RIFAMPIN

Gian G. Gallo and Pietro Radaelli

 GlAN G. G A L L 0 AND PIETRO RADAELLI

TABLE OF CONTENTS
DESCRIPTION
1.1 Name
1.2 Formula and Molecular Weight
1.3 P o t e n t i a l Stereoisomerism
1.4 Appearance, Color and Odor
PHYSICAL PROPERTIES
2.1 Spectra
2.11 Ultraviolet Spectra
2.12 Infrared Spectra
2.13 Nuclear Magnetic Resonance Spectra
2.131 Proton Magnetic Resonance
Spectra
2.132 Carbon Magnetic Resonance
Spectra
2.14 Mass Spectra
2.2 Ionization Constants
2.3 Polarography
2.4 Optical Rotation
2.5 C r y s t a l Properties
2.51 X-ray Diffraction
2.52 Thermal Analysis
2.521 Melting Range
2.522 D i f f e r e n t i a l Scanning Colorime-
try
2.6 Distribution Properties
2.61 Solubility
2.62 Lipid-Water P a r t i t i o n
2.621 Organic S o l v e n t d a t e r P a r t i t i o n
2.622 Silicon O i l d a t e r P a r t i t i o n
2.623 Surface Activity
STABILITY
3.1 S t a b i l i t y a s Powder
3.2 S t a b i l i t y i n Solution
4. SYNTHESIS
 RlFAMPlN

5. METHODS OF ANALYSIS
5.1 Elemental Analysis
5.2 I d e n t i f i c a t i o n Tests
5.3 Spectrophotometric Methods
5.31 Spectrophotometric Assay on B u l k
Product
5.32 Spectrophotometric Assay on Pharma-
c e u t i c a l Preparations
5.33 Spectrophotometric Assay on Biological
Fluids
5.4 F luorome t r i c De termina t ion
5.5 Analysis by Complex F ormation
5.6 Volumetric Methods
5.7 Chromatographic Methods
5.71 T h i n Layer Chromatography
5.72 Column Chromatography
5.73 Paper Chromatography
5.74 High Pressure Liquid Chromatography
5.8 Microbiological Methods
5.81 Diffusion P l a t e Assay Methods
5.82 S e r i a l Tube Dilution Methods
5.83 Turbidime t r i c Methods
6. PROTEIN BINDING
7. PHARMACOKINETICS AND METABOLISM I N MAN
8. REFERENCES

469
 GlAN G. GALL0 AND PIETRO RADAELLI

1. DESCRIPTION
1.1 NAME
Rifampin (1) i s designated by IUPAC r u l e s as 2,7-
(Epoxypentadeca[l ,11,13]trienimino)naphtho~2,1 - b l f u r a n =
,
1 11(2H) -dione, 5,6,9,17,19,21 -hexahydroxy-23-methoxy-
2,4,12,16,18,20,22-heptamethyl=8-CN-(4-methyl-l-pipe=
r a z i n y l ) f o r m i m i d o y l l - 2 1 -acetate. However, i n t h e li-.
t e r a t u r e , r i f a m p i n has been p r e f e r e n t i a l l y known as
3- [[( 4-methyl-1 - p i p e r a z i n y l ) iminolmethyl] r i f a m y c i n SV,
according t o t h e o r i g i n a l nomenclature o f r i f a m y -
c i n s (2-4).
Rifampin (5,6) i s t h e USAN name f o r t h e compound,
r i f a m p i c i n i s t h e i n t e r n a t i o n a l n o n p r o p r i e t a r y name (7)
and other t r i v i a l names used a r e r i f a m y c i n AMP and r i -
f a l d a z i n e (8,9).
1.2 FORWIA AND MOLECULAR WEIGHT

36 35

C43H58N4012 Mol.Wt. = 822.95

470
 R I F AMP IN

T h i s numbering system, according t o t h e s p e c i a l

nomenclature o f rifamycins, i s adopted i n t h e present
profile.
The numbering system according t o IUPAC r u l e s i s :

The molecule o f r i f a m p i n i s u s u a l l y described as

c o n s i s t i n g o f a naphthohydroquinone chrornophoric p a r t
spanned by an a l i p h a t i c c h a i n c a l l e d t h e ansa w i t h a
p i p e r a z i n e s i d e c h a i n attached.
1.3 POTENTIAL STEREOISOMERISM
Rifarnpin, a semi-synthetic a n t i b i o t i c (8) , even
though i t c o n t a i n s 9 asymmetric carbon atoms and 3
double bonds, i s found o n l y as one isomer because a l l
t h e isomerogenic centers belong t o t h e n a t u r a l p a r t o f
t h e molecule and o n l y one isomer i s s p e c i f i c a l l y pro-
duced by t h e microorganism ( 4 , I O ) .

471
 GlAN G. G A L L 0 AND PIETRO RADAELLI

1.4 APPEARANCE, COLOR AND ODOR

Rifampin i s a red-orange, odorless, c r y s t a l l i n e
powder.
2. PHYSICAL PROPERTIES
2.1 SPECTRA
2.1 1 U l t r a v i o l e t Spectra
UV-VIS spectrophotometry has been used f o r
s t r u c t u r a l determination o f various rifamycins t o obtain
s p e c i f i c i n f o r m a t i o n on t h e chromophoric p a r t o f t h e
molecule. I n p a r t i c u l a r , t h e V I S maximum, which under-
goes a hypochromic e f f e c t and a s m a l l hypsochromic s h i f t
w i t h s t r o n g acids, i s c h a r a c t e r i s t i c o f t h e naphthohydrg
quinone form c a r r y i n g t h e a c i d i c i o n i z a b l e f u n c t i o n (1 1 -
13), and t h e auxochromic e f f e c t on t h e same V I S maximum
depends on t h e nature o f t h e s u b s t i t u e n t s i n p o s i t i o n 3
( 14-1 7).
The UV spectrum o f r i f a m p i n , recorded on a
P e r k i n Elmer model 4 0 0 0 4 spectrophotometer i n aqueous
phosphate b u f f e r pH 7.38 (8), e x h i b i t s t h e a b s o r p t i o n
maxima given i n Table I.
Table I
U l t r a v i o l e t absorption o f ri f ampi n

E
X,ax' nm
237 33 ,200
255 32,100
334 27,000
475 15,400

The v a r i a t i o n o f t h e UV-VIS spectrum o f

r i f a m p i n w i t h pH ( f i g u r e 1) i n d i c a t e s t h e presence o f an
i o n i z a b l e f u n c t i o n , a t t r i b u t e d t o t h e a c i d i c 8-hydroxyl
group i n p e r i p o s i t i o n t o t h e hydroquinonic h y d r o x y l
(13,18,19).
2.12 I n f r a r e d Spectra
The i n f r a r e d s p e c t r a o f v a r i o u s r i f a m y c i n s
u s u a l l y have been r e p o r t e d i n t h e l i t e r a t u r e b u t o n l y

472
ELP

215 210 230 1.0 yyI lbo 270 PBO 2vo 1rm 310
WAVUENGIH r*ILLIMICRONS

Figure 1 - W spectra of
I-*

rifampin i n methanol-water (2:3) solution a t different pH's

cc1
C

-.-.-.-.-.=
I

P
0
m

pH 0.5; pH 1.8;
. -..
W

...........
I
---------_ 3.5-1 1 .o

.-.
4
0

0
pH 2.5;
01

01
-%
N
5.

