]Services covers a number of issues starting with the selection of

building materials and finishes; the flow of equipment, personnel, and products; determination of key

parameters like temperature, humidity, pressures, filtration, and airflow; and classification of clean

rooms. It also governs the level of control of various parameters for quality assurance, regulating the

acceptance criteria, validation of the facility, and documentation for operation and maintenance.

An HVAC system performs four basic functions[57]:

1. Controls airborne particles, dust, and micro-organisms– Through air filtration that uses high

efficiency particulate air (HEPA) filters.

2. Maintains room pressure differentials (ΔP) – Areas that must remain “cleaner” than surrounding

areas must be kept under a “positive” pressurization, meaning that air flow must be from the

Manufacturing of Dosage Form

45

“cleaner” area towards the adjoining space (through doors or other openings) to reduce the

chance of airborne contamination. This is achieved by the HVAC system introducing more air

into the “cleaner” space than is mechanically removed from that same space.

3. Maintains space moisture (RH) – Humidity is controlled by cooling air to dew point temperatures

or by using desiccant dehumidifiers. Humidity can affect the efficacy and stability of drugs and is

sometimes important to effectively mold the tablets.

4. Maintains space temperature– Temperature can affect production directly or indirectly by

fostering the growth of microbial contaminants on workers. The temperature also has to be

maintained within the product’s labeled storage condition.

Manufacturing Process The capsules for CFZ are recommended to be prepared under controlled temperature, light, and

humidity conditions. Major unit operations in fabrication include milling, sieving, preparation of fill

material, filling, and packing of soft gelatin capsules (See Figure 11). The manufacturing process is

discussed in the Dosage Form section.

It is important to control the quality of the gelatin raw material to produce soft gelatin capsules.

Specifications of bloom strength, viscosity, and iron content are conventionally applied to control the

quality of the finished product.

Process Controls The API aspects for manufacturing of CFZ dosage form can include parameters of polymorphic form or

particle size distribution (D90 value using standard equipment like Malvern Mastersizer® – which is a

laser diffraction technique), to ensure consistent performance of the drug product.

Based on API properties, the following may be may be Critical Quality Attributes (CQAs) for CFZ

capsules particle size, chemical stability, photostability, moisture content, cross linking of gelatine, and

dissolution of soft gelatine capsule. The pharmaceutical company should understand the role of

formulation factors and process parameters on the CQAs. A risk assessment using a standard tool like

Failure Mode Effect Analysis (FMEA) can be used to identify the most important parameters. A control

strategy that consists of raw material specifications, process monitoring, IPQC, and finished product

testing must be developed.

The quality of soft gelatine capsules should be controlled with additional IPQC tests that assess the

leakage of soft gelatine capsules by visual inspection or vacuum test and moisture content in dried soft

gelatine capsules.

46

Cleaning Validation

During validation, companies must demonstrate that the routine cleaning procedure of the

manufacturing equipment, are able to limit potential carryover of CFZ to an acceptable level[58]. The

limits established must be calculated based on sound scientific rational.

This EMA draft position is an improvement over the ISPE Risk-MaPP Guide. This EMA draft position

allows manufacturers to justify using traditional cleaning limit approaches and could allow

manufacturers to leverage their existing CV work to meet the recent Health Based Exposure Levels

(HBEL) based cleaning limit requirements.

For the risk assessment-based approach to Cleaning Validation (CV), the manufacturer could use the

strategy matrix approach. The following points may be considered for the approach:

• Create a grouping of products or APIs manufactured on the same equipment train and cleaned

using the same validated cleaning method.

• Risk Identification: To minimize CV study to one product or API for each equipment train, score

the risk as high before the mitigation strategy in the protocol.

• Mitigation Strategy: Identify the “worst case” product/API accounting for the solubility, hardest

to clean product/API, and the most toxic candidate. Develop and validate a detailed cleaning

procedure for that product/API. Score the risk as theoretically low in the protocol if your CV

strategy would be successful with the worst-case product/API. If the CV strategy meets the

acceptance criteria, you have successfully used the risk-based approach.

Options like manual and automatic cleaning are available depending on the individual manufacturing

facility. Based on available infrastructure and expertise, a suitable cleaning method may be adopted.

The acceptance criteria for cleaning of equipment, preferably should be based on the ADE or PDE value

calculations whenever this data is available[59]. In many cases industrial hygienists and toxicologists OEL

will define OELs for APIs, Intermediates, and Industrial Chemicals. The OEL data is then used to define

containment measures such that operators are adequately protected while working with the chemicals.

