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CLOFAZIMINE

Caitriona M . ODriscoll and Owen I . Corrigan

University of Dublin
Department of Pharmaceutics
School of Pharmacy
Trinity College, Dublin, Ireland

ANALYTICAL PROFILES OF DRUG SUBSTANCES


AND EXClPlENTS - VOLUME 21

75

Copyright 0 1992 by Academic Press, Inc


All rights of reproduction reserved in any form

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CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

CLOFAZIMINE
Caitriona M. ODriscoll and Owen I. Corrigan
University of Dublin, Department of Pharmaceutics,
School of Pharmacy, Trinity College Dublin, Ireland.

1.

2.

3.
4,

5.

Introduction
Description
2.1
Structural and Molecular Formulas and Molecular
Weight
2.2
Nomenclature
2.3
Official Compendia
2.4
Other Compendia
Synthesis
Physical Properties
Ultraiiolet Absorbance Spectrum
4.1
Infrared Absorbance Spectrum
4.2
Mass Spectrum
4.3
Proton Nuclear Magnetic Resonance Spectrum
4.4
Carbon-13 Nuclear Magnetic Resonance Spectrum
4.5
X-Ray Diffraction
4.6
Melting Point
4.7
Differential Scanning Calorimetry
4.8
Dissociation Constants
4.9
4.10 Solubilities
4.11 Par tition Coefficients
Methods of Analysis
5.1
Elemental Analysis
5.2 Identification
5.3 Ultraviolet and Visible Spectrophotometry
5.4
Spectrofluorometric Analysis

CLOFAZIMINE

6.
7.

5.5
Thin Layer Chromatography
5.6
High Pressure Liquid Chromatography.
Pharmacokinetics
6.1
Bioavailability Considerations
6.2
Distribution, Metabolism and Elimination
Pharmacology
7.1
Mechanisms of Action
7.2
Structure - Activity Relationships
7.3
Toxicity
7.4
Dose Schedules
Acknowledgements
References

1. INTRODUCTION
Clofazimine is active against Mycobacterium leprae and is used
clinically to treat leprosy (Hansen's disease). It was synthesised in
1957 by Barry et al., Laboratories of the Medical Research Council
of Ireland, Trinity College Dublin. The precise mechanism of the
antileprotic action of clofazimine has not been established. The
World Health Organisation classify clofazimine as an "essential
drug" and recommend its use, in combination, with other agents
to treat all cases of leprosy (WHO, 1982).
Clofazimine is also used to treat Mycobacteriurn avium
infections which frequently occur in patients with AIDS (acquired
immunodeficiency syndrome), (Masur et al., 1987; Woods and
Washington, 1987; Gangadharam et al., 1988; Lindholm - Levy
and Heifets, 1988; Young, 1988).
Clofazimine also displays anti inflammatory activity which is
clinically useful in controlling erythema nodosum leprosum
(ENL) reactions which occur in multibacillary forms of leprosy
(Gidoh and Tsutsumi, 1979; Yawalkar and Vischer, 1979; Browne
et al., 1981). A study using animal models of rheumatoid arthritis
has indicated that clofazimine may be potentially useful to treat
this disease (Currey and Fowler, 1972). Although the exact
mechanism of clofazimine mediated anti-inflammatory activity is
unknown, it may be related to the ability of the drug to increase

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CAITRIONA M. ODRISCOU AND OWEN 1. CORRlGAN

the synthesis of anti-inflammatory immunosuppressive


prostaglandin E2 (PGE2) by human polymorphonuclear leucocytes
(Anderson, 1985;Zeis et al., 1987;Yawalkar, 1988).

2. DESCRIPTION

Clofazimine is a dark red or orange - red fine powder, odourless


or almost odourless.
2.1 Structural and Molecular Formulas and Molecular Weight

Q
CI

Molecular Formula:

C27H22C12N4

Molecular Weight:

