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Cephalexin
CEPHALEXIN
Louis P.MarreUi
LOUIS P. MARRELLI
TABLE OF CONTENTS Page 1. Description 1.1 Name: Cephalexin 1.2 Formula and Molecular Weight 1.3 Isomers 1.4 Hydrates 1.5 Appearance 2. Physical Properties 2.1 Spectra 2.11 Infrared Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Ultraviolet Absorbance 2.2 Crystal Properties 2.21 X-Ray Powder Diffraction 2.22 Differential Thermal Analysis 2.3 Solubility 2.4 Dissociation Constant 2.5 Optical Rotatory Dispersion 3 . Cephalexin Stability 4. Synthesis 5. Methods of Analysis 5.1 Identification Tests 5.2 Quantitative Methods 5.21 Titration 5.22 Colorimetric Determination 5.23 Thin Layer Chromatography 5.24 Paper Chromatography 5.25 Column Chromatography 5.26 Electrophoresis 5.27 Microbiological Assays 5.3 Assay Methods for Intermediates and Imp uritie8 5.31 7-Aminodesacetoxycephalosporanic Acid (7-ADCA) 5.32 Phenylglycine 6. Protein Binding 7. Pharmacokinetics 8. References
22
3 3 3 4 4
4 4 4 4 b
7 7 7 7 7 10 10 10 10 11 11 11 11 12 14 15 15 16 16 16 16 17 18 18 20
CEPHALEXIN
1.
Description 1.1
Name: Cephalexin Chemical Abstracts
designates cephalexin as 5-thia1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, 7-( 2-amino2-ph eny1-ac etamido) -3-methyl-8-ox0 Ce halexin monohydrate is also known as 5-thia-1azabicyclo f4.2.01 oct-2-ene-2-carboxylic acid, 7-[(aIUinophenylacetyl) amino]-3-methyl-8-oxo monohydrate' * 7-( D-2amino-2-phenylacetamido) -3-me thyl-3-cephem-4-carboxylic acid monohydrat3 * and 7-( D-cr-aminophenylacetamido)-3-methyl-3cephem-4-carboxylic acid monohydrate'
.
1.2
Formula and Molecular Weight
c16
365.41
17 N304S'H20
23
LOUIS P. MARRELLI
1.3 Isomers
The s y n t h e s i s of t h e L epimer o f cephalexin has been r e p o r t e d The D-isomer e x h i b i t s considerably more b i o l o g i c a l a c t i v i t y than t h e L-isomer. P e n i c i l l i n s derived from D-cr-amino a c i d s a l s o show more b i o l o g i c a l a c t i v i t y than t h e i r L-epimerg ' 6 .
.
1.4
Hydrates P f e i f f e r e t . a1.' provided x-ray powder d i f f r a c t i o n d a t a f o r the monohFdrac and dihydrate of cephlexin. Cephal e x i n w a s found t o c r y s t a l l i z e from aqueous s o l u t i o n s a t room temperature as t h e dihydrate b u t converted t o t h e monohydrate when t h e r e l a t i v e humidity was below 70%. Refer t o Section 2.21. Appearance Cephalexin i s a white t o cream-colored c r y s t a l l i n e powder, having a c h a r a c t e r i s t i c odor.
1.5
2.
Physical P r o p e r t i e s 2.1
Spectra
I n f r a r e d Spectrum The i n f r a r e d spectrum of cephalexin monohydrate recorded as a potassium bromide d i & i s presented i n Figure 1. I n t e r p r e t a t i o n of t h e spectrum is given i n Table 18. Changes i n the B-lactam carbonyl s t r e t c h i n g region (1760 cm-l) can i n d i c a t e opening of the 6-lactam ring. Morin' and coworkers have shown a r e l a t i o n s h i p between t h e B-lactam carbonyl s t r e t c h i n g frequency and b i o l o g i c a l a c t i v i t y . The importance of t h i s s t r e t c h i n g frequency has been discussed i n a recent reviewlo. 2.11
Nuclear Magnetic Resonance Spectrum Figure 2 shows the proton magnetic resonance spectrum of cephalexin monohydrate. The solvent used was deuterium oxide containing a small amount of t r i f l u o r o a c e t i c a c i d t o enhance s o l u b i l i t y . 3-( Trimethylsilyl) -propanes u l f o n i c acid, sodium s a l t w a s added as t h e i n t e r n a l r e ference. "he spectrum was recorded on a Varian "60-A i n s t r u ment. The assignment of t h e spectrum i s shown i n Table IIe A most c h a r a c t e r i s t i c region of t h e NMR spectrum i s t h a t o r i g i n a t i n g from the two 6-lactam r i n g protons, H(6) and 2.12
.
H(7)
24
4000
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
WAVENUMBER CM-'
FIGURE 1. Infrared spectrum of cephalexin monohydrate (potassium bromide disc).
600
400
7.0
5 .O
4.0 3.0 2.0 1.0 PPM (a) FIGURE 2. NMR spectrum of cephalexin monohydrate (020 + trifluoroacetic acid). 8.0
6.0
0
TABLE I
Infrared Spectrum of Cephalexin Monohydrate
-1
Wavelength (cm )
- 3000 (series of broad bands)
3500
2600 (broad)
Assignment OH from H 0 and amide NH stretch 2
+
Y3
1760
@-lactam c
1690
h i d e c = o stretch
(1600 [very broad] (1400
C-0
1550 (unresolved) 820
- 690
0
-
=
o stretch
(carboxylate stretching)
h i d e I1 band Mainly skelectal vibrations including out-of-plane aromatic hydrogen bending, characteristic of monosubstituted aromatic ring
TABLE I1
Proton Magnetic Resonance Spectrum Peak Assignments p .p .m. (6)
Relative Intensity
2.07
Mu1t ip 1icity
Assignment
3
singlet
CH3
(3)
3.30
2
quartet (AB)
(2)
4.85
-
CH*
singlet
HOD
(solvent)
4.97
1
doublet (J=4Hz)
H
(6)
5.34
1
singlet
H
(benzyl)
5.67
1
doublet (J=4HZ)
H
(7)
7.60
5
sing let
‘SH5
CEPHALEXI N
2.13
U l t r a v i o l e t Absorbance An aqueous s o l u t i o n of cephalexin e i b i t s a Pcm W absorption maximum a t 262 nm (Figure 3 ) . The El% reported f o r cephalexin (on an anhydrous b a s i s ) was 23611 The u l t r a v i o l e t absorbance of cephalexin a6 w e l l as o t h e r cephalosporins has been a t t r i b u t e d t o t h e 0 = C-N-C = Cchromophore of t h e ring1*. Chou” u t i l i z e d t h e W absorption a t 262 nm t o determine t h e cephalexin content of s o l u t i o n f r a c t i o n s i s o l a t e d from human urine.
.
2.2
C r y s t a l Properties 2.21
X-Ray Powder Diffraction Cephalexin was found t o occur i n s e v e r a l solvated c r y s t a l forms, and o f t e n i n widely varying mixtures of these f o r m d 4 . Some of t h e solvated c r y s t a l forms prepared were the dihydrate, monohydrate, d i a c e t o n i t r i l a t e , formamid a t e , methanolate, and a c e t o n i t r i l e hydrate. X-ray powder d i f f r a c t i o n data f o r cephalexin monohydrate i s presented i n Table 111. 2.22
D i f f e r e n t i a l Thermal Analysis D i f f e r e n t i a l thermal analysis’ of cephalexin monohydrate was conducted on a W o n t Model 950 thermal analyzer i n a nitrogen atmosphere. A heating r a t e of 2OoC p e r minute w a s u t i l i z e d . An endotherm w a s noted at 123OC i n d i c a t i n g the loss of water, and an exotherm of 203OC i n d i c a t i n g decomposition. 2.3
Solubility The s o l u b i l i t y of cephalexin monohydrate i n t h e following solvents has been reported’ : Solvent
Mg. Cephalexin Monohydrate Per M l . Solvent, 25OC.
13-5 3- 4
Water Methanol N-oc tan01 Chloroform Ether
0.03 4.01 4.01
Table I V r e l a t e s t h e s o l u b i l i t y of cephalexin monohydrate i n water as a function of pH.
29
LOUIS P. MARRELLI
2.0
1.5
1.0
O.!
0.0 I
I
I
250
300
monohydrate. 49 mcg per ml in H20
30
I
350
CEPHALEXIN
TABLE I11
of Cephalexin Monohydrate Norelco De Bye-Scherrer Camera
X-Ray Powder Diffraction Pattern
Radiation; Cu/Ni.
Cephalexin Monohydrate
d
1/11 0.40 1.00 0.30 0.20 0.50 0.50 0.20 0.40 1.00 0.50 0.40 0.40 0.60 0.60 0.70 0.70 0.80 0.30 0.60 0.60 0.40 0.60 0.40 0.20 0.40 0.10 0.30 0.15 0.30 0.30 0.10 0.05 0.02 0.05 0.05 0.05 0.10 0.05 0.02 0.02
-
-
15.15 11.85 11.00 9.36 8.55 7.86 6.89 5.98 5.39 4.97 4.76 4.57 4.39 4.22 4.00 3.86 3.60 3.46 3.24 3.10 2.98 2.90 2.81 2.73 2.68 2.63 2.47 2.41 2.31 2.25 2.12 2.09 2.01 1.93 1.87 1.85 1.82 1.72 1.66 1.62 31
TABLE IV
Solubility
PH
- pH Profile of Cephalexin in Water
(37°C)
.
Cephalexin Monohydrate mg I d .
. .