The OEL data can also be used to calculate the ADE / PDE for setting the acceptance criteria for

cleaning of equipment. The ADE and OEL of CFZ are 30 µg/day and 7 µg/m3 respectively.

47

Conclusion

CFZ is a drug used in the treatment of leprosy and tuberculosis. It is currently the only core second‐line

medicine for the treatment of multidrug-resistant tuberculosis (MDR-TB), which is not yet included in

the WHO Model List of Essential Medicines as an anti-tuberculosis medicine. CFZ is a reddish-brown

powder and, as such, proper cleaning procedures are critical to control and demonstrate prevention of

cross-contamination. In practice, this requires that the product be manufactured using dedicated

equipment and rooms. This requirement can be financially, technically, and operationally challenging for

manufacturers interested in such low-margin products. The API is susceptible to temperature and light

and suggested to be formulated in controlled conditions. The RLD formulation dosage form supplied

commercially is a soft gelatin capsule, which is sensitive to moisture and is therefore recommended to

be stored under controlled conditions.

This Product Information Report (PIR) summarizes the available literature and provides expert scientific

analysis of the physicochemical, pharmaceutics, pharmacokinetics, and toxicological properties. The PIR

also provides a summary of literature API synthesis, analytics, and formulation and provides

recommendations about CFZ capsule manufacturing. The report is expected to provide critical

information and guidance to manufacturers, as well as stakeholders concerned with access and supply

of priority essential medicines.

A dosage form switch from the soft gel to an OSD form concept note is also provided in Appendix 1

based on theoretical considerations, including development activities, i.e., pre--formulation studies,

compatibility studies, prototype development, scale up, and commercialization. Appropriate

experimental evidence would be required to substantiate theoretical discussion.

48

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46. Guidance document, WHO/PQT medicines, Notes on the Design of Bioequivalence Study:Clofazimine , 2016,https://extranet.who.int/prequal/sites/default/files/documents/133%20BE%20clofazimine_Nov%202016.pdf, Accessed on 01.07.2019.

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Appendix 1

Concept Note for Development of Tablet/Capsule Dosage Form for Clofazimine (CFZ)

Introduction The current drug product of CFZ is a soft gelatin capsule prepared for oral administration. Oral

absorption of a drug molecule is governed by fundamental properties like aqueous solubility, intestinal

permeability, and first pass metabolism through the liver. A formulation switch from a soft gelatin

capsule formulation to an OSD (Oral Solid Dose – tablet or capsule) has been herein proposed

by assessment of pre-formulation parameters such as- solubility, pH-solubility profile, intestinal

permeability, BCS class, log P, pKa value, solid-state stability, pH stability profile, and melting point.

Parameters like Tdiss (dissolution time) and maximum absorbable dose (Dabs) of the drug can be

calculated from the values of aqueous solubility and permeability. They can help identify whether the

drug molecule has ‘solubility limited’ or ‘dissolution rate limited’ oral bioavailability.

Careful assessment of the qualitative composition of currently marketed soft gelatin capsule facilitated

the understanding of challenges in this drug’s oral dosage form. It also provided information on the

performance altering excipients used in the formulation. A strategy to switch the formulation to OSD

(tablet/capsule) has been conceptualized, and accounts for the pre-formulation profile, qualitative

composition of the commercially available RLD (Reference Listed Drug) soft gelatin capsule, and

additional parameters like compactability and flow behavior. The need for solubilization enhancement

strategy such as the use of particle size reduction, the use of surfactant and enabling technologies (e.g.

amorphous solid dispersion or lipidic systems), has been suggested based on log P (Oil/Water Partition

Coefficient), the API melting point, and the dose.

This section provides a theoretical concept note and development guidance of the soft gel to OSD

switch including development activities, (i.e. pre--formulation studies, compatibility studies, prototype

development, scale up, and commercialization). Appropriate experimental evidence would be required

to substantiate theoretical discussion.

Macleods Pharma had submitted an application for CFZ 50 mg and 100 mg tablets to WHO PQ. As per

‘List of Tuberculosis Pharmaceutical Products classified according to the Global Fund Quality Assurance

Policy’i.The Expert Review Panel (ERP) has reviewed the application but more details are not available in

public domain.

Appendix 1

53

Pre-Formulation Properties The following (Table 1) has important CFZ pre-formulation properties that help identify the

development challenges of oral solid dosage form like tablet or capsule.

