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Aztreonam
AZTR E0 NAM Klaus Florey
ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 17
1
Copyrisht Q 1988 by Academic Press, Inc All rights of reproduction in any form reserved.
KLAUS FLQREY
2
TABLE OF CONTENTS 1. Description 1.1 Name, Formula and Molecular Weight 1.2 Appearance, Color, Odor 1.3 History 2. S nthesis 3. P ysical Properties 3.1 Infrared Spectra 3.2 NMR Spectra 3.3 Mass spectra 3.4 Ultraviolet Spectra 3.5 Optical Rotation 3.6 Melting Range 3.7 Differential Scanni n g Calorimetry 3.8 Thermogravimetric Analysis 3.9 Ionization Constant, pK 3.10 Solubility 3.1 1 Crystal Properties, Polymorphism 4. Methods of Analysis 4.1 Elemental 4.2 Microbiological Assay 4.3 lodometric 4.4 Ultraviolet 4.5 Colorimetric 4.6 Chromatographic 4.61 Thin-Layer 4.62 High Performance Liquid 4.63 Electrophoretic
K
3
5. Stability-De radation 5.1 Soli Stability 5.2 Solution Stability 5.3 Light Stability 5.4 Pink Discoloration 5.5 Stability in Biological Fluids 6. Drug Metabolism, Pharmacokinetics 7. References 8. Acknowledgement
3
AZTREONAM
1.
Description 1.1
Name, Formula and Molecular Weiqht Aztreonam, also azthreonam and SQ 26,776 in the early literature. (1) Propanoic acid, 2-[ [[ 1-(2-amino-4-thiazolyl)-2-[(2methyI-4-0~0-1-suIfo-3-azetidin I)amino]-2-oxoethylidene] a mino1oxy ] - 2 -methy I- , [2 S - [ 2 a,3DrZ)]I - ; ( 2) (Z)- 2 - [[ [(2 -Amino -4thiazolyl)[[(2S,3S)-2-meth I - 4 - o x o - l - s u l f o - 3 - a z e t i d i n y l ] ca rbamoy I] methy Ie ne la mino ox y ] -2-met hy Ipro pion ic acid. CAS-78110-38-0. INN; BAN.
r
M.W. 435.43 1.2
Appearance, Color and Odor White crystalline, odorless powder.
1.3
History Aztreonam is a synthetic, monocyclic beta-lactam antimicrobial agent, active against gram-negative organism and belonging to a new class of antibiotics, the monobactams. It was developed in the Squibb Laboratories. The events leading t o discovery of the monobactams and synthesis of aztreonam have been described 1-6. 2.
Synthesis
A stereospecific synthesis, starting with L-threonine, of the key nucleus intermediate (2S-trans)-3 amino-2-methyl-4-oxo-lacetidine sulfonic acid) was developed in the Squibb Laboratories 7. It is presented in Figure 1. By coupling with the side chain, this zwitterion is converted to aztreonam. For synthesis variations, see reference 8.
mm
-
U
0
9
f-1
a x
ru
E u
v
Y
=-5
Y
I
m
%
S-=
+It
II N
b
n
5
AZTREON A M
lK-Aztreonam, labelled as shown, has been preparedg.
9-43
N
\
H3cTc00H I
3.
Physical Properties
Infrared5 ectra Til efin 5rare mectrum of aztreonam in KBr/MeOH i s presented in Figure 2. Infrared spectra (KBr pellets) of the t w o pol morphic forms a and fl (see 3.1 1) are presented in Figures 3 an 410. 3.1
cy
NMR S ectra h z proton NMR spectrum of aztreonam in DMSO-db (Figure 5) is described in Table 1. The spectrum was obtained on a Varian XL-100-15 NMR spectrometer equipped with a Nicolet TT-100 data system. Instrumental settings: sweep width 1600Hz (quadrature detection); pulse width, 300; pulse delay, 2 sec.; data points, 8192; acquisition time, 2.56 sec.; and probe temperature, 300 C. The 2.6 Hz coupling constant between the protons of the beta-lactam ring confirms their relative (trans) stereochemistry. The chemical shift of the thiazole proton (6 = 6.82) confirms the 2-configuration for the oxime double bond1 1.
3.2
The proton decoupled 1 5 . 1 MHz carbon-13 NMR spectrum of aztreonam in DMSO-d6 (Figure 6) is assigned in Table 2, obtained on a JEOL FX-270 spectrometer using a Smm C/H dual probe. Spectral parameters; sweep width, 15,500 Hz; 500 pulses; 3.5 p sec. pulse (450); pulse delay, 1.5 sec.; bilevel decoupling; 16384 data pointsll.
6 -,
i'.. .
k FIGURE 2.