Cephalexin Monohydrate mg Iml
~
W
N
2.3
13
2.5
16
3 .O
.o
24
3.5
40
4.0
75
5.0
100
CEPHALEXIN
2.4
Dissociation Constant (pKa) The following d i s s o c i a t i o n constants were reported:
Solvent
rn
66% 66% DMF
Carboxyl
Amino
5-2
7.3
5.3
h0
7.3
7.1
Reference 4
17
18
2.5
Optical Rotatory Dispersion Optical r o t a t i o n has been used as an a u x i l i a r y method f o r the q u a n t i t a t i o n of cephalexir?. The s p e c i f i c r o t a t i o n [@ID reported f o r cephalexin, calculated on an anhydrous b a s i s , w a s +153O (C = 1.0 i n & 0)l 9 .
3.0
Cephalexin S t a b i l i t y The s t a b i l i t y of cephalexin i n solution i s dependent on pH, degrading r a p i d l y i n b a s i c media and remaining s t a b l e under mild a c i d i c conditions. No loss i n cephalexin a c t i v i t y occurred i n 72 hours a t 25OC i n the pH range from 3 t o 5. The r a t e of degradation found at pH 6 and pH 7 (25OC) was approximately 3% and 18%per day, r e s p e c t i v e l y 0 . With r e f r i g e r a t i o n , no appreciable loss occurs between pH 3 and pH 7 a f t e r 72 hours. I n U.S.P. hydrochloric a c i d b u f f e r (pH 1.21, cephalexin l o s t 5% a c t i v i t y i n 24 hours a t 37OC as compared t o a 45% loss i n phosphate b u f f e r at pH 6 . 9 l The a n t i b i o t i c r e t a i n s a c t i v i t y well i n serum and u r i n e as no loss i n a c t i v i t y was noted a f t e r storage a t -2OOC f o r 14 Cephalexin i n serum w a s found t o l o s e lo%, 5% and dayg2 75% a c t i v i t y , respectively, a f t e r storage a t 5'C, 25OC, and 37OC f o r 48 h o u r d l ' z z . Some organisms have been found t o produce a @-lact m a s e ( cephalosporinase) which can r a p i d l y degrade cephalexid' Degradation of cephalexin a l s o r e s u l t s from heat, s t r o n g alkali, strong a c i d s and u l t r a v i o l e t l i g h t (260 nm)
.
.
.
4.0
.
Synthesis Two synthetic r o u t e s of general a p p l i c a b i l i t y have been proposed f o r cephalexin'
.
The f i r s t method i s based on the cleavage of t h e acetoxyl group from cephaloglycid (I) by hydrogenation o r more s a t i s f a c t o r i l y , from N-t-butoxy-carbonylcephaloglycin (11) t o profuce the corresponding desacetoxy analogs V (cephalexin) and I11 as shown i n Figure 4, Scheme I. The t-
'
33
,
LOUIS P. MARRELLI
SCHEME I &lfON::@ !-
N
I, R-H II. A=Boc
/
CHzOAc
@:b.lii> N
C02H 111. R-Boc
/
COzH
V. R-H
SCHEME I1
CO2H
COZH
V
IV
FIGURE 4. Synthasis routes for eaphelexin.
34
CH3
CEPHALEXIN
butoxy-carbonyl (BOC) group was removed from I11 with trifluoroacetic a c i d and the r e a c t i o n product was converted t o desacetoxycephaloglycin ( cephalexin, V) by treatment with Amberlite LA-1 r e s i n .
.
The second method w a s s i m i l a r t o t h a t previously The nucleus, 7-aminodesused t o obtain cephaloglycid' acetoxycephalosporanic a c i d ( 7-ADCA)25 was prepared, acylated with BOC-protected D-phenylglycine employing a mixed anhydride synthesis, and then deblocked with t r i f l u o r o a c e t i c a c i d as shown i n Figure 4, Scheme 11.
5.0 Methods of Analysis 5.1 I d e n t i f i c a t i o n Tests
Cephalexin may be i d e n t i f i e d by i n f r a r e d spectroscopy. B r i t i s h Pharmacopoeia' u t i l i z e s two charact e r i s t i c color r e a c t i o n s f o r i d e n t i t y . Thin l a y e r (Sec. 5.23), paper (Sec. 5.24), and column chromatography (Sec. 5.25) have been u t i l i z e d f o r i d e n t i t y purposes.
5.2
Q u a n t i t a t i v e Methods 5.21
--.T i t r a t i o n
The iodometric t i t r a t i o n procedure has The been used f o r the determination of c e p h a l e x i d 6 method i s based on the f a c t t h a t t h e i n t a c t cephalexin molecule does not consume iodine, whereas the alkali-hydrolysis product of cephalexin does. Alkaline hydrolysis of cephalexin r e s u l t s i n cleavage of t h e @-lactam ring. V a r i a t i o n s i n hydrolysis time, temperature, pH of t h e iodine solut i o n and concentration o f cephalexin present influence t h e consumption of iodine by t h e test solution. The method compares favorably t o t h e microbiological cylinder-plate method (Sec. 5.27) i n accuracy, and is much more rapid. Possible intermediates used i n the s y n t h e s i s such as 7-ADCA w i l l also respond t o the test.
'*'.
An automated iodometric assay has been used r e c e n t l y f o r t h e assay of cephalexin and formulations thereof4 The procedure incorporates a sample hydrolysis step N, at 37OC f o r 10 minutes followed by a 5-minute iodine consumption s t e p (pH 5.3-5.5, 37°C). Concentration of the sample i s r e l a t e d t o the decrease i n iodine color measured a t 350 MI. A reference standard i s run concurrently through the analyzer f o r comparative purposes. The automated system gives excellent l i n e a r i t y of response f o r t h e
!A;:(
35
LOUIS P. MARRELLI
-
recommended concentration range of cephalexin (0 1.5 mg. per m l . sample s o l u t i o n ) , with a l l standard curve p l o t s passing through t h e origin. The r e p r o d u c i b i l i t y of t h e method on t h e same sample o r standard s o l u t i o n on a given day i s generally b e t t e r than +l% r e l a t i v e standard deviation (RSD). Cephalexin can be titf;ated with p e r c h l o r i c a c i d in a g l a c i a l acetic acid m e d i d 9 . Crystal v i o l e t indicator (2% i n g l a c i a l a c e t i c acid) may be used t o determine t h e endpoint.
Moll and D6keio have reported using a formol t i t r a t i o n procedure f o r t h e determination of cephal e x i n , a m p i c i l l i n and r e l a t e d compounds. I n t h i s procedure 4 m l . of d i l u t e formaldehyde s o l u t i o n ( n e u t r a l i z e d t o the phenolphthalein end p o i n t ) is added t o 10 m l . of an aqueous s o l u t i o n containing 15.0 mg. of cephalexin. After 2 minutes t h e s o l u t i o n is t i t r a t e d with 0.02N sodium hydroxide. A p r e c i s i o n of +O.% RSD could be achieved i n t h e t i t r a t i o n o f cephalexin mozohydrate raw m a t e r i a l samples. Acidic compounds as well as amino a c i d s must not b e present as i m p u r i t i e s i n t h e sample. The formol t i t r a t i o n takes advantage of the r e a c t i o n between an amino a c i d and formaldehyde as a means of suppressing t h e b a s i c i t y o f the amino group and thus making p o s s i b l e t h e t i t r a t i o n of t h e acid. Colorimetric Determination Reaction with hydroxylamine has been u t i l i z e d f o r t h e colorimetric determination of cephaThe method i s based on t h e f a c t t h a t hydroxylexid’ ’32 lamine cleaves t h e 8-lactam r i n g (pH 7.0) t o form a hydroxamic a c i d which forms a colored complex with f e r r i c ion. Degradation products o r intermediates having an i n t a c t 8lactam r i n g r e a c t as well. 5.22
.
Kirschbaud’ has described a procedure f o r the colorimetric determination of t h e a n t i b i o t i c cephradine’ and r e l a t e d cephalosporins. An aqueous s o l u t i o n of t h e compound (1 t o 30 mcg. p e r ml.) i s r e a c t e d with sodium hydroxide, p a r t i a l l y n e u t r a l i z e d , and then r e a c t e d with 5*5’dithiobis-( 2-nitrobenzoic acid) resulting i n t h e formation of a y llow chromophore (412 nm.). The molar a b s o r p t i v i t y E x lo-$ reported f o r cephalexin when c a r r i e d through t h i s procedure w a s 1.29. The formation of the yellow chromophore w a s a t t r i b u t e d t o the presence of t h e R C q -CO-cephalo-
’Cephradine i s t h e generic name
-
f o r 7-[D-2-amino-2-(lq4c yclohexadienyl) ace tamido] desac e toxycephalosporanic acid 36
CEPHALEXIN
sporin nucleus, i n which R i s a mono-, di- or tri- enyl cyclohexyl ring. A s p e c i f i c colorimetric t e s t w a s developed f o r t h e determination of cephalosporin d e r i v a t i v e s having the following i n t a c t s i d e chain i n t h e 7- p o s i t i o n : R (3%-CO-cephalosporin nucleus, R being a heterocyclic o r aromatic r i n 8 ' . The D-phenylglycine d e r i v a t i v e s of both 7-ADCA (cephalexin) and 7-ACA ( cephaloglycin)2 respond well. These compounds (0.5 1.0 mg. per m l . i n %O) react with acetone and sodium hydroxide a t 1 0 0 ° C t o form c h a r a c t e r i s t i c A t the 1 mg. per ml. l e v e l , t h i s red chromophores (520 nm.). t e s t w i l l v i s u a l l y d i f f e r e n t i a t e cephalexin from cephradine.