.
I
 GlAN G. G A L L 0 AND PIETRO RADAELLI

occasionally have been discussed (1 9/20). Exhaustive

s p e c t r a l assignments have only recently been made (21).
The infrared spectrum of rifampin i n chloro-
form solution was reported by Maggi e t al. (8). Figures
2 and 3 are the spectra of rifampin recorded as mineral
o i l m u l l and i n deuterochloroform solution, respectivelg
w i t h a Perkin-Elmer mod.421 spectrophotometer. Inter-
pretation of the spectrum o f rifampin (21) i s given i n
Table 11. I n CDCl solution, rifampin does not e x i s t
3
a s the zwitterion, while i n solid s t a t e it seems to.
Table I1
Infrared spectra of rifampin

IR absorption band, cm-1 Interpretation

lineral o i l m u l l CDCl solution
3
35003300 3480 u0H-bonded
* 2970, 2930 uCH3
and 2880
n 2820 uCH30
n 2800 uCH3-N
3200-2300 3300-2300 uNH and vNH-bonded
and vOH-bonded
1715 1715 vC0 a c e t y l
1735 1640 uCO furanone ( i n so.
lution i n t r a H-
bonded)
1670 and 1610 1 620 amide I
1570 1570 vc=c
1540 and 1520 1540 and 520 amide I1
1255,1050 and 1255,1040 and vC-0-C a c e t y l
1020 1020

Non-transparen region of the m neral o i l

2.13Nuclear Maanetic Resonance Spectra
2.131 Proton Magnetic Resonance SDectra
'H-NMR spectroscopy has been widely
used as a fundamental t o o l for s t r u c t u r a l determination
of various rifamycins (18,19,22-24).

474
 WAVELENGTH [MICRONS)

25 3 4 5 6 7 8 9 10 12 15 20 25

3500 2500 Moo 1900 1800 17$XJOJE@&~ 1400 1300 1200 1100 loo0 900 800 700 600 500 400

Figure 2 - IR spectrum o f rifampin i n mineral o i l m u l l .

 WAVELENGTH 'MICRONS)
2.5 3 4 5 6 7 8 9 10 12 15 20 25

3500 3000 2500 zoo0 1900 1400 1300 1200 1100 lo00 900 8 b 700 603 5'20 AX

Figure 3 - IR s p e c t r u m of r i f a m p i n i n CDCl s o l u t i o n .
3
 R I FAMP IN

1
The H-NMR spectrum o f r i f a m p i n i n
C D C l a t 60 MHz has been r e p o r t e d and some assignments
3
g i v e n (8). F i g u r e 4 i s t h e spectrum o f r i f a m p i n i n
C D C l recorded a t 100 MHz w i t h a V a r i a n )(L-l00-15 NMR
specqrometer. Complete i n t e r p r e t a t i o n o f t h e spectrum
has been made (25) and i s g i v e n i n Table 111.
2.132 Carbon Magnetic Resonance S p e c t r a
%NMR spectroscopy has been r e -
c e n t l y used f o r s t r u c t u r a l d e t e r m i n a t i o n i n t h e f i e l d
o f r i f a m y c i n s (24,26-28).
13
F i g u r e 5 i s t h e FT C proton-noise-
decoupled NMR spectrum o f r i f a m p i n i n CDCl3 recorded a t
25.2 MHz w i t h a V a r i a n XL-100-15 NMR spectrometer,
equipped w i t h an FT accessory. Interpretation o f the
spectrum has been made (25) and i s g i v e n i n T a b l e I V .
2.14 Mass Spectra
The mass s p e c t r a o f some r i f a m y c i n s have
been s t u d i e d and f r a g m e n t a t i o n p a t t e r n s have been i n -
t e r p r e t e d (29). I n p a r t i c u l a r , t h e most c h a r a c t e r i s t i c
peaks correspond t o M?, CMICH30Hlt and t o t h e chromo-
p h o r i c ions.
F i g u r e 6 i s t h e spectrum o f r i f a m p i n ob-
t a i n e d under 70eV e l e c t r o n impact i n DIS a t 200OC w i t h a
P e r k i n Elmer 270 spectrometer. Interpretation of this
spectrum has been p u b l i s h e d (30). The spectrum was o n l y
p a r t i a l l y c h a r a c t e r i s t i c as t h e compound decomposes i n
t h e i o n source and M'f and CMLH30HIf were absent, w h i l e
t h e peak a t m/e 398 corresponded t o t h e chromophoric ion.
The mass spectrum o f r i f a m p i n was a l s o ob-
t a i n e d under f i e l d d e s o r p t i o n i o n i z a t i o n c o n d i t i o n s a t
w i r e c u r r e n t 12 mA w i t h a V a r i a n MAT 731 spectrometer
(31). I t e x h i b i t e d t h e m o l e c u l a r i o n a t m/e 822 and
t h e [M+1 I peak corresponded t o t h e i s o t o p i c c o n t r i b u t i o n .
No f r a g m e n t a t i o n peaks were observed.
2.2 IONIZATION CONSTANTS
The i o n i z a t i o n p r o p e r t i e s o f r i f a m y c i n s have been
used i n con j u n c t i o n w i t h UV-VIS spectrophotometry (see
S e c t i o n 2.1 1) t o o b t a i n i n f o r m a t i o n on t h e chromophoric

477
 PPU
1%
I I 1 I I I I 1 ' I ' I I I I I I I I I 1

Figure 4 - 'H4MR spectrum a t 100 MHz of rifampin i n C K l , solution.

 RlFAMPlN

Table I11
'H-NMR d a t a o f r i f a m p i n i n C K l n s o l u t i o n [ M u l t i p l i c i t y ,
c h e m i c a l s h i f t s (6, ppm) and v i c i n a l i n t e r p r o t o n coup-
l i n g c o n s t a n t s (J,Hz)l.