I.R. Spectrum of Aztreonam Research Standard AZ028. KBr/MeOH Instrument: PE983
I
8
8 ¶
8
L
a
I.R. Spectrum of Aztreonam Research Standard A2028 (p - Form). KBr Pellet Instrument: PE 983 Y
FIGURE 4.
I
8
8 ¶
8
L
a
I.R. Spectrum of Aztreonam Research Standard A2028 (p - Form). KBr Pellet Instrument: PE 983 Y
FIGURE 4.
W
FIGURE 5.
Proton NMR Spectrum of Aztreonam Research Standard A2028 Instrument: Varian XL-100-15
FIGURE 6.
Carbon-13 NMR Spectrum of Aztreonam Instrument: JEOL FX-270
in OMSO at 60° C.
11
AZTREONAM
TABLE 1
100 MHz Proton NMR of Aztreonam in DMSO-d6
Chemical Shift (ppm from TMS)
Number of Protons
Assignment
1.42 d (J = 6.2)
3
CH3CH
1.49 s
6
OC(CH3)2
3.72 d,q (J = 6.2,2.6)
1
C H3-CH-CH
4.38 d,d(J = 9.0,2.6)
1
NH-a-CH
6.86 s
1
Th iazole-H
9.33 d(J = 9.0)
1
NH-CH
-7.6 broad
>4
NH363, COOH, XH2O
_.
The proton and carbon-13 NMR spectra of aztreonam are consistent with the postulated structure! 1. 3.3
Mass5 ectra h g u r e 7) and negative (Figure 8) mass spectral& 13 were obtained on a double-focusing magnetic sector instrument Model ZAB-1 F, VG Analytical Ltd., Altrincham, U.K., equipped with a fast atom bombardment source using 4-8 kV xenon neutral atoms. Aztreonam gave a very prominent [M-HIion (base peak) in the negative ion detection mode and a significant MH + ion in the positive ion detection mode. Weak, but perceptible, dimeric ions were also observed.
KLAUS FLOREY
12
TABLE 2
Carbon-13 NMR Data for Aztreonam in DMSO at 600 C.
12
Chemical Shifta
Assiqnmentb
174.1
12
170.2 161.7
9 1
160.0 144.8 132.8 111.1
5 6 7 8 10 2 3 11,lla 4
82.6 60.6 56.9 23.7 17.9
a PPM from TMS with DMSO at 39.5 b Assignments based on long range C-H coupling constants.
Carbon numbering as shown above.
I26
AZTREONAM
I
i
POSITIVE
ION
FAB
I
210 142
209
I00
200
7
313 356
4 36
-
FIGURE 7.
.
I,.. .
.I
300
. L*
1.
:.
,
I.
I,.
. ” ..
,
,. . 400
Positive Ion FAB-MS Spectrum of Aztreonam. Instr u ment : ZAB-I F-VG An a Iy t icaI Ltd .
._
,
II :
,.
I.
.
.
.
. 500 m/z
-l
-
80
122 AZTREONAM NEGATIVE
-
332 J
300
FIGURE 8.
ION
FAB
96
348
\IL 400
Negative Ion FAB-BS Spectrum of Aztreonam. Instrument: ZAB-IF-VG Analytical Ltd.
~
.
,.
.
Y .
.
,
.
.
, 500 m/z
AZTREONAM
15
In the ne ative ion spectrum, the ions resulting from the direct N - 0 bon cleavage yields the [m/z 3321- ion and i t s [mlz 1031- ion complement. The principal high mass fragment ion in the positive ion mass spectrum results from the loss of sulfur trioxide from the MH + ion. Cleavage of the monobactam ring of I gives rise to the 313 + ion in the positive ion detection mode and its [m/z 1221- complement in the negative ion detection mode.
8
The fragmentation patterns for the positive and negative mode 13,14 have been schematized in Figures 9 and 15. Thermospray mass spectra of aztreonam have been produced 107, but due to extensive degradation, off-line HPLC, combined with FAB, has been found more usefull6.
+ H3N
(M + H)+ 436+ (M+Na)+458+ (M + H - S03)+ 356 +
2
313+ + O = C = N H 313+
+
\
CH3-CH=NH
fl
OH
209+ + H02C-C-CH3
FIGURE 9. Positive Ion FAB Spectra of Aztreonam
CH3
KLAUS FLOREY
16
Ii3r+
/
311'
-5
122-
(M-HI- 434(M-H-SO3)- 354-
.-+
311-
+
1 O = C = N H /or CH3-CH = NH
FIGURE 10. Negative Ion FAB Spectra of Aztreonam Ultraviolet Spectra The ultraviolet spectra of Research Standard Batch AZ028 in water (conc. 50.91 mg/100 x 4/100 (pH.4)) and in methanol (conc. 50.41 mg/100 x 6/100 ml) are presented in Figure 11 17. 3.4
The absorptivities are as follows:
Water max
E(1%;1 cm)
Methanol max
E(l%; 1 cm)
233
248
235
244
2 58
237
264
278
285
222
280
251
The ultraviolet characteristics of aztreonam are p H dependent18 (Figure 12).