473.4

CLOFAZIMINE

19

2.2 Nomenclature
2.21 Generic Name
Clofazimine (BAN, USAN, rI")
2.22 Chemical Names

3-(4-chloroanilino)-10-(4-ehlorophenyl)-2,1
O-dihydrophenazin-2ylideneisopropylamine.
N,5-Bis(4-chlorophenyl)-3,5-dihydro-3-[(l-methylethyl)
iminol-2phenazinamine,
or 3-(p-chloroanilino)-1O-(p-chlorophenyl)-2,10-dihydro2(isopropylimino) phenazine,
or 2-(4-chloroanilino)-3-isopropylimino-5-(4-chlorophenyl)-3,5dihydrophen azine,
or 2-p-chloroanilino-5-p-chlorophenyl-3,5-dihydro-3isopropy liminophenazine.
2.23 Trade name
Clofazimine is marketed by Ciba Geigy under the proprietary
name "Lamprene".
2.24 Other Names, Abbreviations and Drug Codes
Riminophenazine, 8663, G30320, NSC 141046, chemical abstracts
service registry number (CAS no.) 2030 - 63-9.
2.3 Official Compendia
A monograph on clofazimine is included in the British
Pharmacoepia and the Indian Pharmacoepia.
2.4 Other Compendia
Clofazimine is included in the Merck Index (19891, the
Pharmaceutical Codex (1979), and in Martindale (1989). Clarke
(1986) gives a useful summary of physical and chemical data.

CAITRIONA M. O'DRISCOU AND OWEN 1. CORRIGAN

80

i, ii

+
NHR

NHz

('1

R = aryl

iii

NHR

NHR

(3)
R = Ph, 4-CI-C6H4-

iv

I V

Reagents: i, FeCl3, H+; ii, NH3; iii, R*NH2, alkyamines;


iv, benzoquinone/carbonyl compound RkOR3; v, Pt @/H or Pt/C (lO%)/H2;
vi, air; vii, Pd/C (lO%)/Hz.

Figure 1. Principal synthetic routes to riminophenazines (Hooper,


1987)
3. SYNTHESIS

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. The modifications have
been summarised by Hooper (1987) (Figure I) as follows; N-aryl
ortho -phenylenediamines (1) undergo regiospecific oxidative
dimerization to yield the parent iminophenazines (2) which react
further with alkylamines to give substituted iminophenazines (3).
Alternatively, oxidation with benzoquinone in the presence of a

CLOFAZIMINE

81

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). A
more 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.
4. PHYSICAL PROPERTIES
4.1 Ultraviolet Absorbance Spectrum
The ultraviolet spectrum of clofazimine (0.001% w/v) is shown
in Figure 2. The spectrum was obtained using a Hewlet Packard
845 2A diode array UV visible spectrophotometer and 1 em quartz
cells. The spectrum, in the range 230 to 600nm, in 0.01m
methanolic hydrochloric acid, exhibits two maxima, at 284nm and
486nm. The absorbance at 284nm is about 1.30 and at 486nm is
about 0.64.

220

300

400

500

WAVELENGTH

Figure 2. Ultraviolet spectrum of clofazimine.

1
600

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CAlTRlONA M. ODRISCOLL AND OWEN I. CORRIGAN

4.2 Infrared Absorbance Spectrum


The infrared absorbance spectrum of clofazimine is shown in
Figure 3. The spectrum was recorded with a Nicolet 5ZDX FT-IR
spectrophotometer, from a compressed potassium bromide disc.
Structural assignments for some of the characteristic absorption
bands in the spectrum are listed in Table I.
Table I. Infrared assignments for clofazimine

W avenumber (cm-l)
1587,1560,1510,1460,1300
1389,1360,1130

Assignment
aromatic CH stretching
CH(CH3)2 stretching

4.3 Mass Spectrum


The mass spectrum of clofazimine, shown in Figure 4, 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. Major peaks were detected at m / z (%) 474
(66.17),473 (36.22), 472 (1001,457 (93.04), 455 (70.87), 456 (24.13),431
(19.57), 414 (30.43), 380 (22.17), 345 (17.83), 331 (30.43).
4.4 Proton Nuclear Magnetic Resonance Spectrum (IH-NMR)
The IH-NMR spectrum of clofazimine, shown in Figure 5, was
obtained in deuterated chloroform containing tetramethylsilane
(TMS) as internal standard, using a Joel GX- 270 MHz instrument.
A 2D COSY spectrum was also obtained (Figure 6a and 6b). Figure
6b is an expansion showing coupling in the aromatic regions.
4.5 Carbon - 13 Nuclear Magnetic Resonance Spectrum
The carbon-13 NMR spectrum of clofazimine was obtained in
deuterated chloroform containing TMS as internal standard using
a Joel GX-270 MHz instrument at a frequency of 67 MHz. The
carbon-13 NMR spectrum, with DEPT, is shown in Figure 7.