Proton M u 1t i p l a ). 6
I
J
1
I
I

NI-i S
I -
CH4l S 8.22 -
CH2-2' ,6' m 2.9-3.3 c)
CH2-3 ' ,5 '
I
m 2.4-2.8 c)
N-CH3 S 2.34 -
OH-8
bs 11.4-14.0 b) -
, OH-4 13.16 b)
I
-
-
S
CH3-1 3 S 1.82
:CH~-14 S 2.22 I -
m 6.3-6.8 c)
H-19 dd 5.92 ~ 15 and 5
H-20 ddq 2.26 5 and 9 and 7
H-21 dd 3.78 I 9 and 1

OH-23
bs 3.2-4.2 b) - I

H-22 ddq 1.70 1 and 1.5 and 71

H-23 dd 3.04 1.5 and 10 I
H-24 ddq 1.52 10 and 1 and 7 1
H-25 dd 4.96 1 and 10 ,
H-26 dds 1.22 10 and 1.5 and 7j
H-27 dd 3.58 1.5 and 7 I

H-28 dd 5.00 7 and 13 '

H-29 d 6.20 13
CH3-30 S 2.10 -
CH3-31 d 0.88 7
CH3-32 d 1.01 7
CH3-33 d 0.58 7
CH3-34 d -0.33 7
CH3-36 S 2.06 -
CH3-37 S 3.05 -
a ) s z s i n g l e t ; d = d o u b l e t ; d d = d o u b l e t of d o u b l e t s ; d d q = d o u b l e t
of d o u b l e t s o f q u a r t e t s ; m = m u l t i p l e t ; bs=broad s i g n a l ;
b) it e x c h a n g e s w i t h D20
c ) not d e t e r m i n e d
479
 A
T
480

- 13

.rl
.rl
4-

4-
hl

hl

.rl
z
UJ

S
NMR s p e c t r u m a t 25.2 MHz of r i f a m p i n i n

c,
C proton-noise-decoupled

c,

S
Figure 5

Q
C
FT

0
E
0

I4
N
0
E
P

3
Q,
0
I4
u)
CDCl s o l u t i o n .
3
 RlFAMPlN

Table IV
'IC-NMR data o f rifampin i n CDC13 solution. [Chemical s h i f t s
16, ppm) and one-bond coupling constants ( I J , H a .
1

1 138.6 -
-
I

2 105.9"'
3 110.8') -
4 147.8 - I
/
5 112.8a) -
6 174.3 - I

7 1 20.3a) -
-
I

8 169.3
I

- I
I

9 104.4') I

I 10 117.8") -
I 11 195.3 -
I 12 108.7 -
13 21.5 130
14 7.6 130
15 169.6 -
I 16 129.4 -
17 135.0 1 50
18 123.2 150
I
19 142.6 1 50
20 38.6 125
21 70.7 140
22 33.4 125
23 76.7 140
24 37.6 125
25 74.4 140
26 39.5 125
27 76.7 140 I
28 118.7 155
29 142.6 190 ,
30 20.7 130
31 17.8 130 1

32 10.9 130 I

33 8.5 130
34 8.8 130
-
I

35 171.9 I

36 20.7 130
37 57.0 140 ,
CH=N- 134.4 1 70
C-2'- 50.2 130 I
I
C-3'- 53.9 130 I

C-5'- 53.9 130

I

1
C-6'- 50.2 130
I CH,-N- 45.8 I 130
I J
a) These assignments may be interchanged

481
 L
o = s s " = o
I
AlISN31NI 3AIlVl3H

I
482

I
I
II

Figure 6 - MS spectrum of rifampin a t 70 eV i n DIS a t 200%.

cc
0
 R I FAMPlN

p a r t o f t h e molecule and as a q u a n t i t a t i v e method o f

,
a n a l y s i s ( 1 1 - 1 3 18).
The pK v a l u e s f o r r i f a m p i n have been determined
s p e c t r o p h o t o m e t r i c a l l y (see f i g . 1 ) and p o t e n t i o m e t r i -
c a l l y i n s o l u t i o n i n water and i n m e t h y l c e l l o s o l v e -
water ( 4 : l ) and a r e r e p o r t e d i n T a b l e V. Rifampin
e x i s t s i n w a t e r s o l u t i o n as t h e z w i t t e r i o n w i t h i s o -
e l e c t r i c p o i n e q u a l t o 4.8 (8,13).
Table V
I o n i z a t i o n constants o f r i f a m p i n

Attribution IReference
pKa *KS
proton l o s t 1.7 3.6 h y d r o x y l a t C-8 I,13,18,19
p r o t o n gained 17.9 6.7 p i p e r a z i n e N-4 113

R i f a m p i n i o n i z e s i n non-aqueous s o l v e n t s , i.e., in
g l a c i a l a c e t i c a c i d t h e b a s i c p i p e r a z i n e n i t r o g e n can be
t i t r a t e d w i t h p e r c h l o r i c a c i d (32).
2.3 POLAROGRAPHY
The p o l a r o g r a p h i c behaviour o f r i f a m y c i n s has been

wave a t about 0 v o l t s =.
d e s c r i b e d and t h e presence o f an o x i d a t i o n o r r e d u c t i o n
X E i n d i c a t e s t h e hydroquinone
o r quinone ( 1 4/33-35) system, r e s p e c t i v e l y . These pola-
r o g r a p h i c p r o p e r t i e s p e r m i t amperometric t i t r a t i o n o f
r i f amyc i n s (36).
R i f a m p i n i n methanol-acetate b u f f e r s o l u t i o n , pH
5.9, shows an o x i d a t i o n wave w i t h El12 = 4 . 1 0 v o l t s.
SCE, a t t r i b u t e d t o t h e hydroquinone system ( 8 ) , and i n
aqueous phosphate b f f e r , pH 6.88, t h e r e i s a l s o a r e -
d u c t i o n wave w i t h E 2 y/ -1.66 v o l t s s. SCE, n o t a t -
t r i b u t e d (37). Sano e t a l . ( 3 8 ) r e p o r t e d an o x i d a t i o n
p o t e n t i a l o f E1/2 = 4 . 0 6 v o l t E. SCE, w i t h o u t o t h e r
details.
2.4 OPTICAL ROTATION
The o p t i c a l r o t t i o n f o r r i f a m p i n was r e p o r t e d by
Sano e t a l . (38) : C a l k +10.60 ( G O . 5% i n CDC13).

483
 GlAN G. G A L L 0 AND PIETRO RADAELLI

2.5 CRYSTAL PROPERTIES

2.51 X-ray D i f f r a c t i o n
S i n g l e - c r y s t a l X-ray d i f f r a c t i o n was used t o
deduce the s t r u c t u r e of the p-iodoanilides of rifamycin
B and rifamycin Y (10,39-42). T h e s i n g l e - c r y s t a l X-ray
d i f f r a c t i o n method was applied t o rifampin c r y s t a l l i z e d
w i t h 5 water molecules i n t h e orthorombic system (43).
The X-ray powder d i f f r a c t i o n p a t t e r n of ri-
fampin, i s reported i n Figure 7 (44) and Table V I g i v e s
the values according t o the ASTM rules. The grinding
causes the c r y s t a l l i n i t y of rifampin t o disappear and
an amorphous form t o o r i g i n a t e , a s shown i n Figure 8.
2.52 Thermal Analysis
2.521 Melting Range
Rifampin melts w i t h decomposition a t
183-1 88% (open c a p i l l a r y g l a s s tube),
2.522 D i f f e r e n t i a l Scanning Calorimetry
U n t i l 1 now, DSC has not been applied
t o the f i e l d of rifamycins.
T h e heating curve of rifampin has
been obtained on a Du Pont D i f f e r e n t i a l Thermal Analyzer
mod.990 w i t h a temperature r i s e of 100C/min and i s re-
ported i n f i g . 9 (45). I t shows an endotherm a t 193OC
corresponding t o t h e melting, immediately followed by an
exotherm corresponding t o t h e r e c r y s t a l l i z a t i o n of t h e
melt, which t h e n decomposes exothermically a t about
240%.
2.6 DISTRIBUTION PROPERTIES
2.61 S o l u b i l i t y
The approximate s o l u b i l i t i e s of rifampin i n
various s o l v e n t s have been determined a t room tempera-
t u r e (32). The r e s u l t s a r e reported i n Table VII,
according t o t h e d e f i n i t i o n used by t h e US Pharmacopeia
X I X , Rifampin i s s o l u b l e i n a c i d i c and a l k a l i n e
water (8).
Q u a n t i t a t i v e s o l u b i l i t y d a t a f o r rifampin
were reported, a s shown i n Table VIII.