FIGURE 11.
Ultraviolet Spectra of Aztreonam (Batch AZ028) in Water and Methanol.
KLAUS FLOREY
18
1.0
0.8
0.6 Y
V
z
C
m
E 0 v)
s0.4
0.2
FIGURE 12.
UV-Absorption Spectra of Aztreonam as a Function of pH. (250 C, 5.7 x 10-5 M,ionic strength 0.5 M
KLAUS FLOREY
20
f 3 BATCH #b: BETA
[
4.211 #I
1
360
FIGURE 13.
180
418
I50
460
200
DSC Curves of Alpha and Beta Forms of Aztreonam.
510
250
OK
't
AZTREONAM
21
These pK values are in good agreement with values obtained by potentiometric, spectrophotometric and kinetic methodsls. 3.10 Solubility In aaueous solution, aztreonam distdavs minimum solubility near itiisoelectricpHof2.25 (- 10mg/ml)jl.
-
The pH-solubility profile shown i n Figure 14 demonstrates that the solubility of the zwitterion of aztreonam is limiting a t pH 1-3, but as ionization occurs, solubility increases dramatically a t pH > 3. Solubilities of 40-50% w/v may be achieved a t pH's as low as 4-5 and maintained even under refrigerator conditions. The curve in Figure 14 a t RT is described as follows: S = So'[ 1
+
10 (pH-pKa2')
+ 10 (2pH-pKa2'-pKa3')
+
10 (PKa3'-PH) ]
where 5 is the total solubility and So' is the apparent intrinsic solubility of the uncharged or zwitterionic form of aztreonarn (see also 3.9). Solubility of the P-form in organic solvents: Methanol - 0.35% w/v19 Ethanol - 0.02% wh19 Solubility according t o USP terminology:22 Methanol Ethanol DMF DMSO To1uene CHC13 EtOAc
-
slightly soluble very slightly soluble soluble soluble insoluble - insoluble - insoluble
3.1 1 Crystal Properties, Polymorphism (see also Sections 3.1, 3.7 and 5.1)
Aztreonam has been observed i n three distinct crystalline forms: a, and E. All are pseudopolymorphs. The aform is obtained from aqueous solutions and is not very stable. It contains about 10-14%water (by K.F.). The crystals are fluffy rods and needles. The powder x-ray diffraction pattern is presented in Figure 15 and Table 323. Small amounts (>11%) of a-form in pform can be detected semiquantitatively by x-ray powder diffraction24. For a DSC pattern, see Section 3.7.
0 D
18.39
10.64
<
<
5
8.04
5.60
<
9.20
5.43
FIGURE 15.
Powder X-Ray Diffraction Pattern of Aztreonam, a - Form.
KLAUS FLOREY
24
TABLE 3 Powder X-Ray Diffraction Pattern of Artreonam, a -Form
Deq. 20
I
d)(Peak Height) - (
1/10
4.8
18.39
63 (lo)
1.oo
6.5
13.80
3
0.05
8.3
10.64
11
0.17
9.6
9.20
8
0.13
11.0
8.04
2
0.03
12.8
6.91
25
0.40
13.2
6.70
1
0.02
15.3
5.43
9
0.14
15.7
5.60
1
0.02
16.7
5.30
11
0.17
17.7
5.01
12
0.19
18.9
4.69
21
0.33
19.5
4.55
8
0.13
20.0
4.44
2
0.03
20.4
4.33
2
0.03
21.2
4.19
5
0.08
22.4
3.97
4
0.06
22.8
3.90
8
0.13
23.9
3.72
10
0.16
24.4
3.64
7
0.11
26.1
3.41
26
0.41
26.9
3.31
8
0.13
27.3
3.26
11
0.17
3.20
4
0.06
3.20
25
AZTREONAM
The p-form, which is obtained from a-material by recrystallizationfrom ethanol, is very stable and contains about 12 % ethanol. The crystals are dense aggregates and clusters. The powder x-ray diffraction pattern is presented in Figure 16 and Table 423. For a DSC pattern, see Section 3.7. The &-form is an orthorhombic pseudopolymorph, consisting of a 1:1 solvate of aztreonam with dimethylacetamide. It is relatively stable but will not normally be encountered, since dimethylacetamide is not used for recrystallization. A single crystal x-ray analysis of the r-form has been made26. In this form, aztreonam is zwitterionic with a proton on the cyclic nitrogen atom of the aminothiazole ring. An intramolecular H-bond occurs between the amide proton and the carbonyl oxygen atom of the carboxylic acid. Each molecule of dimethylacetamide i s the receptor of an intermolecular H-bond from the carboxyl proton of aztreonam.