CLOFAZIMINE

Figure 3.

83

Infrared spectrum of clofazimine.

Y
0

. . .

2000

I BOO

1so0

1400

1200
1000
W ~ v m u n b r r Cpm-1)

800

SO0

400

84

Figure 4.

CAlTRlONA M . O'DRISCOLL AND OWEN 1. CORRIGAN

Electron-impact mass spectrum of clofazimine.

CLOFAZIMlNE

Figure 5 .

Proton nuclear magnetic resonance spectrum of


clofazimine.

85

86

Figure 6a.

CAITRIONA M. O'DRISCOLL AND OWEN I. CORRICAN

2-D proton nuclear magnetic resonance spectrum of


clofazimine.

CLOFAZIMINE

Figure 6b.

2-D proton nuclear magnetic resonance spectrum of


clofazimine.

87

88

Figure 7.

CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN

l3c1 nuclear magnetic resonance spectrum of clofazimine,


with DEPT.

CLOFAZIMINE

89

4.6 X-Ray Diffraction


The powder X-ray diffraction pattern of clofazimine was
obtained on a Siemens D-500 X-ray diffractometer, using a Cu
X-ray tube, at 40 kV and 40 mA. The diffraction pattern is shown
in Figure 8, indicating the crystalline nature of clofazimine.
Rychlewska et al. (1985) reported two different crystalline forms
of clofazimine, a monoclinic form with a density of 1.3 g cm-3, and
a triclinic modification with a density of 1.29 g cm-3. The former
was prepared by recrystallization from acetone, and the latter by
recrystallization from 12 N-methylformamide/acetone. Cell
constants were also calculated. The values obtained for the
monoclinic form were a = 7.788 A, b = 22.960 A, c = 13.362 A, p =
98.580. The values for the triclinic form were a = 10.507 A, b =
12.852 A, c = 9.601 A, a = 95.960, p = 97.220, y = 69.730.
4.7 Melting Point
Melting points reported in the literature are in the temperature
range of 210 - 215OC, with degradation (Barry et al. 1956a; Clarke
1986; Merck Index 1989; Pharmaceutical Codex 1979).
4.8 Differential Scanning Calorimetry (DSC)
The DSC thermogram of clofazimine obtained using a Mettler
DSC 20, scan speed lOOC min-1, is shown in Figure 9. A single
sharp melting endotherm was obtained with onset temperature at
214OC. This value is in good agreement with the melting points
previously published (Section 4.7). The estimate of the heat of
fusion (AH) was 740 joules/gram. However, with some samples
there was evidence of degradation on melting.
4.9 Dissociation Constant
The values for the dissociation constant reported for
clofazimine are summarised in Table 11.

YO

Figure 8.

CAlTRlONA M . O'DRISCOLL AND OWEN 1. CORRIGAN

X-ray powder diffraction pattern of clofazimine.

TWO

- THETA

IOEGREESI

CLOFAZIMINE

Figure 9.

200.0-

2lO.O-

220.0-

--

230.0-

240.0-

--

91

Differential scanning calorimetry thermogram of


clofazimine.

2 0 . 0 0 0 nU

CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

92

Table 11. Dissociation constant of clofazimine (pKa)


PKa

Method of determination

8.35

Not stated

8.37
8.51

Potentiometric
Spectropho tome tric

Reference
Morrison a n d Marley
(1976a)
Canavan et al. (1986)
Fahelelbom et al. (1989)

4.10 Solubilities
Clofazimine is practically insoluble in water, estimates in the
range of 1.03 - 0.49 pg ml-1, at 37OC, have been reported
(Fahelelbom, 1989; OReilly, 1991). It is soluble 1 in 700 of ethanol,
1 in 15 of chloroform, and 1 in 1000 of ether. It is also soluble in
dilute acetic acid and dimethylformamide (Clarke, 1986).

PH
Figure 10. pH solubility profile of clofazimine.