484
Figure 7 - X-ray powder d i f f r a c t i o n pattern of rifampin before grinding.
 T a b l e VI
X-rav powder d i f f r a c t i o n p a t t e r n of r i f a m p i n before q r i n d i n q , a c c o r d i n q t o ASTM r u l e s

Rifampin ( s a m p l e before g r i n d i n g )

d(i) 1/11 hkl d(i) 1/11 hkl

Rad. Cuka x = 1.5418 A
0

17.16 2 5.18 13
Filter N i 12.43 8 4.90 17
11.11 3 4.63 2
I/I Spectrometer 10.76 1 4.43 loo
1 9.40 1 4.12 5
8.80 23 4.05 2
7.86 35 3.95 3
7.51 2 3.82 6
6.91 14 3.67 3
6.51 4 3.44 8
6.21 2 3.38 8
5.60 44 2.96 5
 3a
_L, d

Figure 8
-
- X-ray
-_-
d
-
Y --
20
I.a
10

powder diffraction pattern of rifampin after grinding

3" 2w
Figure 9 - Heating curve of rifampin
 R I F AMP IN

Table VII
Approximate s o l u b i l i t i e s of rifampin

P a r t s of s o l v e n t
Descriptive
Solvent required f o r 1
term
p a r t of rifampin
chloroform from 1 t o 10 freely soluble
methanol from 10 t o 30 soluble
dimethylformamide from 10 t o 30 soluble
dimethylsulphoxide from 10 t o 30 soluble
e t h a n o l 95 per c e n t from 100 t o 1,000 s l i g h t l y soluble
acetone from 100 t o 1,000 s l i g h t l y soluble
benzene from to00 t o 10,000 very s l i g h t l y soluble
c o r bon t e t r ac h l o r i d e practically insoluble
n-hexane practically insoluble
cyclohexane practically insoluble
n-butanal practically insoluble
propyleneglycol practically insoluble
glycerol practically insoluble
carbowax 400 practically insoluble
I I

Table VIII
--
Solubilities of rifampin

I
I chloroform 1 349 I I
' dichloromethane I 216
, ethyl acetate 108
i
I

dioxane 39 25oc I 38
methanol 16 ,
I acetone 14 I
l n-hexane 0.43 I I
j petroleum e t h e r 0.33
water pH 7.3 2.5
w a t e r pH 4.3 1.3
water pH 7.5 2.8
w a t e r pH 2.0 1 99.5
'room j 46
O.tN Hcl
1 phosphate b u f f e r pH 7.4
!2O0.O
, 9.9
;37oc
,
1 47
d
'

489
 GlAN G. G A L L 0 AND PIETRO RADAELLI

2.62 Lipid-water p a r t i t i o n
2.621 Organic solvent-water P a r t i t i o n
The study of the p a r t i t i o n between
water and organic s o l v e n t s a s an i n d i r e c t measure of t h e
lipid-water p a r t i t i o n has not been generally applied t o
t h e f i e l d of rifamycins. T h e p a r t i t i o n of rifampin i n
the system n-octanol/aqueous phosphate buffer, pH 7.4
was determined by Seydel (47) t o be K=15.6, w h i l e C u r c i
e t al. (48) have measured i t i n the system benzene/
aqueous b u f f e r s i n the range pH 5.5-7.0, andKequaled
9.7; a t pH 7.5, K=9.0; a t pH 8.0, K=4.6.
2.622 S i l i c o n oil-water p a r t i t i o n
The lipid-water p a r t i t i o n has been
obtained f o r v a r i o u s rifamycins from Rf values i n par-
t i t i o n reverse-phase t h i n l a y e r chromatography on
S i l i c a g e l p l a t e s impregnated w i t h s i l i c o n o i l as sta-
t i o n a r y phase and water-acetone a s mobile phase (49-51 ).
The f r e e energy parameter RM=log(- 1 -1) (52), was used
R
f o r q u a n t i t a t i v e c o r r e l a t i o n s between s t r u c t u r e and
a n t i b a c t e r i a l (49,50) and a n t i v i r a l (51) a c t i v i t i e s .
RM values were obtained f o r rifampin
and a r e reported i n Table IX.
Table I X
RM values f o r rifampin

I RM 1 mobile phase l s t a t i o nary phase I Ref. I

0.029 30% acetone i n silicon oil 49
water (v/v)

-0.293 40% acetone i n

water (v/v)
silicon oil
I 50

2.623 Surface a c t i v i t y
Rifamycins have been shown t o have
s u r f a c e a c t i v i t y , from the v a r i a t i o n of t h e surface
tension of b u f f e r water s o l u t i o n s w i t h concentration
(53 I 54).

490
 RlFAMPlN

The s u r f a c e p r o p e r t i e s of rifampin
depend on t h e pH of t h e medium. I n the a l k a l i n e t o
n e u t r a l range, rifampin i s a weak s u r f a c t a n t and no
a s s o c i a t i o n s a r e observed; i n the a c i d i c range (pH 4-0)
a pronounced lowering of the s u r f a c e t e n s i o n w i t h con-
c e n t r a t i o n i s observed, and m i c e l l e formation i s
apparent a t a c o n c e n t r a t i o n of about 10-5 m o l e / l i t e r
(55)
3. STABILITY
3.1 STABILITY AS POWDER
Rifampin i s very s t a b l e i n the s o l i d s t a t e i n
s e a l e d c o n t a i n e r s a t room temperature, a s described i n
Table X (56). Rifampin i n t h e s o l i d s t a t e i s s t a b l e
a l s o a t temperatures u p t o 70OC, a s reported by Sano e t
a l . (38).
3.2 STABILITY IN SOLUTION
The s t a b i l i t y of rifampin i n aqueous s o l u t i o n has
been widely i n v e s t i g a t e d and t h e c o n d i t i o n s and t h e
transformation products a r e reported i n Table XI. L i k e
o t h e r rifamycins, rifampin undergoes d e s a c e t y l a t i o n on
a l k a l i n e t r e a t m e n t , y i e l d i n g t h e corresponding 25-des-
a c e t y l d e r i v a t i v e without s u b s t a n t i a l l o s s of a n t i -
b a c t e r i a l a c t i v i t y (57).
I n mildly a l k a l i n e aqueous s o l u t i o n s and i n the
presence of atmospheric oxygen a t room temperature,
rifampin transforms i n t o rifampin quinone; t h i s
oxidation can be prevented by sodium ascorbate. Under
the same c o n d i t i o n s and a t 60-70OC, rifampin y i e l d s
t h r e e main transformation products, which were iden-
t i f i e d by Maggi e t a1.(22) a s 25-desacetyl-rifampin,
25-desacetyl-23-acetyl-rifampin and 25-desacetyl-
21 - a c e t y l - r i f ampin,
Sano e t a l . (38) have s t u d i e d t h e s t a b i l i t y o f
rifampin a t 250 i n aqueous s o l u t i o n s a t d i f f e r e n t pH's:
it decomposes r a p i d l y i n a c i d i c o r a l k a l i n e c o n d i t i o n s ,
but slowly i n n e u t r a l ones. 3-Formylrifamycin SV i s
the main decomposition product of rifampin i n aqueous