Crystal properties for the dimethylacetamide (1: 1) complex are as follows26: 0
a = 11.726 (5) A, a = 900,
0
b = 22,139(8) A,
= 900, y = 900,
0
c = 9.920(3) A 0 3
v = 2575(3) A
dobs = 1.32 g cm-3. Method or comments: Flotation in hexaneKCl4 dcalc. = 1.359 cm-3 for 2 = 4 and formula of asym. unit:
c17H26N609S2
Formula: C13H17N50852. C4HgNO Space Group: P212121
100
100
10
90
SO
110
w
70
i n
(D
&-
60
50
40
w
t deg20 W h)
N
h)Q
N N
N
A
A
m
-+ A Q
d
m
o
0 ~ - - ~ ~ ~ ~ - ~ - - - - ~ - ~ - - - = - ~ ~
FIGURE 16.
m
a
0
al
P
N
0)
N
Powder X-Ray Diffraction Pattern of Aztreonam, fi - Form, Batch AZ028.
21
AZTREONAM
TABLE 4 Powder X-Ray Diffraction Pattern of Aztreonam, BATCH #A2028
Deq. 20
rnl
I
[Peak Heiaht)
fl -Form
1/10 [Relative Peak Heiqht)
27.8
3.21
13
.17
27.0
3.30
7
.09
26.8
3.32
7
.09
26.0
3.42
8
.11
25.5
3.49
1
.01
24.7
3.60
16
.21
23.9
3.72
14
.18
23.5
3.78
18
.24
23.0
3.95
2
.03
21.8
4.07
76
1.00
21.2
4.19
53
.70
20.2
4.39
4
.05
18.9
4.69
55
.72
18.2
4.87
9
.12
17.8
4.98
17
.22
16.0
5.21
46
.61
15.8
5.60
8
.11
15.4
5.75
6
.08
14.0
6.32
3
.04
11.5
7.69
58
.76
9.6
9.20
6
.08
8.9
9.93
14
.18
lo
KLAUS FU)REY
28
4.
Methods of Analysis 4.1
Elemental Analysis
Calculatedfor CI~HI~NSO~S~
%
Found for Batch #A2028 (Res. Standard) [After Drying1
C
35.8
35.77
H
3.9
4.07
N
16.1
15.82
5
14.7
14.58
Microbioloqical Assay The basic microbiological agar diffusion assay method for aztreonam uses E. coli S.C. #12155 as organism, U.S.P. agar Medium #1, and phosphate buffer pH 6 (U.S.P. #6) as diluent27. U.S.P. Medium #2 hasalso been employed28. 4.2
The assay can be used f o r c o n f i r m a t i o n o f chromatographic assays of bulk and formulation. It has found its greatest use in bod fluid assays when high sensitivity (0.06 mcg/ml) is required 2 ,28.
7
lodometric Analysis The well-known iodometric analvsis for O-lactam was tried unsuccessfully for aztreonam29. 4.3
Ultraviolet Analysis Ultraviolet absorbance a t 310 nm has been used t o follow the dissolution of attreonam capsules in 0.1M HCW. 4.4
Colorimetric Analysis The alkaIine hydroxyla mine-ferr ic nitrate a ut o mated method for P-lactams has been adapted t o aztreonam t o assay powders and solutions. I t was shown t o be linear over a concentration of 377 to 1887 mg/m131. 4.5
29
AZTREONAM
4.6
Chromatoqraphic Analysis
4.61 Thin-La er &stems t o detect aztreonam are shown in Table 5. Aztreonam can be detected under short wave U.V. light.
Hiqh Performance Liquid Several svstems have been developed f o r aztreonam. They are basedbn: 4.62
1) Reversed phase columns (CIS) with a mobile phase consisting of a mixture of low pH (mostly pH3) phosphate buffer containing tetrabutyl ammonium hydrogen sulfate (TBAHS) with acetonitrile in approximately 80:20 ratio 27,35,54. A c8 column has also been used36, and so has been methanol instead of acetonitrile in the mobile phase 37. 2) A reversed phase column (c18) with a mobile phase consisting of acetonitrile, ammonium acetate and tetrabutylammonium bromide (TBAB) at a ratio o f 33:10:5 at a pH of 738. 3) A normal phase (silica) column, using 0.1% orthophosphoric acid and 3% acetonitrile in water39A0. This sytem has also been used preparativelyls.