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CLOFAZIMINE

The effect of pH (range 5.15 - 7.8) on the solubility of


clofazimine, shown in Figure 10 (OReilly, 19911, is consistent
with the basic nature of the compound. The solubility of the drug
is 5.68 and 0.278 mg ml-1 x 10 -3 at pH 5.15 and 7.8 respectively.
Values for intrinsic solubility in the range of 2.0 - 2.3 x 10-5mg
ml-1 (Fahelelbom, 1989; OReilly, 1991) have been reported.
The solubility of clofazimine was enhanced in aqueous micellar
systems, containing both naturally occuring surfactants e.g bile
salts, and synthetic surfactants, e.g the non ionic Cremophor EL
and Triton X100, and the anionic sodium dodecyl sulphate. The
incorporation of fatty acids to form mixed micelles brought about a
further enhancement in drug solubility in the case of naturally
occuring surfactants (approximately 300 fold with sodium cholate:
linoleic acid relative to buffer). In contrast, with synthetic
surfactants this enhancement decreased (Fahelelbom et al., 1991;
ODriscoll et al., 1991).
4.11 Partition Coefficients (Log P)
Partition coefficients for clofazimine have been determined
using different solvents and temperatures. The data is
summarised in Table 111.
Table 111. Partition coefficients of clofazimine
Solvents
Octanol: water

Temp (OC) LogP

Isooctane: buffer pH 5.15

20

N-octanol: buffer pH 5.15

20

N-octanol: buffer pH 5.15


N-octanol: buffer pH 5.15
N-octanol: buffer pH 5.15

37
45
55

* Estimated

Reference

+7.48* Morrison and


Marley
(1976a,b)
5.01
Canavan et al.
(1986)
4.30
Quigley
et al. (1990)
4.40
Ibid
4.48
Ibid
4.54
Ibid

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CAITRIONA M.O'DRISCOLL AND OWEN 1. CORRIGAN

5. METHODS OF ANALYSIS
5.1 Elemental Analysis
Carbon
Hydrogen
Nitrogen
Chlorine

% Calculated

68.50
4.68
11.83
14.98

% Found

68.68
4.52
11.48
15.32

5.2 Identification
The B.P. (1988) outlines three methods of identification:
(A) By the infrared absorption spectrum, outlined in section 4.2.
(B) The light absorption, the UV spectrophotometry is described
in section 4.1.
(C) A colour test, dissolve 2mg clofazimine in 3ml of acetone
and add O.lml of hydrochloric acid, an intense violet colour is
produced. Add 0.5ml of 5M sodium hydroxide, the colour changes
to orange - red.
5.3 Ultraviolet and Visible Spectrophotometry
Quantitative ultraviolet analysis of clofazimine has been
performed, in a range of aqueous and nonaqueous media, at
280nm (Canavan et al., 1986; O'DriscolI et al., 1990a,b), and
colorimetrically at 482nm (Quigley et al., 1990).
A colormetric assay was developed by Barry et al. (1960) and
modified by Mansfield (1974) to analyse plasma and tissue levels of
clofazimine. The drug was extracted using benzene and
concentrated hydrochloric acid, and the absorption read at 540nm.
The limit of detection reported was 0.2pg/ml in plasma and
O.lmg/gram in tissue.
5.4 Spectrofluorometric Analysis
A fluorescent derivative of clofazimine was formed following
reduction with titanous chloride (Dill et al., 1970). The
fluorescence was measured at 366mp emission. The limits of

CLOFAZIMINE

95

detection reported for this method were in the range of 0.1 - 0.2
pg/ml in plasma (Banerjee et al., 1974; Levy, 1974).
5.5 Thin Layer Chromatography
A thin layer chromatographic (TLC) system suitable for
determination of clofazimine in plasma has been developed
(Lanyi and Dubois, 1982). The plasma samples were acidified
using acetate buffer pH 5 and extracted with toluene, evaporated to
dryness under nitrogen, reconstituted in toluene and applied to
the TLC plate. The adsorbent used was HPTLC silica gel 60. The
plates were developed in toluene - acetic acid - water (50 : 50 : 4),
allowed to stand for 30 min at room temperature, the Rf value of
clofazimine was 0.36. Detection and quantitation is carried out
using a densitometric method. The limit of detection reported for
this method was 5ng/g.
5.6 High Performance Liquid Chromatography
Gidoh et al. (1981) developed a high performance liquid
chromatographic (HPLC) method with ultraviolet detection to
separate and quantify clofazimine (287nm) from other antileprosy
drugs, dapsone and rifampicin, in serum on a pBondapak c18
column. This method involved a complicated extraction
procedure with the switching of 2 different mobile phase (i.e
acetonitrile - water, 20 : 80; and tetrahydrofluran - water containing
PIC B-5,50 : 50, the latter reagent contains 1 - pentanesulfonic acid
and glacial acetic acid) in order to allow complete resolution of
clofazimine from related components. The limit of detection for
this method was long ml-1. Recently a modification of this
technique was used to study clofazimine and its derivatives
(O'Sullivan et al., 1990).
Another HPLC method, was described by Peters et al. (1982), for
measuring clofazimine in plasma, with a limit sensitivity of 10 ng
ml-1. This method involved extraction of clofazimine into
organic solvents and quantifation on a reversed-phase Ultrasphere
- octyl column, using a mobile phase of 0.0425M phosphoric acid
in 81% methanol and UV detection at 285nm.
The gastrintestinal absorption of clofazimine, using a rat gut
perfusion technique, was determined by HPLC (O'Driscoll et al.,
1990a,b). The column used was Partisil lOPAC, the mobile phase