491
 Table X
Stability
- of rifampin i n t h e s o l i d s t a t e a t room temperature (56)
st o r t i n g 1 2 months 21 months 30 months 41 months

' UV Microbig UV Microbig UV Microbig UV Microbig UV Microbig

kssay l o g i c a l Assay l o g i c a l Assay l o g i c a l Assay l o g i c a l Assay l o g i c a l
% Assay % % Assay % % Assay % % Assay % % Assay %
c

96.4 99.0 100.0 100.2 95.4 97.3 95.9

1
I

I
I

I
I
TLC TLC I TLC TLC TLC
P
jI
t ~forrnvlrifa- I ,
(D
N
i I

rifampin 1 absent 1-1.5% 1 1.5-2%

i1 1.5-2% I 2.54%
-
: rifampin N-
oxide
!
! traces 1-1.5% 11 1-1.5%
!
1 1-1 .!j% I
I
1-1.5%

25-desacetyl
rifampin
,1
1
0.5 - 1% j 1.5 - 2% 1.5 - 2% 1 1 - 1.5%
I
1
i
1-1.5%
i
acetyl-rifampin I
I I
 R I F AMP IN

Table XI
S t a b i l i t y o f r i f a m p i n i n aqueous s o l u t i o n s

+ +
3-formyl-rifamycin SV r i f ampin-quinone

pH 2.3, 20-22OC (8)

pH 8.2, 20-22% (8)

-i
tic1 0,1N, 37% (47)

r i f ampin

NaOH 5% i n e t h a n o l
pH 8 . 2 (22)
water ( 1 : l ) 20-
60-70%
22% (57)

t
25-desacetyl-21-acetylrifampin

I 1
bl 25-desace t y l - 2 3 - a c e t y l r i f am p i n I

493
 GlAN G. GALL0 AND PIETRO RADAELLI

acidic medium (8). Seydel (47) has determined the de-

composition r a t e of rifampin i n 0.1N tC1 vs. temperature
and, from the Arrhenius plot, calculated the activation
energy, &la=l9.2 Kcal/mole/degree.
Rifampin does not lower i t s microbiological activ-
i t y i n the various conditions reported i n Table XII.
Table XI1
S t a b i l i t y of the a n t i b a c t e r i a l a c t i v i t v of rifamPin
i n solution.

Conditions
’ Concentra-
tion( mg/ml)
Time Ref .
water-dimethyl-
f ormamide ( 95 :5) 1 5% days 58
a t 5%

d imet hyl-
10 8 months 59
sulphoxide a t 150
t I
I
I
I
I
I i
water-ethanol
(8:2) a t 4% 1 8 weeks 6O,6l
o r 20%

4. SYNTHESIS
Rifampin i s a semi-synthetic rifamycin, obtained
by condensation o f 1-amino-4-methylpiperazine w i t h 3-
formyl-rifamycin SV i n peroxide-free tetrahydrofuran a t
100-150C. The 3-formyl-rifamycin SV (14) was obtained
from rifamycin B, the fermentation product (62-64) , by
the chemical procedure reported i n t a b l e XIII. Gianan-
tonio e t al. (65) have patented the production of ri-
fampin by reacting rifamycin S d i r e c t l y w i t h form-
aldehyde, tert-butylamine and MnO and condensing w i t h
1 -amino-4-methylpiperazine (see t a2b l e XIII).

494
 R I F AMP IN

Table X l l l
Synthesis of r if a mpin
STREPTOMYCES MEDITERRANEI

1
oxidation
w H3C*
reduction 0 0
I!
CH2-COOH '0
RlFAMYClN B(62-64)
(fermentation product) \ in aerated
RlFAMYClN 0 ( 66)
Acidic
aqueous hydrolyais
solution
OH OH
oxidation
\ OH 0

reduction
RlFAMYClN SV(67)
Mannich
reaction
and
reduction
tart-but lamine,
dn02 in OH OH
tetrahydrofuran
2) l-amino- H3c&
f CH2-N /RI
4 -methyl-
piperazine (65) OH 'R2

/
MANNICH DERIVATIVE OF RlFAMYClN SV (15)
Oxidation
in acidjc
conditions

OH OH
"3c@cH>-am~
;ih
t:yl

OH i-NnN-CH3 OH
v
RIFAMPIN ( 8 ) 3-FORMYL RlFAMYClN SV (14,681

495
 GlAN G. G A L L 0 AND PIETRO RADAELLI

5. METHODS OF ANALYSIS
5.1 ELEMENTAL ANALYSIS
Element Theoretical %
C 62.76
H 7.10
N 6.81
0 23.33
5.2 IDENTIFICATION TESTS
R i f a m p i n i s i d e n t i f i e d and d i f f e r e n t i a t e d from
t h e o t h e r r i f a m y c i n s by i n f r a r e d and n u c l e a r magnetic
resonance spectroscopy, f i e l d d e s o r p t i o n mass s p e c t r o
metry, p o t e n t i o m e t r i c t i t r a t i o n , d i f f e r e n t i a l scanning
c a l o r i m e t r y and e l e m e n t a l a n a l y s i s , I n o r d e r t o d e f i n e
t h e homogeneity o f t h e r i f a m p i n sample, chromatographic
procedures (paper and t h i n l a y e r ) and t h e r m a l a n a l y s i s
a r e used.
C o l o r i m e t r i c t e s t s based on o x i d a t i o n were de-
scribed, To 5 m l o f aqueous r i f a m p i n s o l u t i o n ( a b o u t
1 mg/ml), I m l o f 10% (w/v) ammonium p e r s u l p h a t e i n
phosphate b u f f e r , pH 7.38, i s added: t h e c o l o r t u r n s
from orange-yellow t o v i o l e t - r e d (69). A s i m i l a r me-
thod, employing f e r r i c n i t r a t e as o x i d i z i n g agent, was
d e s c r i b e d by Eidus e t a l . (70,71), who used i t t o i d e n -
t i f y t h e presence o f r i f a m p i n and d e s a c e t y l r i f a m p i n i n
biological fluids.
5.3 SPECTROPHOTOMETRIC METHODS
5.31 S p e c t r o p h o t o m e t r i c assay on b u l k p r o d u c t
The V I S maximum a t 475 nm i n aqueous phos-
phate b u f f e r s o l u t i o n pH 7.38 w i t h an a b s o r p t i v i t y
(g/a) v a l u e o f 18.7 (see S e c t i o n 2.11) enables r i f a m p i n
t o be q u a n t i t a t i v e l y assayed.
The main i m p u r i t i e s o f r i f a m p i n a r e 3-
f o r m y l r i f a m y c i n SV and rifampin-quinone. The two
p r o d u c t s can be s p e c t r o p h o t o m e t r i c a l l y q u a n t i t a t e d : t h e
f i r s t one by an e x t r a c t i o n method u s i n g n-hexane:ethyl-
a c e t a t e ( 2 : l ) (13) and t h e second one by a d i f f e r e n t i a l
s p e c t r o p h o t o m e t r i c method which t a k e s advantage o f t h e
r e d u c t i o n by sodium ascorbate o f t h e quinone ( 1 3).