Retention times in these systems vary from 1 to 10 minutes. Detection by U.V. absorption has been carried out at 210,220,254,280 and 293 nm. Detection limits of 0.1 p/ml have been achieved. The various systems have been used t o determine s t a b i l i t y o f b u l k d r u g and dosage form, as w e l l as pharmacokinetics in biological fluids. 4.63
Electrophoretic
Three electrophoretic systems were used to determine aztreonam33:
TABLE 5
Rf Values of SQ 26,776 after Thin-LayerChromatoaraphy in Different Solvent Systems Solvent System
Rf Value ---
Ref
1 0.25mm Silica Gel GF (Analtech)
n-Propanol/acetic acid/water/ethyl acetate (70: 2:35: 60)
0.68
32
2 0.25mm Silica Gel GF (Analtech)
n-Propanol/acetic acid (9: 1)
0.53
32
3 0.25mm Silica Gel GF (Analtech)
Chloroform/methanol/arnmonia (50: 50: 5)
0.38 (very broad)
32
4 0.25mm Silica Gel GF (Analtech)
Chloroform/methanol/acetic acid/ethyl acetate (50:50:2: 50)
0.26
32
5 0.25mm Silica Gel (Analtech)
Chloroform/methanol/methylisobutyl ketone (1 :1:1)
0.21 (very broad)
32
6 0.25mm SilicaGel GF (Analtech)
Ethyl acetate/methanol/acetic acid/methyl isobutyl ketone (50:50:2:50)
0.46
32
7 0.25mm Silica Gel GF (Analtech)
b-ButanoVethyl acetate/water/acetic acid (1 :1:1: 1)
0.62
33
8 0.25mm Silica Gel GF (Analtech)
n-Butanol/acetic acidlwater (3:1:1)
0.50
32
9 0.25mm Silica Gel 60 F254(Merck)
n-PropanoVacetic acid/water/ethyl acetate (70:2:35:60)
0.32
34
10 0.25mm Silica Gel 60 F254 (Merck)
n-ButanoVethyl acetate/water/acetic acid (1 :1: 1:1)
0.50
33
11 0.25mm Silica Gel G (Analtech) impregnated with tetradecane
Mcllvaine's buffer pH 6.Yacetone (200:3)
-0.5
34
12 0.25mm Silica Gel (Merck)
n-Propanol-ethyl acetate-pH 7.0 phosphate buffer (70:60 :35)
-0.50
34
13 Analtech RPS
Water-acetonitrile-sodi um perchlorate (995: 5: 0.7)
-0.80
33
Plate
AZTREONAM
Svstern
5.
31
SUDDO~~
BufferhH
Voltslcrn Minutes
1
Celldose (Eastman)
Pyridine acetate 0.05M. pH 4.0
20
60
2
CelIdose (Eastman)
2M Phosphate
10
45,81
3
Celldose (Eastman)
Phosphate 0.05M. pH 6.9
20
60
+
Stability - Deqradation
5.1 Solid Stabilit T e sta ility of a and fl crystalline forms has been studied (see 3.1 1 Crystal Properties). 5.1 1 a-Form
T h e - f o r m (crystallization f r o m aqueous ethanol or methanol) is not very stable. A 1% loss at RT and an 80% loss at 800 C. after a one-week stora e has been reported (Figure 17)41. An energy of activation of 25 cal/mole, assuming a first-order model and 20 kcal/mole, assuming a zero-model was calculated. Both energies o f activation were in the range indicative of hydrolysis reactions.
1
w
5.12 T e -form (recrvstallized from anhvdrous ethanol) is stable. Aft&' a 12-monthastorageat -200, + 5%: + 330, + 400, + 220, 80% RH and 400 C./75% RH batches s t i l l pass specifications at all temperatures and humidity stations42. Even under the most rigorous storage conditions (40% C./75% RH), the samples were found to have undergone only a slight increase (<2% respectively by TLC) in impurities and a small drop (3.0to 3.51% by HPLC) in potency.
Solution Stabilit In aaueous solttion, the most imDortant source of instability of azireonam over the whole pH range is hydrolysis o f the beta-lactam ring19. 5.2
In weakly acid solutions (pH 2 t o 5), hydrolysis i s preceded by isomerization of the side chain as shown in Figure 18 (conversion of Z (syn) t o E (anti) isomer)1619. Care has t o be exercised that E-isomer formation does not occur as an artifact
KLAUS FJBREY
32
0 1 2 3 . ( 5 6 7 8 9 1 O I I I 2 l 3
TI ME
FIGURE 17.
(weeks)
Aztreonam Alpha Form: Solid-state Stability: Temperature Profile
I m 0 In
d N
1.:
4,
m
X
V
X-Z
N X
5: N
L? z
I
N
W
I N
\ /
X
/
a
KLAUS FLOREY
34
9
durin chromatographic separation43. The pH-rate profile for the hydro ysis of aztreonam at 350 C. and constant ionic strength (l.~ = 0.5M KCI) is presented in Figure 1919. It shows that aztreonam is most stable in the pH 5-7 range. The Arrhenius plot for aztreonam degradation is presented in Figure 2019. It has also been shown41 that in aqueous, buffer-free solution, aztreonam is about 5 times more stable in the pH 4-7 range than most penicillins and cephalosporins, including ampicillin and cephradine. At pH 5-7, aztreonam shows 10% de radation in 300-500 hours. Phosphate, tris, borate and car onate buffers accelerate degradation .