-
-
Cephaloglycin is the generic name f o r 7-( D-a-aminophenylacetamido) cephalosporanic a c i d 31
LOUIS P. MARRELLI
5.23
Thin Layer Chromatography The following t h i n l a y e r chromatographic systems have been reported:
TABLE V Adsorbent
Solvent System
Ref.
Rf -
-
Silica G e l
Ace t o n i t r i l e / W a ter
0.67
13
Silica Gel
Ethyl Acetate/Acetone/Acetic Acid/ Water (5:2:2:1)
0.22
35
Cellulose Chromatogram Sheet
Butanol/Acetic Acid/ Water (3:l:l)
-
36
Sheet
Ethyl Acetate/Acetic A c i o a t e r (3:l:l)
-
36
Sheet
Acetonitrile/Ethyl Acet at e/Water ( 3 :1 :1)
-
36
Cellulose
Acetonitrilefiater (3:1)
0.50
37
Cellulose
Butanol/Acetic Acid/ 0.70 Water (3:l:l)
37
(3:l)
Cellulose is t h e p r e f e r r e d sorbent s i n c e i t i s i n e r t toward cephalexin. Additional t h i n l a y e r chromatography systems used f o r cephalexin and o t h e r cephalosporins have been t a b u l a t e d ? . Cephalexin may be detected by u l t r a v i o l e t absorbance and quenching, ninhydrin, i o d o p l a t i n a t e , a l k a l i n e permanganate, and phosphomolybdic a c i d sprays. Iodine detection and vanillin-phosphoric a c i d spray have a l s o been u t i l i z e d . O f t h e microorganisms used, Sarcina l u t e a i s p r e f e r r e d over B a c i l l u s s u b t i l i s o r Staphylococcus aureus.
-
38
CEPHALEXIN
5.24
Paper Chromatography The following paper chromatographic systems have been reported: Solvent Systems Butanol/Ac e t i c AcidJWate r (3:l:l)
Rf -
Ref. -
0.60
13, 36, 38
-
36
Ethyl Acetate/Acetic Acid/ Water (3:l:l)
Whatman No. 1*,untreated, was used with both solvent systems and the e q u i l i b r a t i n g solvent w a s t h e same as t h e developing solvent. Additional paper chromatographic systems f o r cephalexin and o t h e r cephalosporins have been t a b u l a t e d 8 . The butanol/acetic aciuwater (3 :l:l) system w i l l separate cephaloglycin from cephalexin, cephaloglycin being l e s s mobile. An acetonitrile/water (9:l) s y s t e d 9 using Whatman No. 3 paper buffered a t pH 5.0 (16 hour development) h a s been u t i l i z e d t o s e p a r a t e cephradine from cephalexin, cephradine being l e s s mobile. The developed chromatogram can be examined under u l t r a v i o l e t l i g h t , dipped i n ninhydrin, or bioautographed, using Sarcina lutea'o
.
5.25
Column Chromatography The Moore-Stein amino acid analyzer has been used f o r the determination of cephalexin i n u r i n e samplesb1 Beckman Custom Research Resin Type PA-35, packed t o a height of 9.0 cm. i n a water-jacketed column (0.9-cm. i.d. x 2'3-cm. length) w a s used f o r t h e separation. The u r i n e sample was d i l u t e d with an equal volume of sodium c i t r a t e buffer (pH 2.2) and 100 h applied t o t h e column. The e l u t i o n time for cephalexin was approximately 61 minutes. Excellent agreement was found between the analyzer method and the microbiological method (Section 5.27) on a s e r i e s of u r i n e samples tested. I n a d d i t i o n , t h i s technique has been u s e f u l i n determining low l e v e l s of 7-ADCA and phenylglycine i n cephalexin'l (Section 5.3). Determination of t h e l e s s a c t i v e ( b i o l o g i c a l l y ) L-isomer i n cephalexin by t h e Moore-Stein amino a c i d analyzer has been r e p o r t e 8 . Chou" used the anionic r e s i n , Bio-Rad AG2 x8 ( a c e t a t e form), t o i s o l a t e cephalexin from human urine.
.
-
*Whatman chromatography' paper, Reeve Angel, 9 Bridewell Place, C l i f t o n , New Jersey 39
LOUIS P. MARRELLI
Electrophoresis Paper e l e c t r o p h o r e s i s has been u t i l i z e d test f o r impurities by the B r i t i s h Pharmacopoeia' as present i n cephalexin (Section 5.3).
5.26
Microbiological Assays Microbiological assays f o r cephalexin have been discussed by Marrelli'*, Wick" Mann'' and Simmond' and are l i s t e d i n the Federal R e g i s t e r C 6 . B r i e f l y , the two p l a t e systems well s u i t e d f o r t h e determination of cephalexin i n pharmaceutical formulations a r e t h e cylinder p l a t e methods u t i l i z i n g S t a hylococcus aureus ( ATCC 6535) and Bacillus s u b t i l i s (A--e-ac range f o r both i s approximately 2.5 t o 20 mcg. of cephalexin p e r ml.'2 '" The B. s u b t i l i s p l a t e test h a s an advantage over t h e S. aureus p l a t e t e s t i n t h a t b e t t e r defined zones of inhTbition a r e obtained, thereby increasing t h e assay precision4 Since degradation product6 of cephalexin possess p r a c t i c a l l y no antimicrobial a c t i v i t y 2 r a p i d and p r e c i s e photometric microbiological assays a r e p o s s i b l e with cephalexin. The t e s t organism f o r the photometric assay i s Staphylococcus aureus 9144, 3 t o 3.5 hours being required f o r incubation. I n Antibiotic No. 3 Broth, t h e concentration range is 0.2 t o 2.0 mcg. p e r m l . of broth. I f t h e automated AUTOTU@ System is used, p r e c i s i o n i n the order o f 1 2 % i s possible". Cephalexin i n b i o l o g i c a l f l u i d s may be assayed by a Sarcina l u t e a p l a t e system. The concentration range f o r assay i s 0.2 t o 3.5 m~g./ml.~+
5.27
.
5.3
Methods f o r Intermediates and Impurities
5.31 7-Aminodesacetoxycephalosporanic Acid
( 7-ADCA) Cole'' u t i l i z e d the Moore-Stein amino a c i d analyzer f o r the determination of 7-ADCA i n cephalexin. Column s p e c i f i c a t i o n s were o u t l i n e d i n Section 5.25. Twenty-five milligrams of cephalexin sample were dissolved w i t h sodium c i t r a t e b u f f e r (pH 2.2) t o a t o t a l volume of 10.0 m l . and 1.0 m l . applied t o t h e column. The e l u t i o n time f o r 7-ADCA w a s approximately 45 minutes. 'The s e n s i t i v i t y of the assay w a s 0.B 7-AKA.
A colorimetric procedure was developed by M ~ ~ r r e l l iwhich '~ permitted t h e d i r e c t determination of The prolow l e v e l s of 7 - A K A i n cephalexin (0.4 1.5%). cedure i s based on t h e i n t e r a c t i o n of 7-ADCA with ninhydrin under c o n t r o l l e d conditions t o produce a s p e c i f i c chromophore
-
40
CEPHALEXIN
.
( A max. a t 480 nm.) Compounds having an a-amino group adjacent t o a B-lactam r i n g respond i n general t o t h e t e s t . B r i t i s h Pharmacopoeia' u t i l i z e d a paper e l e c t r o phoresis technique which estimated t h e 7-ADCA content i n cephalexin a t the l% l e v e l . The b u f f e r s o l u t i o n s p e c i f i e d i n the t e s t consisted of a mixture of 5 m l . of formic acid, 25 m l . of g l a c i a l a c e t i c a c i d , 30 m l . of acetone and water t o a t o t a l volume o f 1000 m l . A 2.0 pl. a l i q u o t of the cephalexin sample s o l u t i o n (5.0% w/v i n 0.5N HC1) along with the s p e c i f i e d amounts of reference standards and markers were applied t o the paper. The voltage was adjusted t o about 20 v o l t s per cm. of paper and e l e c t r o p h o r e s i s w a s allowed t o proceed u n t i l the c r y s t a l v i o l e t spot moved 9 cm. from the base l i n e . Both ninhydrin spray and W were used f o r detection purposes.
5.32
Phenylglycine The ChromatonraDhic Drocedures o u t l i n e d i n Section 5.31 f o r the determination o f 7 - m A i n cephal e x i n have been concurrently used for the determination of phenylglycine i n cephalexin. The e l u t i o n time f o r phenylglycine i n the amino a c i d analyzer assay was approximately '56 minutes. The s e n s i t i v i t y of the assay w a s 0.01%. The paper electrophoresis technique permitted estimation of phenylglycine a t the 1%l e v e l . Hussepe has u t i l i z e d a chromatographic procedure f o r phenylglycine similar t o t h a t of the amino a c i d analyzer assay but incorporating a Fluramm detection system'. I
t
&
Hoffman-LaRoche Inc., Nutley, New J e r s e y
41
LOUIS P. MARRELLI
6.