496
 RlFAMPlN

5.32 Spectrophotometric assay on p h a r m a c e u t i c a l

preparations
R i f a m p i n can be determined i n pharmaceuti-
c a l p r e p a r a t i o n s by u s i n g t h e d i f f e r e n t i a l spectropho-
t o m e t r i c method (72), m o d i f i e d by P a s q u a l u c c i e t a l .
(73)
5.33 Spectrophotometric assay i n b i o l o n i c a l
fluids
Numerous s p e c t r o p h o t o m e t r i c methods f o r de-
t e r m i n i n g r i f a m p i n i n b i o l o g i c a l f l u i d s have been de-
s c r i b e d i n t h e l i t e r a t u r e , b u t owing t o t h e i r low sen-
s i t i v i t y t h e y were s a t i s f a c t o r i l y a p p l i e d o n l y t o t h e
d e t e r m i n a t i o n o f r e l a t i v e l y h i g h l e v e l s (more t h a n
5pg/ml). The methods were based on t h e e x t r a c t i o n o f
r i f a m p i n and i t s m e t a b o l i t e s w i t h o r g a n i c s o l v e n t s and
on t h e d e t e r m i n a t i o n o f t h e V I S absorbance o f t h e o r -
ganic e x t r a c t . The e x p e r i m e n t a l c o n d i t i o n s a r e sum-
marized i n T a b l e XIV.
5.4 FLUORQMETRIC DETERMINATION
R i f a m y c i n s do n o t e x h i b i t n a t u r a l fluorescence. A
q u a n t i t a t i v e method was developed f o r r i f a m y c i n B (82),

ti v e .
based on i t s t r a n s f o r m a t i o n i n t o t h e t r i a c e t y l d e r i v a -

R i f a m p i n was determined f l u o r e m e t r i c a l l y by t r a n s -
f o r m i n g i t w i t h hydrogen p e r o x i d e (83) i n t o a f l u o r e -
scent product. The maximum f l u o r e s c e n c e develops i n an
aqueous carbonate-bicarbonate b u f f e r , pH 9.2 a t 4-80 nm,
when t h e e x c i t a t i o n wavelength i s 370 nm. The r e l a t i v e
fluorescence i n t e n s i t y i s l i n e a r w i t h concentrations o f
r i f a m p i n i n t h e range 0.1 t o 10 pg/ml.
5.5 ANALYSIS BY COMPLEX FORMATION
R i f a m y c i n s complex w i t h m e t a l l i c ions, as r e p o r t e d
f o r r i f a m y c i n L (34) and f o r r i f a m y c i n S (84).
R i f a m p i n i n methanol s o l u t i o n g i v e s a complex when
t r e a t e d w i t h an aqueous s o l u t i o n o f A l C l i n t h e r a t i o
2:l (85); t h e i n t a b i l i t y c o n s t a n t i n waqer-methanol
( 1 : l ) i s 3.4.10 8 . Complex f o r m a t i o n was demonstrated
t o t a k e p l a c e a l s o w i t h Hg*, Cu", Ag' and Fe- (86).

497
 GlAN G. G A L L 0 AND PIETRO RADAELLI

Table X I V
Spectrophotometric methods f o r t h e d e t e r m i n a t i o n o f r i f a m p i n and
i t s m e t a b o l i t e s i n body f l u i d s .

1
*-
1 Compound Analytical
Assayed ar
Body Extraction wavelength
stated i n eference
fluid solvent
the r e f 2
:abrorpt i v i t y ,
I I rence 9/11

bile, urine benzene RtDA

I R+DA
475nm and
urine, iso-amyl
335nm ( c a l i -
serum alcohol
b r a t i o n curve) 75

urine
I$;;ne-hexane
I R 335nm ( c a l i -
b r a t i o n curve)

urine,
butanol-hexane 478nm ( c a l i -
serum, b i l e RtDA 76
(4:l) b r a t i o n curve)

1
and t i s s u e s

I R
1 I
:;;p;ol-hexane
urine 478nm (19.6) 77

teeth
butanol-hexane II R
478nm ( C a l i -
(4:l) t i o n curve)
/
I
serum 1;;;oLheptane I R 482nm (15.6) 79

ethylalcohol-
aerum ethylacetate R 475nm (15.5) 80
(1 :1)
~~ ~

cyclohexane- blue l i g h t f i
urine b u t y l acetate
I(i :i)
j R
' t i o n curve) I

498
 RlFAMPlN

The f o r m a t i o n o f t h e complex w i t h A1C13 was em-

ployed f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f r i f a m p i n i n
bulk, i n pharmaceutical f o r m u l a t i o n s and i n u r i n e (87),
by measuring t h e cherry-red c o l o r a t 507nm i n methanol-
water (49:l) o r i n methanol-water-butanol-hexane (1 9:
1 :4:1),
5.6 VOLUMETRIC METHODS
Rifampin i n capsules was determined by a volu-
m e t r i c method (88): t h e a n t i b i o t i c was o x i d i z e d w i t h
an excess o f f e r r i c c h l o r i d e , which was then t i t r a t e d
iodometr i c a l l y .
5.7 CHROWTOGRAPHIC METHODS
5.71 T h i n l a y e r chromatography
TLC has been w i d e l y used i n t h e f i e l d o f
r i f a m y c i n s (89,90) and t h e c o l o r e d spots a r e d i r e c t l y
located.
Many TLC procedures were developed f o r t h e
a n a l y s i s o f r i f a m p i n and i t s m e t a b o l i t e s i n body f l u i d s .
The Rf values were shown t o be dependent on t h e concen-
t r a t i o n s (91). The spots were q u a n t i t a t e d by u s i n g t h e
bioautographic technique (92) o r t h e d e n s i t o m e t r i c one
(93) o r by v i s u a l e s t i m a t i o n (94) or, f i n a l l y , by t h e
e l u t i o n method f o l l o w e d by m i c r o b i o l o g i c a l assay (95).
A reverse,,phase p a r t i t i o n TLC procedure has been de-
s c r i b e d f o r t h e d e t e r m i n a t i o n o f r i f a m p i n (96) :s i l a n i z -
ed S i l i c a g e l p l a t e s were used as s t a t i o n a r y phase, w i t h
phosphate b u f f e r , pH 7 c o n t a i n i n g 0.1 % sodium ascorbate
as mobile phase ( o t h e r mobile phases are reported), The
c o l o r e d spot was e l u t e d and measured spectrophotome-
trically. Reverse-phase p a r t i t i o n TLC has been used
f o r s t r u c t u r e - a c t i v i t y c o r r e l a t i o n s (see S e c t i o n 2.622).
5.72 Column chromatography
The c o n t e n t o f r i f a m p i n and i t s m e t a b o l i t e s
i n urine, b i l e and serum, was determined by e x t r a c t i o n
w i t h c h l o r o f o r m and by column chromatography, f o l l o w e d
by spectrophotometry (97). The l i q u i d - s o l i d chromato-
graphy was c a r r i e d o u t u s i n g a g l a s s column 7 cm long,
4 mm I.D., packed w i t h S i l i c a g e l G, b u f f e r e d a t pH 6,
and chloroform and chloroform-methanol i n p r o g r e s s i v e l y