%
3
Other de radation products that have been observed are the desulfonate productsu:
H '.R
Lf3
and
0
H
R\'
NrB"3 NB2
0-C,
ow
Degradation of aztreonam t o dimers and trimers in aqueous solution has also been describedss. Li ht Stabilit M e x o o s e d one week t o 400 and 900 footcandles of fludrestent illumination and 33-35% C. temperature yellowed and showed a 6% conversion t o the 'E' isomer. Samples stored in the dark a t 330 C. or exposed t o room light and temperature f o r one week showed n o y e l l o w i n g or chromatographic evidence of significant '2' t o 'E' isomerizationl9. 5.3
Pink Discoloration After long standing at a pH of <5, a pink discoloration of aztreonam has been observed. Solids exposed t o moisture may also exhibit this phenomenonlg. 5.4
PH
FIGURE 19: AZTREONAM. pH Rate Profile for Degradation of Aztreonam at 3 5 O C. an p = 0.5M. The rate constants have been extrapolated to zero buffer concentration.
1o'lr
FIGURE 20: AZTREONAM. Arrhenius Plots for Aztreonam De radation. Key: [51 pH 3.66;A pH 5.40; pH 7.92
0
KLAUS FLOREY
36
5.5 Stability in Bioloqical Fluids
Aztreonam is stable in human serum and urine for at least 10 months when kept a t -780 C.46. However, aztreonam is unstable in human serum even at -200 C. where a loss of 5% was observed after 7 days. However, immediate dilution (1120)in pH 6.0 phosphate buffer great1 increased stability a t -200 C. t o a loss of
i
Aztreonam is more stabile in urine. It loses about 2.5%/24 hours at 250 C., 1.5%124 hours a t 50 C. and only 0.515%12 weeks a t -200 C. It, therefore, should be stored a t -200 C. and assayed within 4-6 weeks47. Aztreonam was found to be stable in hemodialysis and peritoneal dialysis fluids for at least four days a t room temperature to -200 C.43. There was no loss of activity in waste dialysate buffered to pH 6 after storage of -200 C. for seven weeks. However an activity lossof -9% occurred in the unbuffered dialysate after two weeks' storage46. 6.
Drua Metabolism, Pharmacokinetics
Metabolism and pharmacokinetics of aztreonam have been studied with 14C labeled aztreonam in the rat, dog and monkeya as well as in human volunteers48A9. Distribution in male and female ratsso, in rat tissue51 and fetuses and milk of rats52 has also been described. In human volunteers, after intravenous administration, the pharmacokinetic properties of aztreonam in serum were described by an open, linear, two-compartment model. After intramuscular administration of aztreonam t o human volunteers, the serum concentration vs. time data was described by a biex ponent ia I equat ion w h 3 represented a one-compartme nt model with first-order absorption and elimination. Bioavailability after an intramuscular dose was 100%. After either intravenous or intramuscular administration, aztreonam was eliminated primarily by urinary excretion of unchanged aztreonam (about 66% of dose); only 1% of the dose was found as unchanged aztreonam in the feces, presumably as a result of bilia secretion. The values determined for the average elimination alf-life of aztreonam were 1.6 and 1.7h, respectively, after intravenous and intramuscular administration. Aztreonam did n o t undergo extensive metabolism; the most prominent biotransformation roduct of aztreonam was the derivative resulting from the lydrolytic opening of the beta-lactam ring. Excretion o f this compound in urine and feces accounted for 7 and 3% of the
7l
AZTREONAM
37
administered dose, respectively. It was eliminated a t a considerably slower rate than was aztreonam53. Other metabolites, accounting for 4 t o 5% of the radioactivity in urine, have not yet been identified53. 7. 1.
2. 3.
4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21.