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CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

was ethanol : N-heptane (50 : 50) and detection was by UV at


283nm.The limit of sensitivity was 0.1pg ml-I.

6 . PHARMACOKINETICS
6.1 Bioavailability Considerations
Clofazimine absorption following oral administration is
incomplete and varies significantly from patient to patient.
Following administration as coarse crystals only about 20% is
absorbed, if however, the drug is given as a microcrystalline
suspension in an oil wax base an absorption rate of 70% can be
achieved (Yawalkar and Vischer, 1979).
The gastrointestinal absorption of clofazimine in the
anaestheised rat, using an in situ rat gut perfusion model (Komiya
et al., 19801, was enhanced by co-administration of simple and
mixed micellar systems (O'Reilly et al., 1988; O'Driscoll et al.,
1990a,b). The simple micellar systems included various bile salts,
and the synthetic emulgents, Cremophor EL (non ionic) and
sodium dodecyl sulphate (anionic). The mixed micelles were
formulated by the incorporation of various fatty acids. A mixed
micellar system containing sodium cholate: linoeleic acid
enhanced the rate of absorption of clofazimine by a factor of 840
compared to a buffered solution of the drug. The enhancements
were due to a combination of increased solubility and increased
membrane permeability. There is also evidence that clofazimine is
transported in part via the lymphatic system (Barry et al., 1960;
Atkinson et al., 1967).
Clofazimine has a reported pKa of 8.35 and consequently it is
highly ionised under physiological conditions. This high degree
of ionization, together with its high molecular weight, may be
significant factors in the poor oral bioavailability.
Schaad - Lanyi et al. (1987) studied the pharmacokinetics of
single oral doses of clofazimine over 11 days following
administration. They examined the effect of food on the
bioavailability. Following administration with food the area
under the plasma concentration versus time curve (AUC) and the
peak plasma concentration C,
were 62 and 30% higher
respectively compared to results obtained in the fasted state. The

CLOFAZIMINE

97

median time (tmx) to reach Cmaxwas 8 hours with food and 12


hours without food.
6.2 Distribution, Metabolism and Elimination

Plasma levels of the drug are approximately 0.5mg 1-1 but


increase with the dose and at 300mg daily levels of 1.0 - 1.5 mg 1-1
have been achieved (Banerjee et al., 1974;Levy,1974).
Administration of 50mg of clofazimine daily for 8 days did not
achieve steady state (Schaad - Lanyi et al., 1987). The time to reach
steady state has been theoretically estimated to be in the range of 30
- 70 days (Schaad - Lanyi et al., 1987; Holdiness, 1989). There is no
data available on loading doses. Likewise, there is no information
currently available on the pharmacokinetics of clofazimine
following intravenous administration.
The appearance of clofazimine in the plasma following
absorption is short lived (Banerjee et al., 19741,it rapidly passed
out of the circulation and is deposited in various tissues and
organs, particularly the fatty tissue, the spleen, lymph nodes, and
the cells of the reticulo - endothelial system. Concentrations of 2.15.3 mg g-1 have been reported in the subcutaneous fat (Mansfield,
1974),and 0.6-1.0mg g1 in the spleen (Desikan and Balakrishnan,
1976;Mansfield, 1974). It is taken up by the macrophages
throughout the body (Conalty et al., 1971;Yawalkar and Vischer,
1979). Electrophoretic studies of serum from orally treated mice
have shown almost complete binding of clofazimine to the
lipoproteins of the a and globulin fractions, these lipoprotein are
then phagocytosed by the macrophages (Conalty et al., 1971).
Clofazimine crystals have been found at autopsy in the small
intestine and in the macrophages of mesenteric lymph nodes
(Conalty et al., 1971;Aplin and McDougall, 1975;Desikan and
Balakrishnan, 1976;Jopling, 1976).Clofazimine does not appear to
cross the intact blood-brain barrier (Mansfield, 1974;Desikan and
Balakrishnan, 1976). It does, however, appear to cross the placenta
causing pigmentation of the foetus (Holdiness, 1989). There is no
data available on the volume of distribution of clofazimine.
Feng et al. (1981;1982)have used mass, ultraviolet and visible
spectrometry to identify three metabolites in the urine of leprosy
patients (Figure 11). Metabolite I is the unconjugated compound 3
(p-hydroxyanilino)-lO-(p-chlorophenyl)-2,lO-dihydro-2-