P r o t e i n Binding Various values have been reported f o r t h e percentage of cephalexin bound t o serum protein. Wicp' concluded t h a t serum i n a c t i v a t i o n o r p r o t e i n binding of cephalexin i s low. Addition of serum t o broth medium did n o t a f f e c t -.i n -v .- i t r o minimal i n h i b i t o r y concentration determinations. Cephalexin assays i n pH 7 b u f f e r and human serum r e s u l t e d i n i d e n t i c a l standard curves when 6-mm d i s c s were satur a t e d with s o l u t i o n s and t e s t e d by a Sarcina l u t e a microb i o l o g i c a l assay. U t i l i z i n g a similar method, G r i f f i t h and Black!) found t h a t p r o t e i n binding of cephalexin i n human berum was % a t concentrations above 1.0 bg./ml. Naumann and Fedde9O a l s o found and 4196 a t 0.2 yg./ml. t h a t the amount o f cephalexin bound t o serum p r o t e i n s varied with t h e cephalexin concentration. Using an u l t r a f i l t r a t i o n method, Kind e t . al.51 estimated t h e serum binding as being 15%. O v a l r q h a n and Muggletor?2 obtained a value of 43% by u t i l i z a t i o n of t h e u l t r a f i l t r a t i o n technique.
7.
Pharmacokinetics Oral doses of cephalexin are r a p i d l y absorbed by animals and man, r e s u l t i n r a t h e r high blood serum l e v e l s , and a r e excreted unchanged i n t h e urine. Wick?* found t h a t a 20-mg./kg. o r a l dose of cephalexin i n mice gave Wells e t . al.5' a blood serum l e v e l of 18 pg./ml. reported a similar value (17 Fg./ml.) with a 10 mg./kg. o r a l dose i n dog. The r a p i d i t y of o r a l absorption was demonstrated by t h e f a c t t h a t a n t i b a c t e r i a l a c t i v i t y i n t h e serum was t h e same within 1.5 hours a f t e r o r a l or intramuscular administration; t h e r e a f t e r , the l e v e l s were higher f o r t h e o r a l dose. I n man, a f t e r a 500-mg. dose t h e mean of t h e peak a n t i b i o t i c a c t i v i t i e s found '5' 'J and by s e v e r a l i n v e s t i g a t o r s w a s 15 pg./ml.' u s u a l l y occurred a t 1-1.2 hours a f t e r treatment. The compound is almost completely absorbed from the upper small i n t e s t i n e i n both m a n and animals6'J6. I n addition, t h e a n t i b i o t i c i s excreted unchanged i n u r i n e with almost 100% r e c o ~ e r f ' ~ ' 5 9 . A meal j u s t p r i o r t o treatment r e s u l t e d i n lower blood l e v e l s and increased t h e time required for peak t i t e ? ' 6 0
--
*
"'
42
.
CEPHALEXIN
Kirby et. a1.61 c a l c u l a t e d the serum h a l f - l i f e of intravenously administered cephalexin as 36 minutes. Kabins et. al.62 and Naumann and FeddeSO c a l c u l a t e d the serum h a l f - l i f e a f t e r o r a l dosage a s 54 minutes. al.5 found t h a t probenecid increased the Thornhill et. peak serum concentration by 50%. but Meyers e t . al.55 found a l e s s e r e f f e c t . Linquist --e t . a1.6sexamined the disappearance of cephalexin from the blood s e r a of aneph41.0 r i c p a t i e n t s . H a l f - l i f e values ranged from 23.5 hours with a mean of 31 hours, c l e a r l y demonstrating t h e dependence upon the kidney f o r excretion.
-
43
--
LOUIS P. MARRELLI
8. References
1. The United States Pharmacopoeia, XIX, Proof p. 2122. 2. Federal Register, 21CFR148w.6. 3. British Pharmacopoeia, 1973, p. 87. 4. Ryan, C.W., Simon, R.L., and Van Heyningen, E.M., J. Med. Chem., l2, 310 (1969). 5. Doyle, F.P., Fosker, G.R., Naylor, J.H.C., and Smith, H., J. Chem. SOC., 1440 (1962). 6. Analytical Profiles of Drug Substances, Vol. 2 (K. Florey, ed.) p.4, Academic Press, New York and London, 1973. 7. Pfeiffer, R.R., Yang, K . S . and Tucker, M.A., J. Phann. Sci., 59, 1809 (1970). 8. Underbrink, C.D., Eli Lilly Analytical Development, Unpublished Data. 9. Morin, R.B., Jackson, B.G., Mueller, R.A., Lavagnino, E.R., Scanlon, W.B., and Andrews,, S.L., J. her. Chem. SOC., 91, 1401 (1969). 10. Flynn, E.H., ed., Cephalosporins and Penicillins. Chemistry and Biology, Academic Press, New York and London (1972), p. 315. 11. Flynn, op. cit., p. 631. 12. Chawette, R.R., et. al., J. Am. Chem. SOC., 84, 3401 (1962). 13. Chou, T.S., J. Med. Chem., l2, 925 (1969). 14. Pfeiffer, R.R., Eli Lilly Analytical Development, Personal Communication, (1969). 15. Cole, T.E. , Eli Lilly Analytical Development, Personal Communication, (1968). 16. Pfeiffer, R.R., Eli Lilly Analytical Development, Personal Communication, (1970). 17. Flynn, op. cit., p. 310. 18. Hargrove, W.W. , Eli Lilly and Company, Personal Communication, (1967). 19. Flynn, op. cit., p . 633. 20. Winely, C.L., Eli Lilly Analytical Development Laboratories, Unpublished Data. 21. Simmons, R. J. , Anal. Microbiol., 11. , 193 (1972). 22. Wick, W.E., Appl. Microbiol., l5, 765 (1967). 23. Ott, J.L., and Godzeski, C.W., Antimicrob. Ag. Chemother. 1966, 75 (1967). 24. Spencer, J.L., Flynn, E.H., Roeske, R.W., Siu, F.Y., and Chauvette, R.R., J. Med. Chem., 2, 746 (1966). 25. Stedman, R.J., Swered, K., Hoover, J.R.E., J. Med. * .Chem 7, 117 (1964). 26. Federal Register, 21CFR141.506. 44
CEPHALEXIN
27. British Pharmacopoeia, p. 88 (1973). 28. Stevenson, C.E., and Bechtel, L.D. (1971) Publication submitted for review in J. Pharm. Sci. 29. Marrelli, L.P., Eli Lilly and Company, Personal Cmunication (1967). , -~ 30. Moll, F., and Dzker, H., Arch. Phann., Berl., 305 (7), 548 (1972). 31. Federal Register, 21CFR141.507. 32. Plynn, op. cit., p. 615. 33. Kirschbaum, J., J. Pharm. Sci., 63, 923 (1974). 34. Marrelli, L.P., J. Pharm. Sci., 6 l , 1647 (1972). 35. Thomas, P.N., Eli Lilly and Company, Personal Cmunication (1972). 36. Sullivan, H.R., Billings, R.E., and McMahon, R.E., J. Antibio., 22, 195 (1969). 37. Flynn, op. cit., p. 621. 38. Flynn, op. cit., p. 620. 39. Marrelli, L.P., Eli Lilly and Company, Personal Cmunication (1972). 40. Miller, R.P., Antibiot. and Chemother., 12, 689 (1962). 41. Flynn, op. cit., pp. 629, 680. 42. Flynn, op. cit., p. 610. 43. Flynn, op. cit., p. 497. 207 (1972). 44. Mann, J.M. , Anal. Microbiol., Simmons, R.J., Anal. Microbiol., s, 193 (1972) 45. 46. Federal Register, CFR148~6. 47. Kuzel, N.R. and Kavanagh, F.W., J. Phann. Sci., 60, 767 (1971). 48. Hussey, R.L., Eli Lilly Analytical Development, Personal Communication (1974). 49. Griffith, R.S. and Black, H.R., Postgrad. Med. J., 47, February Suppl. , 32 (1971). 50. Kumann, P. and Fedder, J., Int. J. Clin. Pharmacol., Suppl., 2, 6 (1970). 51. Kind, A.C., Kestle, D.G., Standiford, H.C. and Kirby, W.M.M., Antimicrob. Ag. Chemother., 405 (1968). 52. Flynn, op. cit., p. 438. 53. Wells, J.S., Froman, R.O., Gibson, W.R., Owen, N.V., and Anderson, R.C., Antimicrob. Ag. Chemother., 489 (1968). 54. Kunin, C.M., and Finkelberg, Z . , Ann, Inst. Med., 72, 349 (1970). 55. Eyers, B.R., Kaplan, K., and Weinstein, L., Clin. Pharmacol. Ther., 10, 810 (1969). 56. Muggleton, P.W., O'Callaghan, C.H., Foord, R.O., Kirby, S.M., and Ryan, D.M., Antimicrob. Ag.
a,
45
LOUIS P. MARRELLI
57. 58. 59. 60. 61. 62. 63.
Chemother., 353 (1968). Perkins, R.L., Apicella, M.A., Lee, I., Cuppage, F.E., and Saslaw, S., J. Lab. Clin. Med., 7 l , 75 (1968). Thornhill, T . S . , Levison, M.E., Johnson, W.D., and Kaye, D., Appl. Microbiol., l7, 457 (1969). Gower, P.E. and Dash, C.H., Br. J. Pharmac., 37, 738 (1969). O'Callaghan, C.H., Footill, J.P.R., and Robinson, W.D., J. Pharm. Pharmac., 23, 50 (1971). Kirby, W.M.M., de Maine, J.B., and Serrill, W.S., Postgrad. Med. J., 47, February Suppl., 46 (1971). Kabins, S.A., Kelner, B., Walton, E., and Goldstein, E., her. J. Med. Sci., 259, 133 (1970). Linquist, J.A., Siddiqui, J.Y., and Smith, I.M., New Engl. J. Med., 283, 720 (1970).
ACKNOWLEDGEMENTS The author wishes to express his indebtedness to Dr. C. L. Winely for his contribution of the sections on microbiological assays, protein binding and pharmacokinetics.