References C.M. Cimarusti, R.B. Sykes, H.A. Applegate, D.P. Bonner, H. Breuer, H.W. Chang, T. Denzel, D.M. Floyd, A.W. Fritz, W.H. Koster, W.C. Liu, W.L. Parker, M.L. Rathnum, W.A. Slusarchyk, U.D. Treuner, M.G. Young, Am. College Clin. Pharm., 1,35 (1981). R.B. Syk& and I.Phillips (eds.), J. Antimicrob. Chemother. 8 (Supplement E), s(1981). R.B. Sykes, D.P. Bonner, K. Bush and N.H. Georgopapadakou, Antimicrob. Agents and Chemother., a , 8 5 (1982). C.M. Cimarusti and R.B. Sykes, Chem. in Brit., 19,302 (1983). C.M. Cimarusti, Trends Pharmacol. Sci., 4,468fi983). C.M. Cimarusti and R.B. Sykes, Med. Res. Rev., 4,l (1984). D.M. Floyd, A.W. Fritz and C.M. Cimarusti, J. Org. Chem., 47, 176 (1982). R.B. Sykes, W.L. Parker, C.M. Cimarusti, W.H. Koster, W.A. Slusarchyk, A.W. Fritz, D.M. Flo d, U.K. Patent Application GB2071650A, published Septem er 29,1981; also EP048953 (1982). P. Egli and L. Gnacek, Personal Communication. M. Paslawsky, The Squibb Institute, Personal Communication (1982). M. Porubcan, The Sauibb Institute. Personal Communication (1983). A.I. Cohen, P.T. Funke, B.N. Green, J. Pharm. Sci., 7 l , 1065 (1982). S.E. Unger and T.J. McCormick, Trends Anal. Chem., 4, 178 (1985). S.E. Unger, Int. J. Mass Spectrom. Ion Process.& 195 (1985). S.E. Unger and B.M. Warrack, Spectroscopy, 1 , s (1986). J. Adamovics and 5. Unger, J. Liquid Chromatogr., 9, 141 (1986). B. Kline and 5 . Willis, The Squibb Institute, Personal Commun ication. J.D. Pipkin and M. Davidovich, The Squibb Institute, Personal Communication (1983). J.D. Pipkin, Chemical Solubility of Pharmaceuticals, 2nd ed., p. 250, K.A. Connors, et. al. (eds.), John Wiley, New York (1986). J.DeVincentis, The Squib b l n s t i t Ute, Personal Communication. J.D. Pipkin, The Squibb Institute, Personal Communication (1981).
1
KLAUS FLQREY
38
22. A. Pudzianowski, The Squibb Institute, Personal Communication (1983). 23. C. Sachs, The Squibb Institute, Personal Communication. 24. A. Pudzianowski, C. Sachs, The Squibb Institute, Personal Communication (1982). 25. DeIeted . 26. M. Malley and J. Gou outas, The Squibb Institute, Personal Communication (19847. 27. E.A. Swabb, M.A. Leitz, F.G. Pilkiewicz and A.A. Sugerman, J. Antimicrob. Chemotherap. 8 (Sup I. E), 131 (1981). 28. W. Ehret, H. Probst, G. Ruck eschel, J. Antimicrobial Chemother., 19,541(1987). 29. J. Gentile, C. Fassbender, H. Weisblatt, The Squibb Institute, Personal Communication (1981). 30. D. Both, The Squibb Institute, Personal Communication (1981). 31. C. Sherman, The Squibb Institute, Personal Communication. 32. M.S. Bathala and S.H. Weinstein, The Squibb Institute, Personal Communication (1981). 33. 0. Kocy, The Squibb Institute, Personal Communication (1984). 34. H.R. Roberts, The Squibb Institute, Personal Communication (1982). 35. F.G. Pilkiewicz, B.J. Remsburg, S.M. Fisher and R.B. Sykes, Antimicrobiol. Agents Chemother., 3,852(1983). 36. D.W. Marble, J.A. Bosso and R.J. Townsend, Am. J. Hosp. Pharm., 43,1732(1986). 37. M.J. Jamzand C. Riley, Am. 1. Hosp. Pharm., 42,1095. 38. F. Hehl, P. Birckel and H. Monteil, 1. Chromatogr., 413, 109 (1987). 39. J. Adamovics, Liq. Chromatogr. Mag.,& 393 (1984). 40- M.J. James and C.M. Riley, Am. J. Hosp. Pharm., 42, 1984 (1985). 41. J.D. Pipkin and E.P. Barry, The Squibb Institute, Personal Communication (1981). 42. A.R. Restivo and M.C. Blaich, Personal Communication (1983). 43- J.M. Joseph, The Squibb Institute, Personal Communication (1983). 44. S.E. Un er, T.J. McCormick, Analytical R&D Report dated Septem er 30,1983. 45. J.D. Pipkin, S.A. Ranadive, N.H. Chang, S.E. Unger, T.J.
8
%
McCormick, M. Porubcan, E.P. Barr , S.A. Varia and J. Adamovics, Am. Pharm. Assoc. Acad. PYl arm. Sci. Abst., l5(2)
135 (1985). 46. M.A. Leitz, 5 . Wind, The Squibb Institute, Personal Communication (1983).
AZTREONAM
47. 48.
49. 50.
51. 52. 53.
54.
8.