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CAITRIONA M. ODRISCOLL AND OWEN 1. CORRIGAN

isopropyliminophenazine, the other two metabolites are


conjugated, metabolite 11 is 3-(P-D-glucopyransiduronic acid)-lO-(pchloropheny1)-2, 10-dihydro-2- isoproyliminophenazine), and
metabolite 111 is 3 - (p-chlorani1ino)-10- (p-chlorophenyl) - 4, 10dihydro - 4 (PD-glucopyranosiduronic acid) -2isopropyliminophenazine. Metabolite I is reported to be formed
by a hydrolytic dehalogenation reaction, metabolite 11by hydrolytic
deamination followed by glucuronidation, and metabolite III by
hydration followed by glucuronidation.
Following administration of 300mg/day of clofazimine, 0.2% of
metabolite I, 0.25% of metabolite 11, and 0.2% of metabolite I11 were
recovered in the urine over 24 hours (Feng et al., 1981; 1982). No
information is available on the pharmacological activity of these
metabolites, or whether they are found in faeces or bile. The
authors have shown that metabolite I11 may be produced in the
laboratory through metabolism by liver enzymes. However, they
were unable to demonstrate the same hepatic conversion of
clofazamine to metabolites I and 11. In contrast, they suggest that
these metabolites are produced by bacterial degradation in the
intestine prior to absorption and urinary excretion.
Clofazimine accumulates in certain tissues throughout the body
(fatty tissue, skin, lymph nodes, macrophages etc.) and is
eliminated very slowly. The kinetics of the drug has been
described by both one and two compartment models. Data
obtained with relatively low dose, short term administration
indicated a one compartment model, with a a plasma tipof
approximately 7 days (Levy, 1974; Hastings et al., 1976; Holdiness,
1989). A second compartment is evident with long term, high dose
administration and appears to have a ttp of at least 70 days
(Banerjee et al., 1974; Levy, 1974).
Following oral administration of 50mg/day of clofazimine to
health volunteers Schaad - Lanyi et al. (1987) predicted that steady
state (SS) plasma concentrations would occur after approximately
30 days. They calculated an accumulation factor for the drug from
the ratio of AUCss: AUC. A value of 4.85 was obtained suggesting
a slow accumulation towards steady state. The authors suggest
that this may be avoided by administering higher loading doses,
followed by daily maintenance doses.

CLOFAZIMINE

99

CI

N.CH(CH,),

Metabolite I

1. Hydrolytic

N.CH(CH,),

- - -deamination
-- -- ---

2. Glucuronation

Clofazimine
Metabolite II

Metabolite 111

Figure 11. Metabolic pathways of clofazimine in humans (Feng et


al., 1981; 19821)

Up to 50% of a dose of clofazimine is excreted unchanged in the


faeces, indicative of poor oral absorption (Banerjee et al., 1974).
However, high concentrations of the drug have been found in bile
and in the gall bladder. This suggests that part of the ingested drug
recovered from the faeces may represent excretion by means of the
bile rather than simply the failure of absorption from the
gastrointestinal tract (Mansfield, 1974). Urinary excretion in
leprosy patients is negligable accounting for an average of 0.1%

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CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN

(range 0.01 - 0.43%)of the dose in 24 hours (Levy, 1974). A small


amount of the drug is excreted in the sebum and sweat (Vischer,
1969).