46
Louis P.MarreUi
LOUIS P. MARRELLI
TABLE OF CONTENTS Page 1. Description 1.1 Name: Cephalexin 1.2 Formula and Molecular Weight 1.3 Isomers 1.4 Hydrates 1.5 Appearance 2. Physical Properties 2.1 Spectra 2.11 Infrared Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Ultraviolet Absorbance 2.2 Crystal Properties 2.21 X-Ray Powder Diffraction 2.22 Differential Thermal Analysis 2.3 Solubility 2.4 Dissociation Constant 2.5 Optical Rotatory Dispersion 3 . Cephalexin Stability 4. Synthesis 5. Methods of Analysis 5.1 Identification Tests 5.2 Quantitative Methods 5.21 Titration 5.22 Colorimetric Determination 5.23 Thin Layer Chromatography 5.24 Paper Chromatography 5.25 Column Chromatography 5.26 Electrophoresis 5.27 Microbiological Assays 5.3 Assay Methods for Intermediates and Imp uritie8 5.31 7-Aminodesacetoxycephalosporanic Acid (7-ADCA) 5.32 Phenylglycine 6. Protein Binding 7. Pharmacokinetics 8. References
22
3 3 3 4 4
4 4 4 4 b
7 7 7 7 7 10 10 10 10 11 11 11 11 12 14 15 15 16 16 16 16 17 18 18 20
CEPHALEXIN
1.
Description 1.1
Name: Cephalexin Chemical Abstracts
designates cephalexin as 5-thia1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, 7-( 2-amino2-ph eny1-ac etamido) -3-methyl-8-ox0 Ce halexin monohydrate is also known as 5-thia-1azabicyclo f4.2.01 oct-2-ene-2-carboxylic acid, 7-[(aIUinophenylacetyl) amino]-3-methyl-8-oxo monohydrate' * 7-( D-2amino-2-phenylacetamido) -3-me thyl-3-cephem-4-carboxylic acid monohydrat3 * and 7-( D-cr-aminophenylacetamido)-3-methyl-3cephem-4-carboxylic acid monohydrate'
.
1.2
Formula and Molecular Weight
c16
365.41
17 N304S'H20
23
LOUIS P. MARRELLI
1.3 Isomers
The s y n t h e s i s of t h e L epimer o f cephalexin has been r e p o r t e d The D-isomer e x h i b i t s considerably more b i o l o g i c a l a c t i v i t y than t h e L-isomer. P e n i c i l l i n s derived from D-cr-amino a c i d s a l s o show more b i o l o g i c a l a c t i v i t y than t h e i r L-epimerg ' 6 .
.
1.4
Hydrates P f e i f f e r e t . a1.' provided x-ray powder d i f f r a c t i o n d a t a f o r the monohFdrac and dihydrate of cephlexin. Cephal e x i n w a s found t o c r y s t a l l i z e from aqueous s o l u t i o n s a t room temperature as t h e dihydrate b u t converted t o t h e monohydrate when t h e r e l a t i v e humidity was below 70%. Refer t o Section 2.21. Appearance Cephalexin i s a white t o cream-colored c r y s t a l l i n e powder, having a c h a r a c t e r i s t i c odor.
1.5
2.
Physical P r o p e r t i e s 2.1
Spectra
I n f r a r e d Spectrum The i n f r a r e d spectrum of cephalexin monohydrate recorded as a potassium bromide d i & i s presented i n Figure 1. I n t e r p r e t a t i o n of t h e spectrum is given i n Table 18. Changes i n the B-lactam carbonyl s t r e t c h i n g region (1760 cm-l) can i n d i c a t e opening of the 6-lactam ring. Morin' and coworkers have shown a r e l a t i o n s h i p between t h e B-lactam carbonyl s t r e t c h i n g frequency and b i o l o g i c a l a c t i v i t y . The importance of t h i s s t r e t c h i n g frequency has been discussed i n a recent reviewlo. 2.11
Nuclear Magnetic Resonance Spectrum Figure 2 shows the proton magnetic resonance spectrum of cephalexin monohydrate. The solvent used was deuterium oxide containing a small amount of t r i f l u o r o a c e t i c a c i d t o enhance s o l u b i l i t y . 3-( Trimethylsilyl) -propanes u l f o n i c acid, sodium s a l t w a s added as t h e i n t e r n a l r e ference. "he spectrum was recorded on a Varian "60-A i n s t r u ment. The assignment of t h e spectrum i s shown i n Table IIe A most c h a r a c t e r i s t i c region of t h e NMR spectrum i s t h a t o r i g i n a t i n g from the two 6-lactam r i n g protons, H(6) and 2.12
.
H(7)
24
4000
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
WAVENUMBER CM-'
FIGURE 1. Infrared spectrum of cephalexin monohydrate (potassium bromide disc).
600
400
7.0
5 .O
4.0 3.0 2.0 1.0 PPM (a) FIGURE 2. NMR spectrum of cephalexin monohydrate (020 + trifluoroacetic acid). 8.0
6.0
0
TABLE I
Infrared Spectrum of Cephalexin Monohydrate
-1
Wavelength (cm )
- 3000 (series of broad bands)
3500
2600 (broad)
Assignment OH from H 0 and amide NH stretch 2
+
Y3
1760
@-lactam c
1690
h i d e c = o stretch
(1600 [very broad] (1400
C-0
1550 (unresolved) 820
- 690
0
-
=
o stretch
(carboxylate stretching)
h i d e I1 band Mainly skelectal vibrations including out-of-plane aromatic hydrogen bending, characteristic of monosubstituted aromatic ring
TABLE I1
Proton Magnetic Resonance Spectrum Peak Assignments p .p .m. (6)
Relative Intensity
2.07
Mu1t ip 1icity
Assignment
3
singlet
CH3
(3)
3.30
2
quartet (AB)
(2)
4.85
-
CH*
singlet
HOD
(solvent)
4.97
1
doublet (J=4Hz)
H
(6)
5.34
1
singlet
H
(benzyl)
5.67
1
doublet (J=4HZ)
H
(7)
7.60
5
sing let
‘SH5
CEPHALEXI N
2.13
U l t r a v i o l e t Absorbance An aqueous s o l u t i o n of cephalexin e i b i t s a Pcm W absorption maximum a t 262 nm (Figure 3 ) . The El% reported f o r cephalexin (on an anhydrous b a s i s ) was 23611 The u l t r a v i o l e t absorbance of cephalexin a6 w e l l as o t h e r cephalosporins has been a t t r i b u t e d t o t h e 0 = C-N-C = Cchromophore of t h e ring1*. Chou” u t i l i z e d t h e W absorption a t 262 nm t o determine t h e cephalexin content of s o l u t i o n f r a c t i o n s i s o l a t e d from human urine.
.
2.2
C r y s t a l Properties 2.21
X-Ray Powder Diffraction Cephalexin was found t o occur i n s e v e r a l solvated c r y s t a l forms, and o f t e n i n widely varying mixtures of these f o r m d 4 . Some of t h e solvated c r y s t a l forms prepared were the dihydrate, monohydrate, d i a c e t o n i t r i l a t e , formamid a t e , methanolate, and a c e t o n i t r i l e hydrate. X-ray powder d i f f r a c t i o n data f o r cephalexin monohydrate i s presented i n Table 111. 2.22
D i f f e r e n t i a l Thermal Analysis D i f f e r e n t i a l thermal analysis’ of cephalexin monohydrate was conducted on a W o n t Model 950 thermal analyzer i n a nitrogen atmosphere. A heating r a t e of 2OoC p e r minute w a s u t i l i z e d . An endotherm w a s noted at 123OC i n d i c a t i n g the loss of water, and an exotherm of 203OC i n d i c a t i n g decomposition. 2.3
Solubility The s o l u b i l i t y of cephalexin monohydrate i n t h e following solvents has been reported’ : Solvent
Mg. Cephalexin Monohydrate Per M l . Solvent, 25OC.
13-5 3- 4
Water Methanol N-oc tan01 Chloroform Ether
0.03 4.01 4.01
Table I V r e l a t e s t h e s o l u b i l i t y of cephalexin monohydrate i n water as a function of pH.
29
LOUIS P. MARRELLI
2.0
1.5
1.0
O.!
0.0 I
I
I
250
300
monohydrate. 49 mcg per ml in H20
30
I
350
CEPHALEXIN
TABLE I11
of Cephalexin Monohydrate Norelco De Bye-Scherrer Camera
X-Ray Powder Diffraction Pattern
Radiation; Cu/Ni.
Cephalexin Monohydrate
d
1/11 0.40 1.00 0.30 0.20 0.50 0.50 0.20 0.40 1.00 0.50 0.40 0.40 0.60 0.60 0.70 0.70 0.80 0.30 0.60 0.60 0.40 0.60 0.40 0.20 0.40 0.10 0.30 0.15 0.30 0.30 0.10 0.05 0.02 0.05 0.05 0.05 0.10 0.05 0.02 0.02
-
-
15.15 11.85 11.00 9.36 8.55 7.86 6.89 5.98 5.39 4.97 4.76 4.57 4.39 4.22 4.00 3.86 3.60 3.46 3.24 3.10 2.98 2.90 2.81 2.73 2.68 2.63 2.47 2.41 2.31 2.25 2.12 2.09 2.01 1.93 1.87 1.85 1.82 1.72 1.66 1.62 31
TABLE IV
Solubility
PH
- pH Profile of Cephalexin in Water
(37°C)
.
Cephalexin Monohydrate mg I d .
. .