499
 GlAN G. G A L L 0 AND PIETRO RADAELLI

i n c r e a s i n g amounts (5%, 10% and 16.6%) as s o l v e n t , a t a

f l o w r a t e o f 0.15 ml/min. R i f a m p i n and i t s m e t a b o l i t e s
were t h e n q u a n t i t a t e d by r e a d i n g t h e absorbance o f t h e
e l u a t e s a t 475 nm.
5.73 Paper chromatoqraphy
A paper chromatographic t e c h n i q u e f o r de-
t e r m i n a t i o n o f r i f a m p i n and 2 5 - d e s a c e t y l r i f a m p i n was
used f o r u r i n e and b i l e (74). Descending chromatography
was c a r r i e d o u t on Whatman 3MM paper u s i n g methanol: n-
o c t a n o l ( 4 : l ) as s t a t i o n a r y phase and aqueous b u f f e r ,
pl-l 6 , as m o b i l e phase f o r 6 hr. The i n t e n s i t y o f t h e
red-orange s p o t s was measured by an A n a l y t r o l photoden
s i t o m e t e r (mod.Rl3-450 nm f i l t e r ) o r t h e m a t e r i a l s de-
termined b i o a u t o g r a p h i c a l l y .
Akimoto e t a l . (98) r e p o r t e d a paper chro-
matographic method f o r t h e s e p a r a t i o n o f r i f a m p i n and
i t s m e t a b o l i t e s , u s i n g e t h y l acetate-water-dimethyl-
formamide ( 1 O : l O : l ) .
5.74 H i g h pressure l i q u i d chromatography
R i f a m p i n has been f r e q u e n t l y c i t e d as an
emample o f t h e u s e f u l n e s s o f HPLC (99-102). Rifampin,
25desacetylrifampi11, r i f a m p i n quinone and 3 - f o r m y l
r i f a m y c i n SV were separated (99) under t h e f o l l o w i n g
c o n d i t i o n s : DuPont 820 Chromatograph equipped w i t h UV
d e t e c t o r , ODS Permaphase column a t 50% and 1,000 p s i ,
w a t e r t o methanol w i t h l i n e a r g r a d i e n t 8%/min as m o b i l e
phase and l m l / m i n f l o w r a t e .
5.8 MICROBIOLOGICAL METHODS
M i c r o b i o l o g i c a l met hods have been w i d e l y d e s c r i b e d
f o r t h e d e t e r m i n a t i o n o f r i f a m p i n potency i n b u l k prod-
u c t s , i n p h a r m a c e u t i c a l f o r m u l a t i o n s and i n body f l u i d s ,
as l i s t e d i n t h e r e v i e w by B i n d a e t a l . (103). These
methods can be c l a s s i f i e d as a) d i f f u s i o n p l a t e methods,
b) s e r i a l t u b e d i l u t i o n methods and c) t u r b i d i m e t r i c
methods,
5.81 D i f f u s i o n p l a t e assay methods
These methods were used t o d e t e r m i n e r i -
fampin i n serum, b i l e and u r i n e as w e l l as i n o t h e r body
f l u i d s and organs (fragments o f organs and t i s s u e s were

500
 RlFAMPlN

appropriately treated f o r extracting rifampin). They

can be d i v i d e d i n d i f f e r e n t c l a s s e s a c c o r d i n g t o tech-
n i c a l aspects as r e p o r t e d i n T a b l e XV, where o t h e r ex-
p e r i m e n t a l d e t a i l s a r e summarized, The c y l i n d e r - p l a t e
t e c h n i q u e d e s c r i b e d by Grove e t a l . (104) has been
adopted i n L e p e t i t S.p.A. (105)
5.82 S e r i a l t u b e d i l u t i o n methods
T h i s method, f i r s t a p p l i e d by C l a r k e t a l .
(115) t o r i f a m i d e and r i f a m p i n , has been used f o r t h e
d e t e r m i n a t i o n o f r i f a m p i n i n serum by v a r i o u s l a b s .
Some d e t a i l s a r e r e p o r t e d i n T a b l e XVI.
5.83 T u r b i d i m e t r i c met hods
A t u r b i d i m e t r i c m i c r o b i o l o g i c a l assay o f
r i f a m p i n u s i n g an a u t o m a t i c a n a l y z e r was developed by
S i m o n c i n i e t a l . (120). The method i s based on t h e
measurement o f t h e o p t i c a l d e n s i t y o f t h e b a c t e r i a l
suspension ( K l e b s i e l l a Pneumoniae ATCC 10031 o r
E s h e r i c h i a c o l i ATCC 10536 as t e s t microorganism) i n a
D i f c o c u l t u r e medium c o n t a i n i n g r i f a m p i n , a f t e r i n c u -
b a t i o n a t 370 f o r 3.5 hr.
6 PROTEIN BINDING
The b i n d i n g o f r i f a m p i n t o blood serum and plasma
p r o t e i n s i n man and o t h e r a n i m a l species was w i d e l y
studied. The d a t a o b t a i n e d c o v e r a r e l a t i v e l y l a r g e
range, p r o b a b l y because o f t h e d i f f e r e n t methodologies

.
used and o f t h e many parameters t h a t can i n f l u e n c e t h e
phenomenon (1 21 )
I n v i t r o experiments were c a r r i e d o u t by C u r c i e t
a l . w i t h a d i a l y s i s technique, and t h e y r e p o r t e d t h a t
a t t h e c o n c e n t r a t i o n s reached i n v i v o , about 7 5 4 5 % o f
r i f a m p i n b i n d s w i t h serum p r o t e i n s (48,122-125). In-
t e r e s t i n g l y , t h e y found t h a t PAS ( p a r a - a m i n o - s a l i c y l a t e )
i n c o n c e n t r a t i o n s from 100 t o 200 pg/ml decreases t h e
b i n d i n g o f t h e a n t i b i o t i c (122). By an u l t r a c e n t r i -
f u g a t i o n technique, Seydel (47) o b t a i n e d a percentage o f
r i f a m p i n bound t o bovine albumin i n v i t r o i n t h e range
from 50 t o 70%. From t h e s e data, K e b e r l e e t a l . (126)
d e r i v e d t h a t each albumin molecule has two b i n d i n g
s i t e s f o r rifampin. Mashimo (127) has found t h e p r o t e i n