7. PHARMACOLOGY
7.1 Mechanisms of Action

Although the precise mechanism of the antileprotic activity of


clofazimine has yet to be determined several explanations have
been proposed (Hooper 1987).
(a) The drug has been shown to bind to cytosine - guanine DNA
base pairs in vitro (Morrison and Marley, 1976a,b). The binding is
specific for guanine residues only. The DNA of M. Zeprue has a
high guanine - cytosine content, consequently this binding may
disrupt the template function of the DNA, causing inhibition of
protein synthesis.
(b) The redox properties of clofazimine can divert up to 20% of
cellular oxygen (Barry et al., 1957) and thus disrupt normal
mitochondria1 oxidation processes (Rhodes and Wilkie, 1973). In
addition, it has been suggested that cytotoxic oxygen species,
hydrogen peroxide and superoxide, are generated as a result of the
presence of the drug (Hooper and Purohit, 1983; Savage et al.,
1989). If such a reaction occurred within the macrophages it will
enhance the killing of the bacilli which are also found inside the
macrophages.
(c) In addition, it has been suggested that the antileprotic effect
of clofazimine may be due to its action on the macrophage
lysosomal apparatus (Sarracent and Finlay, 1984).
7.2 Structure Activity Relationships (SAR)

The earlier SAR studies, reviewed by Hooper and Purohit (1983),


concentrated on three main areas of molecular modification
(Figure 12). Firstly, the structure of clofazimine was varied by
introducing additional chlorine atoms at positions 4, 7,8 and 9.
This resulted in loss of activity, except for the 7- chloro derivative,
with was equipotent with clofazimine. The second series was

CLOFAZIMINE

101

based on triaryl derivative. A variety of derivatives with a chloroor methoxy substituent in various positions showed only modest
activity. The third series involved variations at R2 coupled with
changes at R1 and R3, and the introduction of various substituents
at positions 7 and 9. In general for optimum activity R2 had to be
alkyl or cycloalkyl, and R1/R3 aryl or substituted aryl. When
hydrophilic salt forming groups were introduced at R2 activity was
greatly reduced.

R
1

Figure 12. Basic structure of iminophenazines


An X-ray crystallographic study (Rychlewska et al., 1985)
described the crystal and molecular structures of two crystal forms
of clofazimine and of its inactive isomer, isoclofazimine (B3857).
The geometric differences between clofazimine and isoclofazimine
were probed by CND0/2 molecular orbital calculations. The
geometry at the exocyclic amino nitrogen atom N(3) is
significantly different in isoclofazimine from that in both forms of
clofazimine and in other active analogues (Figure 13). The
authors suggest that the value for the intramolecular angle a at
N(3) (defined by C(3) - N(3) - C(21)in clofazimine) may play a
significant role in the activity. Molecules with values of a in the
range 125.5 & 10 were inactive, while those with expanded a angles
(i.e 131f 10) were active in vitro. The larger angle in the active
compounds is thought to favour intramolecular hydrogen
bonding between N(3)-H ... N(2). The capacity to form an
intramolecular hydrogen bond was interpreted as evidence of a
capacity for intermolecular hydrogen bonding in solution e.g
between guanine in DNA and clofazimine.

I02

CAlTRlONA M. ODRISCOLL AND OWEN 1. CORRICAN

Figure 13. Crystal structure of clofazimine (Rychlewska et al., 1985)


A wide range of clofazimine analogues have been designed as
follows; (a) to be active against resistant organisms, (b) not to
accumulate in adipose or other tissues, (c) to be rapidly and
adequately absorbed from the gastrointestinal tract, and (d) not to
crystallize within cells (Barry et al., 1959; Franzblau and
OSullivan, 1988; OSullivan et al., 1988; Byrne et al., 1989). These
structural modifications generally involve substitution at the
imino nitrogen atom by an unbranched alkyl or branched alkyl
chain containing a primary, secondary, tertiary, or alicyclic amino
group. Frequently the pKa values of these amine containing side
chains are approximately 9.5 - 10.5 thus ensuring that these
molecules will be substantially ionized under physiological
conditions. To counter act this increased hydrophilicity the
aliphatic part of the substituents usually contain 6 - 8 hydrophobic
methylene groups (Hooper, 1987).
A study, (Canavan et al., 19861, on the influence of lipophilic
and stearic properties on the distribution of a range of clofazimine
analogues to the spleen of mice following oral administration,