Cephalexin Monohydrate mg Iml
~
W
N
2.3
13
2.5
16
3 .O
.o
24
3.5
40
4.0
75
5.0
100
CEPHALEXIN
2.4
Dissociation Constant (pKa) The following d i s s o c i a t i o n constants were reported:
Solvent
rn
66% 66% DMF
Carboxyl
Amino
5-2
7.3
5.3
h0
7.3
7.1
Reference 4
17
18
2.5
Optical Rotatory Dispersion Optical r o t a t i o n has been used as an a u x i l i a r y method f o r the q u a n t i t a t i o n of cephalexir?. The s p e c i f i c r o t a t i o n [@ID reported f o r cephalexin, calculated on an anhydrous b a s i s , w a s +153O (C = 1.0 i n & 0)l 9 .
3.0
Cephalexin S t a b i l i t y The s t a b i l i t y of cephalexin i n solution i s dependent on pH, degrading r a p i d l y i n b a s i c media and remaining s t a b l e under mild a c i d i c conditions. No loss i n cephalexin a c t i v i t y occurred i n 72 hours a t 25OC i n the pH range from 3 t o 5. The r a t e of degradation found at pH 6 and pH 7 (25OC) was approximately 3% and 18%per day, r e s p e c t i v e l y 0 . With r e f r i g e r a t i o n , no appreciable loss occurs between pH 3 and pH 7 a f t e r 72 hours. I n U.S.P. hydrochloric a c i d b u f f e r (pH 1.21, cephalexin l o s t 5% a c t i v i t y i n 24 hours a t 37OC as compared t o a 45% loss i n phosphate b u f f e r at pH 6 . 9 l The a n t i b i o t i c r e t a i n s a c t i v i t y well i n serum and u r i n e as no loss i n a c t i v i t y was noted a f t e r storage a t -2OOC f o r 14 Cephalexin i n serum w a s found t o l o s e lo%, 5% and dayg2 75% a c t i v i t y , respectively, a f t e r storage a t 5'C, 25OC, and 37OC f o r 48 h o u r d l ' z z . Some organisms have been found t o produce a @-lact m a s e ( cephalosporinase) which can r a p i d l y degrade cephalexid' Degradation of cephalexin a l s o r e s u l t s from heat, s t r o n g alkali, strong a c i d s and u l t r a v i o l e t l i g h t (260 nm)
.
.
.
4.0
.
Synthesis Two synthetic r o u t e s of general a p p l i c a b i l i t y have been proposed f o r cephalexin'
.
The f i r s t method i s based on the cleavage of t h e acetoxyl group from cephaloglycid (I) by hydrogenation o r more s a t i s f a c t o r i l y , from N-t-butoxy-carbonylcephaloglycin (11) t o profuce the corresponding desacetoxy analogs V (cephalexin) and I11 as shown i n Figure 4, Scheme I. The t-
'
33
,
LOUIS P. MARRELLI
SCHEME I &lfON::@ !-
N
I, R-H II. A=Boc
/
CHzOAc
@:b.lii> N
C02H 111. R-Boc
/
COzH
V. R-H
SCHEME I1
CO2H
COZH
V
IV
FIGURE 4. Synthasis routes for eaphelexin.
34
CH3
CEPHALEXIN
butoxy-carbonyl (BOC) group was removed from I11 with trifluoroacetic a c i d and the r e a c t i o n product was converted t o desacetoxycephaloglycin ( cephalexin, V) by treatment with Amberlite LA-1 r e s i n .
.
The second method w a s s i m i l a r t o t h a t previously The nucleus, 7-aminodesused t o obtain cephaloglycid' acetoxycephalosporanic a c i d ( 7-ADCA)25 was prepared, acylated with BOC-protected D-phenylglycine employing a mixed anhydride synthesis, and then deblocked with t r i f l u o r o a c e t i c a c i d as shown i n Figure 4, Scheme 11.
5.0 Methods of Analysis 5.1 I d e n t i f i c a t i o n Tests
Cephalexin may be i d e n t i f i e d by i n f r a r e d spectroscopy. B r i t i s h Pharmacopoeia' u t i l i z e s two charact e r i s t i c color r e a c t i o n s f o r i d e n t i t y . Thin l a y e r (Sec. 5.23), paper (Sec. 5.24), and column chromatography (Sec. 5.25) have been u t i l i z e d f o r i d e n t i t y purposes.
5.2
Q u a n t i t a t i v e Methods 5.21
--.T i t r a t i o n
The iodometric t i t r a t i o n procedure has The been used f o r the determination of c e p h a l e x i d 6 method i s based on the f a c t t h a t t h e i n t a c t cephalexin molecule does not consume iodine, whereas the alkali-hydrolysis product of cephalexin does. Alkaline hydrolysis of cephalexin r e s u l t s i n cleavage of t h e @-lactam ring. V a r i a t i o n s i n hydrolysis time, temperature, pH of t h e iodine solut i o n and concentration o f cephalexin present influence t h e consumption of iodine by t h e test solution. The method compares favorably t o t h e microbiological cylinder-plate method (Sec. 5.27) i n accuracy, and is much more rapid. Possible intermediates used i n the s y n t h e s i s such as 7-ADCA w i l l also respond t o the test.
'*'.
An automated iodometric assay has been used r e c e n t l y f o r t h e assay of cephalexin and formulations thereof4 The procedure incorporates a sample hydrolysis step N, at 37OC f o r 10 minutes followed by a 5-minute iodine consumption s t e p (pH 5.3-5.5, 37°C). Concentration of the sample i s r e l a t e d t o the decrease i n iodine color measured a t 350 MI. A reference standard i s run concurrently through the analyzer f o r comparative purposes. The automated system gives excellent l i n e a r i t y of response f o r t h e
!A;:(
35
LOUIS P. MARRELLI
-
recommended concentration range of cephalexin (0 1.5 mg. per m l . sample s o l u t i o n ) , with a l l standard curve p l o t s passing through t h e origin. The r e p r o d u c i b i l i t y of t h e method on t h e same sample o r standard s o l u t i o n on a given day i s generally b e t t e r than +l% r e l a t i v e standard deviation (RSD). Cephalexin can be titf;ated with p e r c h l o r i c a c i d in a g l a c i a l acetic acid m e d i d 9 . Crystal v i o l e t indicator (2% i n g l a c i a l a c e t i c acid) may be used t o determine t h e endpoint.
Moll and D6keio have reported using a formol t i t r a t i o n procedure f o r t h e determination of cephal e x i n , a m p i c i l l i n and r e l a t e d compounds. I n t h i s procedure 4 m l . of d i l u t e formaldehyde s o l u t i o n ( n e u t r a l i z e d t o the phenolphthalein end p o i n t ) is added t o 10 m l . of an aqueous s o l u t i o n containing 15.0 mg. of cephalexin. After 2 minutes t h e s o l u t i o n is t i t r a t e d with 0.02N sodium hydroxide. A p r e c i s i o n of +O.% RSD could be achieved i n t h e t i t r a t i o n o f cephalexin mozohydrate raw m a t e r i a l samples. Acidic compounds as well as amino a c i d s must not b e present as i m p u r i t i e s i n t h e sample. The formol t i t r a t i o n takes advantage of the r e a c t i o n between an amino a c i d and formaldehyde as a means of suppressing t h e b a s i c i t y o f the amino group and thus making p o s s i b l e t h e t i t r a t i o n of t h e acid. Colorimetric Determination Reaction with hydroxylamine has been u t i l i z e d f o r t h e colorimetric determination of cephaThe method i s based on t h e f a c t t h a t hydroxylexid’ ’32 lamine cleaves t h e 8-lactam r i n g (pH 7.0) t o form a hydroxamic a c i d which forms a colored complex with f e r r i c ion. Degradation products o r intermediates having an i n t a c t 8lactam r i n g r e a c t as well. 5.22
.
Kirschbaud’ has described a procedure f o r the colorimetric determination of t h e a n t i b i o t i c cephradine’ and r e l a t e d cephalosporins. An aqueous s o l u t i o n of t h e compound (1 t o 30 mcg. p e r ml.) i s r e a c t e d with sodium hydroxide, p a r t i a l l y n e u t r a l i z e d , and then r e a c t e d with 5*5’dithiobis-( 2-nitrobenzoic acid) resulting i n t h e formation of a y llow chromophore (412 nm.). The molar a b s o r p t i v i t y E x lo-$ reported f o r cephalexin when c a r r i e d through t h i s procedure w a s 1.29. The formation of the yellow chromophore w a s a t t r i b u t e d t o the presence of t h e R C q -CO-cephalo-
’Cephradine i s t h e generic name
-
f o r 7-[D-2-amino-2-(lq4c yclohexadienyl) ace tamido] desac e toxycephalosporanic acid 36
CEPHALEXIN
sporin nucleus, i n which R i s a mono-, di- or tri- enyl cyclohexyl ring. A s p e c i f i c colorimetric t e s t w a s developed f o r t h e determination of cephalosporin d e r i v a t i v e s having the following i n t a c t s i d e chain i n t h e 7- p o s i t i o n : R (3%-CO-cephalosporin nucleus, R being a heterocyclic o r aromatic r i n 8 ' . The D-phenylglycine d e r i v a t i v e s of both 7-ADCA (cephalexin) and 7-ACA ( cephaloglycin)2 respond well. These compounds (0.5 1.0 mg. per m l . i n %O) react with acetone and sodium hydroxide a t 1 0 0 ° C t o form c h a r a c t e r i s t i c A t the 1 mg. per ml. l e v e l , t h i s red chromophores (520 nm.). t e s t w i l l v i s u a l l y d i f f e r e n t i a t e cephalexin from cephradine.