501
 Table XV

M i c r o b i o l o q i c a l ass= of r i f a m p i n by d i f f u s i o n p l a t e assay methods

Technical aspects Medium Microorganism Reference

metal c y l i n d e r s containing
1 05
,ISarcina
Agar ( D i f c o 0263-02) l u t e a (ATCC 9341)
-t h el i q u i d t o be assayed
wells ( l o m m ) c o n t a i n i n g
t h e l i q u i d t o be assayed
wells (9mm)

d i s k s (6mm) soaked i n the

l i q u i d t o be assayed
d i s k s (9mm)
disks

disks

: d i s k s (9mm) Salt-glucose-peptone k o r c i n a l u t e a (IP 5345) 112

b r o t h ( P a s t e u r Inst. 1
1
Codex) I
~ d i s k s (9mm) ' C a s e i n peptone-soya !Staphylococcus c o a g u l a s e 113
1 peptone-agar lnegative
1
1 disks (6mm) ' Agar ( D i f c o 0263-02)
I
' S a r c i n a l u t e o (ATCC 9341)
I
j 114
I-i .
 RlFAMPlN

Table XVI
M i c r o b i o l o q i c a l assays o f r i f a m p i n by s e r i a l t u b e
d i l u t i o n methods.

Medium Microorganism Inoculum i e f e r e nc e

non-spec i f i e d 0.031111o f a
Sarcina l u t e a
broth 1 :250 d i l u t i o n 115
o f 18h c u l t u r e
D i f c o sucrose Staphylococcus 0.05ml o f a
broth w i t h aureus (ATCC 1 :lo0 d i l u t i o n 116
phenol r e d 6538P) o f 18h c u l t u r e
mannite and Staphylococcus 0.05ml o f a
phenol r e d aureus (Oxford, 1 :200 d i l u t i o n 117
selective sensitive t o o f 24h c u l t u r e
broth O.O2ug/ml)
Youmans Mycobacterium 5-1 OO*1O4
tuberculosis viable units 118
(H37RV)
Mycobacterium
Loc kemann
tuberculosis
(H37Rv o r
- 119 j
F u hrmann

b i n d i n g o f r i f a m p i n by d i a l y s i s , u l t r a f i l t r a t i o n and
u l t r a c e n t r i f u g a t i o n , a t a c o n c e n t r a t i o n o f 2Opg/ml, t o
be lo%, 90% and 70%, r e s p e c t i v e l y . No c l e a r i n t e r p r e -
t a t i o n was g i v e n f o r t h e d i f f e r i n g data,
I n v i v o experiments o f p r o t e i n b i n d i n g were
c a r r i e d o u t u s i n g ' ' k - r i f a m p i n (128) and about 75% o f
r i f a m p i n was found t o be bound t o human serum p r o t e i n ,
P i l h e u e t a l . (129) found r a t i o s o f 15 t o 40% between
c e r e b r o s p i n a l f l u i d ( c o n s i d e r e d as a p r o t e i n - f r e e f l u i d )
and plasma l e v e l s . Aoyagi has r e p o r t e d (130) t h e
b i n d i n g o f r i f a m p i n t o t h e serum p r o t e i n s o f v a r i o u s
animals: i n t h e horse 40-46%, i n t h e r a b b i t 13-17% and
i n man 17-38%. A r e c e n t s t u d y by d i a l y s i s u s i n g
r i f a m p i n (131) showed t h a t 86.1% and 88.9% o f r i f a m p i n
were bound t o t h e plasmas o f t u b e r c u l a r p a t i e n t s and

503
 GlAN G. G A L L 0 AND PIETRO RADAELLI

healthy volunteers, r e s p e c t i v e l y . Yokosawa e t a l . (1 32)

have suggested, on t h e b a s i s of e l e c t r o p h o r e s i s and
microbiological assay, t h a t rifampin a s s o c i a t e s w i t h
a1-t 012- and P-globulins of human serum.
7. PHARM4COKINETICS AND METABOLISM I N
T h e pharmacokinetics of rifampin has been widely
s t u d i e d and t h e serum l e v e l s of rifampin a f t e r s i n g l e
and repeated administration of d i f f e r e n t doses were
determined by various i n v e s t i g a t o r s and have been
reviewed (103). To summarize, a f t e r o r a l administration,
rifampin is well-absorbed w i t h t h e maximum plasma lev-
e l s a t 1.5-3 h r over a wide range of s i n g l e dose, i . e . ,
0.1 t o 1.2 g. The l e v e l s a r e s t i l l appreciable (5-10%
of the peak l e v e l ) a f t e r 12 h r only a t t h e high doses
(103).
Rifampin i s widely d i f f u s i b l e i n the t i s s u e s and
i n t h e various body f l u i d s (105), a s indicated i n Table
X V I I . T h i s i s r e l a t e d t o t h e h i g h lipotropism of r i -
fampin (47,413).
Rifampin e l i m i n a t i o n , which m u s t be considered
slow, occurs mainly through t h e b i l e b u t a l s o through the
u r i n e (103,128,133). The t o t a l amount of rifampin elim-
inated i n the b i l e i s not proportional t o t h e dose
administered (48,116,134) while t h e urinary e l i m i n a t i o n
i n c r e a s e s w i t h t h e dose (77,105,109,116,133-136). I n an
i n v e s t i g a t i o n c a r r i e d out over 6 days (105), about 22%
was eliminated i n t h e f a e c e s and about 26% i n t h e u r i n e ,
I n another study, however, Riess(128) found t h a t 96 hrs
a f t e r administration of 300 mg of labeled rifampin, 94%
of t h e t o t a l r a d i o a c t i v i t y had been recovered i n equal
percentages i n the faeces and u r i n e ,
The main metabolite of rifampin i n man was iden-
t i f i e d , a f t e r i s o l a t i o n from b i l e and urine, a s 25-
desacetylrifampin (74,92,94,137). T h i s compound i s much
less l i p o p h i l i c than rifampin and it is e a s i l y excreted
i n urine (46) and not reabsorbed (138,139). Sano e t a l ,
(93) reported t h a t i n a d d i t i o n t o 2 5 - d e ~ a c e t y l r i f a m p i n ~
a second metabolite i s 3-formyl rifamycin SV.

504
 R I F AMP IN

Table XVII
Distribution of rifampin i n human tissues and f l u i d s
a f t e r oral administration of a s i n g l e 450 mg dose (105)

f
adm i n i-
stration
16 spleen
l2 lung

\-I
6 Dliver 36
13 I ]gallbladder wall
colon wall

12 s k i n muscle

12 kidney

15 mammary gland

b ovarian c y s t wall

505
 GlAN G. G A L L 0 AND PIETRO RADAELLI

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