Table 1. Pre-Formulation Properties of CFZ

No. Pre-formulation

Parameter Value Significance

1 Log P 7.66 Highly lipophilic molecule likely to have poor solvation in water

2 pKa Strongest acid Strongest acid

8.5 16.18

Likely to form hydrochloride salt in stomach; these pKa values are not relevant in the pH range encountered in the GIT (Gastro-Intestinal Tract)

3 Melting point 201 to 215°C Relatively high melting point may limit its solubility in water and lipidic excipients

4 Water solubility 0.225 µg/mL Practically insoluble in water

5 Permeability (Peff) 4.38 X 10-4 cm/s Highly permeable

6 Photo-stability Reported to be sensitive to photo-degradation

Need to adopt strategies of photoprotection during manufacturing and primary packaging material selection

7 Flow properties May vary based on particle size and morphology of the API

Unlikely to pose a major challenge due to low dose of the API in the final dosage form. It would be possible to achieve required flow compaction by selection of appropriate excipients.

8 Compactability

CFZ has low solubility and high permeability and is classified as BCS class II. It is a highly lipophilic

compound and has a high melting point. Both these properties confer extremely low solubility in water

due to poor solvation and strong crystal lattice interactions. Highly lipophilic molecules with low melting

points can dissolve in lipids/oils. But lipophilic molecules with high melting point do not dissolve in

lipids/oils because strong inter molecular interactions do not allow molecules to escape the crystal

lattice and dissolve in lipid/oil. Molecules with high log P are typically good candidates for lipidic

systems (self-emulsifying drug delivery systems) but the high melting point of CFZ limits its solubility,

even in oils and lipids.

Low drug solubility is a primary rate limiting step in absorption of CFZii. Because of lipophilicity and

poor water solubility, it is administered as a microcrystalline suspension in an oil wax base in order to

improve its absorption. In humans, significant food effect has been reported for CFZiii.


54

RLD Formulation Lamprene® 50 and 100mg are available as micronized API suspended in an oil-wax vehicle and filled

into soft gelatin capsules. The function of excipients present in Lamprene® has been described in Table

6 of the PIR. The presence of an oil and propylene glycol vehicle along with lecithin likely provides

efficient wetting and solubilization of CFZ in GIT. It is pertinent to mention that CFZ is a weakly basic

molecule and is likely to form a hydrochloride salt in the gastric contents, which may enhance its

solubility. Overall, this could be governed by the inherent properties of the API like pKa, PSD (particle

size distribution), and solid form. This may also lead to inter subject variability due to the conditions like

achlorhydria and pH changes in the stomach due to feeding state.

Determination of Limiting Parameter for Oral Bioavailability Drugs may have ‘solubility limited’ or ‘dissolution rate limited’ oral bioavailability. The following

equationsiv were used to identify the rate limiting step in oral bioavailability.

Tdiss = hr0/3DCS and

Dabs = PeffCSA (Tsi)

Where:

• A = Effective intestinal surface area for absorption

• CS = Aqueous solubility

• D = Diffusion coefficient

• h = Diffusion layer thickness

• Peff= Effective human intestinal permeability

• r0 = Initial radius of particles

• (Tsi) = Mean small intestinal transit time

• = Density of drug

A drug molecule has a dissolution-rate-limited oral bioavailability if Tdiss of un-micronized drug is greater

than (>) 199minutes. Solubility-limited oral bioavailability is inferred if Tdiss of micronized drug is less than

(<) 50 minutes and Dabs is < the dose.

Both dissolution-rate and solubility limited bioavailability is inferred when Tdiss of a micronized drug

molecule is much greater than (>>) 199 minutes and Dabs is much less than (<<) dose.

Dabs for CFZ was calculated as 0.94135 mg (which is << the dose). Tdiss for un-micronized drug

(diameter-100 µm) and micronized drug (diameter-5 µm) was calculated as 96296.3 min and 4814.8 min

respectively. Hence, Tdiss micronized drug is >>199 min.

Appendix 1

55

As is evident from the above calculations, CFZ has both dissolution-rate and solubility limited oral

bioavailability. In such cases, micronization of the API may be insufficient to provide optimal

bioavailability hence it may be necessary to use enabling technologies like amorphous solid dispersion

(ASD), nanocrystals or inclusion complexation. The proceeding section discuss the appropriateness of

these enabling technologies for CFZ.