-
-
Cephaloglycin is the generic name f o r 7-( D-a-aminophenylacetamido) cephalosporanic a c i d 31
LOUIS P. MARRELLI
5.23
Thin Layer Chromatography The following t h i n l a y e r chromatographic systems have been reported:
TABLE V Adsorbent
Solvent System
Ref.
Rf -
-
Silica G e l
Ace t o n i t r i l e / W a ter
0.67
13
Silica Gel
Ethyl Acetate/Acetone/Acetic Acid/ Water (5:2:2:1)
0.22
35
Cellulose Chromatogram Sheet
Butanol/Acetic Acid/ Water (3:l:l)
-
36
Sheet
Ethyl Acetate/Acetic A c i o a t e r (3:l:l)
-
36
Sheet
Acetonitrile/Ethyl Acet at e/Water ( 3 :1 :1)
-
36
Cellulose
Acetonitrilefiater (3:1)
0.50
37
Cellulose
Butanol/Acetic Acid/ 0.70 Water (3:l:l)
37
(3:l)
Cellulose is t h e p r e f e r r e d sorbent s i n c e i t i s i n e r t toward cephalexin. Additional t h i n l a y e r chromatography systems used f o r cephalexin and o t h e r cephalosporins have been t a b u l a t e d ? . Cephalexin may be detected by u l t r a v i o l e t absorbance and quenching, ninhydrin, i o d o p l a t i n a t e , a l k a l i n e permanganate, and phosphomolybdic a c i d sprays. Iodine detection and vanillin-phosphoric a c i d spray have a l s o been u t i l i z e d . O f t h e microorganisms used, Sarcina l u t e a i s p r e f e r r e d over B a c i l l u s s u b t i l i s o r Staphylococcus aureus.
-
38
CEPHALEXIN
5.24
Paper Chromatography The following paper chromatographic systems have been reported: Solvent Systems Butanol/Ac e t i c AcidJWate r (3:l:l)
Rf -
Ref. -
0.60
13, 36, 38
-
36
Ethyl Acetate/Acetic Acid/ Water (3:l:l)
Whatman No. 1*,untreated, was used with both solvent systems and the e q u i l i b r a t i n g solvent w a s t h e same as t h e developing solvent. Additional paper chromatographic systems f o r cephalexin and o t h e r cephalosporins have been t a b u l a t e d 8 . The butanol/acetic aciuwater (3 :l:l) system w i l l separate cephaloglycin from cephalexin, cephaloglycin being l e s s mobile. An acetonitrile/water (9:l) s y s t e d 9 using Whatman No. 3 paper buffered a t pH 5.0 (16 hour development) h a s been u t i l i z e d t o s e p a r a t e cephradine from cephalexin, cephradine being l e s s mobile. The developed chromatogram can be examined under u l t r a v i o l e t l i g h t , dipped i n ninhydrin, or bioautographed, using Sarcina lutea'o
.
5.25
Column Chromatography The Moore-Stein amino acid analyzer has been used f o r the determination of cephalexin i n u r i n e samplesb1 Beckman Custom Research Resin Type PA-35, packed t o a height of 9.0 cm. i n a water-jacketed column (0.9-cm. i.d. x 2'3-cm. length) w a s used f o r t h e separation. The u r i n e sample was d i l u t e d with an equal volume of sodium c i t r a t e buffer (pH 2.2) and 100 h applied t o t h e column. The e l u t i o n time for cephalexin was approximately 61 minutes. Excellent agreement was found between the analyzer method and the microbiological method (Section 5.27) on a s e r i e s of u r i n e samples tested. I n a d d i t i o n , t h i s technique has been u s e f u l i n determining low l e v e l s of 7-ADCA and phenylglycine i n cephalexin'l (Section 5.3). Determination of t h e l e s s a c t i v e ( b i o l o g i c a l l y ) L-isomer i n cephalexin by t h e Moore-Stein amino a c i d analyzer has been r e p o r t e 8 . Chou" used the anionic r e s i n , Bio-Rad AG2 x8 ( a c e t a t e form), t o i s o l a t e cephalexin from human urine.
.
-
*Whatman chromatography' paper, Reeve Angel, 9 Bridewell Place, C l i f t o n , New Jersey 39
LOUIS P. MARRELLI
Electrophoresis Paper e l e c t r o p h o r e s i s has been u t i l i z e d test f o r impurities by the B r i t i s h Pharmacopoeia' as present i n cephalexin (Section 5.3).
5.26
Microbiological Assays Microbiological assays f o r cephalexin have been discussed by Marrelli'*, Wick" Mann'' and Simmond' and are l i s t e d i n the Federal R e g i s t e r C 6 . B r i e f l y , the two p l a t e systems well s u i t e d f o r t h e determination of cephalexin i n pharmaceutical formulations a r e t h e cylinder p l a t e methods u t i l i z i n g S t a hylococcus aureus ( ATCC 6535) and Bacillus s u b t i l i s (A--e-ac range f o r both i s approximately 2.5 t o 20 mcg. of cephalexin p e r ml.'2 '" The B. s u b t i l i s p l a t e test h a s an advantage over t h e S. aureus p l a t e t e s t i n t h a t b e t t e r defined zones of inhTbition a r e obtained, thereby increasing t h e assay precision4 Since degradation product6 of cephalexin possess p r a c t i c a l l y no antimicrobial a c t i v i t y 2 r a p i d and p r e c i s e photometric microbiological assays a r e p o s s i b l e with cephalexin. The t e s t organism f o r the photometric assay i s Staphylococcus aureus 9144, 3 t o 3.5 hours being required f o r incubation. I n Antibiotic No. 3 Broth, t h e concentration range is 0.2 t o 2.0 mcg. p e r m l . of broth. I f t h e automated AUTOTU@ System is used, p r e c i s i o n i n the order o f 1 2 % i s possible". Cephalexin i n b i o l o g i c a l f l u i d s may be assayed by a Sarcina l u t e a p l a t e system. The concentration range f o r assay i s 0.2 t o 3.5 m~g./ml.~+
5.27
.
5.3
Methods f o r Intermediates and Impurities
5.31 7-Aminodesacetoxycephalosporanic Acid
( 7-ADCA) Cole'' u t i l i z e d the Moore-Stein amino a c i d analyzer f o r the determination of 7-ADCA i n cephalexin. Column s p e c i f i c a t i o n s were o u t l i n e d i n Section 5.25. Twenty-five milligrams of cephalexin sample were dissolved w i t h sodium c i t r a t e b u f f e r (pH 2.2) t o a t o t a l volume of 10.0 m l . and 1.0 m l . applied t o t h e column. The e l u t i o n time f o r 7-ADCA w a s approximately 45 minutes. 'The s e n s i t i v i t y of the assay w a s 0.B 7-AKA.
A colorimetric procedure was developed by M ~ ~ r r e l l iwhich '~ permitted t h e d i r e c t determination of The prolow l e v e l s of 7 - A K A i n cephalexin (0.4 1.5%). cedure i s based on t h e i n t e r a c t i o n of 7-ADCA with ninhydrin under c o n t r o l l e d conditions t o produce a s p e c i f i c chromophore
-
40
CEPHALEXIN
.
( A max. a t 480 nm.) Compounds having an a-amino group adjacent t o a B-lactam r i n g respond i n general t o t h e t e s t . B r i t i s h Pharmacopoeia' u t i l i z e d a paper e l e c t r o phoresis technique which estimated t h e 7-ADCA content i n cephalexin a t the l% l e v e l . The b u f f e r s o l u t i o n s p e c i f i e d i n the t e s t consisted of a mixture of 5 m l . of formic acid, 25 m l . of g l a c i a l a c e t i c a c i d , 30 m l . of acetone and water t o a t o t a l volume o f 1000 m l . A 2.0 pl. a l i q u o t of the cephalexin sample s o l u t i o n (5.0% w/v i n 0.5N HC1) along with the s p e c i f i e d amounts of reference standards and markers were applied t o the paper. The voltage was adjusted t o about 20 v o l t s per cm. of paper and e l e c t r o p h o r e s i s w a s allowed t o proceed u n t i l the c r y s t a l v i o l e t spot moved 9 cm. from the base l i n e . Both ninhydrin spray and W were used f o r detection purposes.
5.32
Phenylglycine The ChromatonraDhic Drocedures o u t l i n e d i n Section 5.31 f o r the determination o f 7 - m A i n cephal e x i n have been concurrently used for the determination of phenylglycine i n cephalexin. The e l u t i o n time f o r phenylglycine i n the amino a c i d analyzer assay was approximately '56 minutes. The s e n s i t i v i t y of the assay w a s 0.01%. The paper electrophoresis technique permitted estimation of phenylglycine a t the 1%l e v e l . Hussepe has u t i l i z e d a chromatographic procedure f o r phenylglycine similar t o t h a t of the amino a c i d analyzer assay but incorporating a Fluramm detection system'. I
t
&
Hoffman-LaRoche Inc., Nutley, New J e r s e y
41
LOUIS P. MARRELLI
6.