CLOFAZIMINE

I03

indicated that lipophilicity of the molecule is a significant factor


whereas the stearic properties of the N2 - substituents are not.
The structural features of phenazine derivatives which
contribute to stimulation of PGE2 production by
polymorphonuclear leucocytes (Zeis et al., 1987) and pro-oxidative
interactions with neutophils (Savage et al., 1989), have also been
investigated.
7.3 Toxicity
Clofazimine is a relatively non-toxic drug W.S. Leprosy panel,
1976). The acute LD50 was found to be >5 g/kg in mice rats and
guinea pigs. It was 3.3 g/kg in the rabbit. Daily oral doses of 30 and
50 mg/kg given for six months were generally well tolerated by
monkeys and rats. Reddish discolouration of the skin, faeces and
urine was observed. Temporary diarrhoea was occassionally
reported in rats (Stenger et al., 1970). Experimental studies in
animals did not show any evidence that clofazimine possesses a
primary embryotoxic or teratogenic action (Stenger et al., 1970).
The drug does not exhibit mutagenic activity (Morrison and
Marley, 1976a).
A long term study on 51 patients receiving clofazimine for
periods up to 8 years showed that, despite the deposition of the
drug in various tissues, it appears to be remarkably free from
serious side effects in clinical use (Hastings et al., 1976). Although
clofazimine crosses the placenta, no evidence of teratogenicity has
been found (Schulz, 1972). The most frequently reported side
effects of clofazimine therapy are red-brown hyperpigmentation of
the skin and conjunctiva, and abdominal pain (Hastings et al.,
1976; Jopling, 1976; Yawalkar and Vischer, 1979; Granstein and
Sober, 1981; Moore, 1983; Negrel et al., 1984; Venencie et al., 1986).
Cutaneous pigmentation normally fades within 6 to 12 monthsGeneralised dryness of the skin (xeroderma) ichthyosis, puritis,
phototoxicity, acneiform eruptions, exfoliative dermatitis and non
specific skin rashes have been reported (Yalwalkar and Vischer,
1979; Pavithran, 1985). Discolouration of sweat, hair, sputum,
urine and faeces have also been observed (Yalwakar and Vischer,
1979). Apart from subepithelial pigmentation in the cornea no
other side effects on the eye were recorded. Clofazimine crystals
were found in the tears of 82% of patients studied (Negrel et al.,
1984).

I 04

CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN

Gastrointestinal side effects, nausea, diarrhoea, anorexia,


constipation and weight loss have also been reported (Hastings et
al., 1976; Moore, 1983). These symptoms have been associated with
the deposition of clofazimine crystals in the submucosa of the
small intestine and in the mesenteric lymph nodes (Jopling, 1976;
Harvey et al., 1977).
The occurence of drug interactions involving clofazimine have
also been investigated. Most of the studies show that clofazimine
does not exert any effect on dapsone excretion in leprosy patients
(Balakrishnan and Seshadri, 1981; Zuidema et al., 1986).
Clofazimine has been shown to significantly reduce the absorption
of simultaneously administered rifampicin, resulting in delayed
time to reach peak serum concentration and increased t; . No
significant changes were seen in C,,, or AUC (Mehta et al., 1986).
7.4 Dose Schedules
A dose of 300mg once montly plus 50mg daily or lOOmg on
alternative days has been recommended to treat multibacillary
forms of leprosy (Martindale, 1989).
The World Health Organisation (1982) has published guidelines
for the treatment of leprosy. Dosage schedules are generally not
based on serum/plasma concentrations, or pharmacokinetic data.
Clofazimine is usually used in combination with other
antileprotic agents e.g dapsone and rifampicin, to prevent the
emergence of resistance. It is usually given with food in doses
adjusted according to body weight and the activity of the disease.
The therapeutic activity of clofazimine depends on the
concentration of drug in the immediate environment of M.leprae
in the tissues and not on the serum level. Since the drug in not
evenly distributed through out the tissues it is impossible to
calculate the minimal inhibitory concentration (MIC) in animals
(Yawalkar and Vischer, 1979).

CLOFAZIMINE

10.5

ACKNOWLEDGEMENTS
The authors wish to thank Dr. J. F. OSullivan, formerly of the
Health Research Board, Trinity College, Dublin, Dr. Helen
Sheridan, Department of Pharmacognosy and Dr. Mary Meegan,
Department of Pharmaceutical Chemistry, Trinity College, Dublin
for their advice and assistance, Ciba Geigy, England, for the supply
of clofazimine, Ms. Mary Lally and Ms. Mary Reilly for technical
assistance.

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