Enabling Technologies Discussion on use of enabling technologies have been presented in a question and answer (Q&A)

format.

Question 1. Is a salt form with enhanced solubility feasible?

Answer 1: CFZ is a weakly basic compound with pKa value of 8.5. Theoretically, it can form a salt with

acidic counter ions having more than 3 units difference in the pKa value ( pKa rule). Formation of new

salt can change the biopharmaceutical and pharmacological profile of the molecule which would require

additional toxicological, preclinical, and clinical studies to establish safety and efficacy of the new salt of

the API. In view of significantly enhanced regulatory burden, salt formation as a strategy for the

enhancement of oral bioavailability will not be discussed further

Question 2. Can a lipidic system be used to enhance oral bioavailability?

Answer 2: Lipidic systems are suitable for molecules that have (i) high log P and low melting point or (ii)

high log P, high melting point, and low dose. CFZ has a high log P, high melting point, and an

intermediate dose of 50 and 100 mg, and hence may not be suitable for development of lipidic system.

It is reported that high log P molecules with a high melting point are not soluble in oils despite their

favorable lipophilicity. This rationale is substantiated by the fact that current marketed formulation has

CFZ suspended (not solubilized) in an oil-wax vehicle.

Question 3. Can a conventional OSD be developed?

Answer 3. Micronization or nanonization of the API can be adopted to develop an oral solid dosage

form that is bioequivalent to the currently marketed product. Additionally, a surfactant can be

incorporated to enhance the wettability and increase dissolution kinetics. However, these strategies do

not significantly enhance the apparent solubility and hence may have limited use for enhancing the oral

bioavailability of CFZ. Employing ASD technology (see below) can be a more effective tool for

developing an OSD dosage form. The goal of achieving a bioequivalent OSD to Lamprene® should be

kept in focus whatever technology is employed to enable filing of a WHO PQ or and ANDA application

however.

Question 4. Can enabling technologies like ASD help improve oral bioavailability?

Answer 4. Polymeric ASD are an elegant way of improving aqueous solubility and oral bioavailability of

BCS class II and IV molecules. Numerous products using ASD have been commercialized. CFZ with a


56

high log P, a high melting point, and an intermediate dose can be developed as polymeric ASD. Two

technologies (i.e., hot melt extrusion and spray drying) are more commonly used for manufacturing of

ASD. With its high melting point, CFZ would be suitable for spray drying for the development of ASD.

Polymers like HPMC (hydroxyl propyl methyl cellulose), PVP-VA (poly vinyl pyrrolidone – vinyl acetate),

HPMC AS (hydroxyl propyl methyl cellulose acetate succinate), and PVP (poly vinyl pyrrolidone) can be

evaluated for or the development of ASD. The formulation is expected to provide solubility and

dissolution rate benefits. Development of ASD for CFZ may also help in overcoming variability in oral

absorption of CFZ in fed or fasted state. The developed ASD should be carefully assessed for physical

stability, chemical stability, residual solvent, and compaction mediated re-crystallization.

The following is a decision treevthat can be used for the development of ASD formulation.

Decision Tree for Selection of ASD candidate

Conclusion An OSD bioequivalent to Lamprene® can be developed using micronization or nanonization of the API.

ASD would be the preferred formulation approach for development of an OSD with enhanced oral

bioavailability of CFZ.

It is to be noted that Macleods Pharma had submitted an application for CFZ 50 mg and 100 mg tablets

to WHO PQ, as per ‘List of Tuberculosis Pharmaceutical Products classified according to the Global

Fund Quality Assurance Policy’vi. The ERP reviewed the application, but more details are not available in

public domain.

Appendix 1

57

References

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ii Yawalkar SJ, Vischer W. Lamprene (clofazimine) in leprosy. Basic information. Lepr Rev 1979; 50: 135–44.

iii Schaad-Lanyi Z, Dieterle W, Dubois JP et al. Pharmacokinetics of clofazimine in healthy volunteers. Int J Lepr Other Mycobact Dis 1987; 55: 9–15.

iv Lawrence X. Yu. An integrated model for determining causes of poor drug absorption. Phar Res 1999; 16 (12): 1883-87

v Engers D, Newman A et al., J Pharm Sci. 2010: 99(9):3901-22

vi List of Tuberculosis Pharmaceutical Products classified according to the Global Fund Quality Assurance Policy. April 2019. https://www.theglobalfund.org/media/4757/psm_productstb_list_en.pdf?u=636917017340000000, Accessed on 01.07.2019.

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