P r o t e i n Binding Various values have been reported f o r t h e percentage of cephalexin bound t o serum protein. Wicp' concluded t h a t serum i n a c t i v a t i o n o r p r o t e i n binding of cephalexin i s low. Addition of serum t o broth medium did n o t a f f e c t -.i n -v .- i t r o minimal i n h i b i t o r y concentration determinations. Cephalexin assays i n pH 7 b u f f e r and human serum r e s u l t e d i n i d e n t i c a l standard curves when 6-mm d i s c s were satur a t e d with s o l u t i o n s and t e s t e d by a Sarcina l u t e a microb i o l o g i c a l assay. U t i l i z i n g a similar method, G r i f f i t h and Black!) found t h a t p r o t e i n binding of cephalexin i n human berum was % a t concentrations above 1.0 bg./ml. Naumann and Fedde9O a l s o found and 4196 a t 0.2 yg./ml. t h a t the amount o f cephalexin bound t o serum p r o t e i n s varied with t h e cephalexin concentration. Using an u l t r a f i l t r a t i o n method, Kind e t . al.51 estimated t h e serum binding as being 15%. O v a l r q h a n and Muggletor?2 obtained a value of 43% by u t i l i z a t i o n of t h e u l t r a f i l t r a t i o n technique.
7.
Pharmacokinetics Oral doses of cephalexin are r a p i d l y absorbed by animals and man, r e s u l t i n r a t h e r high blood serum l e v e l s , and a r e excreted unchanged i n t h e urine. Wick?* found t h a t a 20-mg./kg. o r a l dose of cephalexin i n mice gave Wells e t . al.5' a blood serum l e v e l of 18 pg./ml. reported a similar value (17 Fg./ml.) with a 10 mg./kg. o r a l dose i n dog. The r a p i d i t y of o r a l absorption was demonstrated by t h e f a c t t h a t a n t i b a c t e r i a l a c t i v i t y i n t h e serum was t h e same within 1.5 hours a f t e r o r a l or intramuscular administration; t h e r e a f t e r , the l e v e l s were higher f o r t h e o r a l dose. I n man, a f t e r a 500-mg. dose t h e mean of t h e peak a n t i b i o t i c a c t i v i t i e s found '5' 'J and by s e v e r a l i n v e s t i g a t o r s w a s 15 pg./ml.' u s u a l l y occurred a t 1-1.2 hours a f t e r treatment. The compound is almost completely absorbed from the upper small i n t e s t i n e i n both m a n and animals6'J6. I n addition, t h e a n t i b i o t i c i s excreted unchanged i n u r i n e with almost 100% r e c o ~ e r f ' ~ ' 5 9 . A meal j u s t p r i o r t o treatment r e s u l t e d i n lower blood l e v e l s and increased t h e time required for peak t i t e ? ' 6 0
--
*
"'
42
.
CEPHALEXIN
Kirby et. a1.61 c a l c u l a t e d the serum h a l f - l i f e of intravenously administered cephalexin as 36 minutes. Kabins et. al.62 and Naumann and FeddeSO c a l c u l a t e d the serum h a l f - l i f e a f t e r o r a l dosage a s 54 minutes. al.5 found t h a t probenecid increased the Thornhill et. peak serum concentration by 50%. but Meyers e t . al.55 found a l e s s e r e f f e c t . Linquist --e t . a1.6sexamined the disappearance of cephalexin from the blood s e r a of aneph41.0 r i c p a t i e n t s . H a l f - l i f e values ranged from 23.5 hours with a mean of 31 hours, c l e a r l y demonstrating t h e dependence upon the kidney f o r excretion.
-
43
--
LOUIS P. MARRELLI
8. References
1. The United States Pharmacopoeia, XIX, Proof p. 2122. 2. Federal Register, 21CFR148w.6. 3. British Pharmacopoeia, 1973, p. 87. 4. Ryan, C.W., Simon, R.L., and Van Heyningen, E.M., J. Med. Chem., l2, 310 (1969). 5. Doyle, F.P., Fosker, G.R., Naylor, J.H.C., and Smith, H., J. Chem. SOC., 1440 (1962). 6. Analytical Profiles of Drug Substances, Vol. 2 (K. Florey, ed.) p.4, Academic Press, New York and London, 1973. 7. Pfeiffer, R.R., Yang, K . S . and Tucker, M.A., J. Phann. Sci., 59, 1809 (1970). 8. Underbrink, C.D., Eli Lilly Analytical Development, Unpublished Data. 9. Morin, R.B., Jackson, B.G., Mueller, R.A., Lavagnino, E.R., Scanlon, W.B., and Andrews,, S.L., J. her. Chem. SOC., 91, 1401 (1969). 10. Flynn, E.H., ed., Cephalosporins and Penicillins. Chemistry and Biology, Academic Press, New York and London (1972), p. 315. 11. Flynn, op. cit., p. 631. 12. Chawette, R.R., et. al., J. Am. Chem. SOC., 84, 3401 (1962). 13. Chou, T.S., J. Med. Chem., l2, 925 (1969). 14. Pfeiffer, R.R., Eli Lilly Analytical Development, Personal Communication, (1969). 15. Cole, T.E. , Eli Lilly Analytical Development, Personal Communication, (1968). 16. Pfeiffer, R.R., Eli Lilly Analytical Development, Personal Communication, (1970). 17. Flynn, op. cit., p. 310. 18. Hargrove, W.W. , Eli Lilly and Company, Personal Communication, (1967). 19. Flynn, op. cit., p . 633. 20. Winely, C.L., Eli Lilly Analytical Development Laboratories, Unpublished Data. 21. Simmons, R. J. , Anal. Microbiol., 11. , 193 (1972). 22. Wick, W.E., Appl. Microbiol., l5, 765 (1967). 23. Ott, J.L., and Godzeski, C.W., Antimicrob. Ag. Chemother. 1966, 75 (1967). 24. Spencer, J.L., Flynn, E.H., Roeske, R.W., Siu, F.Y., and Chauvette, R.R., J. Med. Chem., 2, 746 (1966). 25. Stedman, R.J., Swered, K., Hoover, J.R.E., J. Med. * .Chem 7, 117 (1964). 26. Federal Register, 21CFR141.506. 44
CEPHALEXIN
27. British Pharmacopoeia, p. 88 (1973). 28. Stevenson, C.E., and Bechtel, L.D. (1971) Publication submitted for review in J. Pharm. Sci. 29. Marrelli, L.P., Eli Lilly and Company, Personal Cmunication (1967). , -~ 30. Moll, F., and Dzker, H., Arch. Phann., Berl., 305 (7), 548 (1972). 31. Federal Register, 21CFR141.507. 32. Plynn, op. cit., p. 615. 33. Kirschbaum, J., J. Pharm. Sci., 63, 923 (1974). 34. Marrelli, L.P., J. Pharm. Sci., 6 l , 1647 (1972). 35. Thomas, P.N., Eli Lilly and Company, Personal Cmunication (1972). 36. Sullivan, H.R., Billings, R.E., and McMahon, R.E., J. Antibio., 22, 195 (1969). 37. Flynn, op. cit., p. 621. 38. Flynn, op. cit., p. 620. 39. Marrelli, L.P., Eli Lilly and Company, Personal Cmunication (1972). 40. Miller, R.P., Antibiot. and Chemother., 12, 689 (1962). 41. Flynn, op. cit., pp. 629, 680. 42. Flynn, op. cit., p. 610. 43. Flynn, op. cit., p. 497. 207 (1972). 44. Mann, J.M. , Anal. Microbiol., Simmons, R.J., Anal. Microbiol., s, 193 (1972) 45. 46. Federal Register, CFR148~6. 47. Kuzel, N.R. and Kavanagh, F.W., J. Phann. Sci., 60, 767 (1971). 48. Hussey, R.L., Eli Lilly Analytical Development, Personal Communication (1974). 49. Griffith, R.S. and Black, H.R., Postgrad. Med. J., 47, February Suppl. , 32 (1971). 50. Kumann, P. and Fedder, J., Int. J. Clin. Pharmacol., Suppl., 2, 6 (1970). 51. Kind, A.C., Kestle, D.G., Standiford, H.C. and Kirby, W.M.M., Antimicrob. Ag. Chemother., 405 (1968). 52. Flynn, op. cit., p. 438. 53. Wells, J.S., Froman, R.O., Gibson, W.R., Owen, N.V., and Anderson, R.C., Antimicrob. Ag. Chemother., 489 (1968). 54. Kunin, C.M., and Finkelberg, Z . , Ann, Inst. Med., 72, 349 (1970). 55. Eyers, B.R., Kaplan, K., and Weinstein, L., Clin. Pharmacol. Ther., 10, 810 (1969). 56. Muggleton, P.W., O'Callaghan, C.H., Foord, R.O., Kirby, S.M., and Ryan, D.M., Antimicrob. Ag.
a,
45
LOUIS P. MARRELLI
57. 58. 59. 60. 61. 62. 63.
Chemother., 353 (1968). Perkins, R.L., Apicella, M.A., Lee, I., Cuppage, F.E., and Saslaw, S., J. Lab. Clin. Med., 7 l , 75 (1968). Thornhill, T . S . , Levison, M.E., Johnson, W.D., and Kaye, D., Appl. Microbiol., l7, 457 (1969). Gower, P.E. and Dash, C.H., Br. J. Pharmac., 37, 738 (1969). O'Callaghan, C.H., Footill, J.P.R., and Robinson, W.D., J. Pharm. Pharmac., 23, 50 (1971). Kirby, W.M.M., de Maine, J.B., and Serrill, W.S., Postgrad. Med. J., 47, February Suppl., 46 (1971). Kabins, S.A., Kelner, B., Walton, E., and Goldstein, E., her. J. Med. Sci., 259, 133 (1970). Linquist, J.A., Siddiqui, J.Y., and Smith, I.M., New Engl. J. Med., 283, 720 (1970).
ACKNOWLEDGEMENTS The author wishes to express his indebtedness to Dr. C. L. Winely for his contribution of the sections on microbiological assays, protein binding and pharmacokinetics.
46