39
J.A. Manning, M.A. Leitz, T.B. Platt, The Squibb Institute , Personal Communication (1982). K.J. Kripalani, S.M. Singhvi, S.H. Weinstein, D.W. Everett, M.S. Bathala, A.V. Dean, C.E. Ita, L. Lawrence, F.S. Meeker, J.M. Shaw, B.D. Walker and B.H. Migdalof, Antimicrob. Agents Chemother., 26,119 (1984). E.A. Swabb, A.A. Sugerman, T.B. Platt, F. Pilkiewicz and M. Frantz, Antimicrob. Agents and Chemother., 21,944 (1982). S.M. Singhvi, C.E. Ita, J.M. Shaw and B.H. Migdalof, Pharmacologist, 2 5 , 1 1 5 (1983).
3
S.M. Singhvi, C.E. Ita, J.M. Shaw and B.H. M i dalof, Antimicrob. Agents and Chemotherap., 26,127 (1984 . S.M. Singhvi, C.E. Ita, J.M. Shaw, G.R. Keim and B.H. Migdalof, Antimicrob. Agents Chemother., 26,132 (1984). €.A. Swabb, S.M. Singhvi, M.A. Leitz, M. Frantz and A. Sugerman, Antimicrob. Agents Chemother., 24,394 (1983). A. Menlemans, J. Mohler, D. Vitteco , G. Haroche, M.A. Rosset, M. Vulpillat and J. Modai, J. C romatogr., 377, 466 (1986).
i?
Acknowledaement
For contributions t o the orofile, 1 am indebted t o G.A. Brewer, C. Cimarusti, F. Dondzija, P. Funke, J.Z. Gougoutas, J. Kirschbaum, 0. Kocy, B. Migdalof, M. Paslawsky, J.D. Pipkin, T. Platt, M. Porubcan, A. Pudzianowski, A. Restivo, H. Roberts and 5. Unger. I also would like to thank Marie Bruno for competent and patient secretarial assistance. .
ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 17
1
Copyrisht Q 1988 by Academic Press, Inc All rights of reproduction in any form reserved.
KLAUS FLQREY
2
TABLE OF CONTENTS 1. Description 1.1 Name, Formula and Molecular Weight 1.2 Appearance, Color, Odor 1.3 History 2. S nthesis 3. P ysical Properties 3.1 Infrared Spectra 3.2 NMR Spectra 3.3 Mass spectra 3.4 Ultraviolet Spectra 3.5 Optical Rotation 3.6 Melting Range 3.7 Differential Scanni n g Calorimetry 3.8 Thermogravimetric Analysis 3.9 Ionization Constant, pK 3.10 Solubility 3.1 1 Crystal Properties, Polymorphism 4. Methods of Analysis 4.1 Elemental 4.2 Microbiological Assay 4.3 lodometric 4.4 Ultraviolet 4.5 Colorimetric 4.6 Chromatographic 4.61 Thin-Layer 4.62 High Performance Liquid 4.63 Electrophoretic
K
3
5. Stability-De radation 5.1 Soli Stability 5.2 Solution Stability 5.3 Light Stability 5.4 Pink Discoloration 5.5 Stability in Biological Fluids 6. Drug Metabolism, Pharmacokinetics 7. References 8. Acknowledgement
3
AZTREONAM
1.
Description 1.1
Name, Formula and Molecular Weiqht Aztreonam, also azthreonam and SQ 26,776 in the early literature. (1) Propanoic acid, 2-[ [[ 1-(2-amino-4-thiazolyl)-2-[(2methyI-4-0~0-1-suIfo-3-azetidin I)amino]-2-oxoethylidene] a mino1oxy ] - 2 -methy I- , [2 S - [ 2 a,3DrZ)]I - ; ( 2) (Z)- 2 - [[ [(2 -Amino -4thiazolyl)[[(2S,3S)-2-meth I - 4 - o x o - l - s u l f o - 3 - a z e t i d i n y l ] ca rbamoy I] methy Ie ne la mino ox y ] -2-met hy Ipro pion ic acid. CAS-78110-38-0. INN; BAN.
r
M.W. 435.43 1.2
Appearance, Color and Odor White crystalline, odorless powder.
1.3
History Aztreonam is a synthetic, monocyclic beta-lactam antimicrobial agent, active against gram-negative organism and belonging to a new class of antibiotics, the monobactams. It was developed in the Squibb Laboratories. The events leading t o discovery of the monobactams and synthesis of aztreonam have been described 1-6. 2.
Synthesis
A stereospecific synthesis, starting with L-threonine, of the key nucleus intermediate (2S-trans)-3 amino-2-methyl-4-oxo-lacetidine sulfonic acid) was developed in the Squibb Laboratories 7. It is presented in Figure 1. By coupling with the side chain, this zwitterion is converted to aztreonam. For synthesis variations, see reference 8.
mm
-
U
0
9
f-1
a x
ru
E u
v
Y
=-5
Y
I
m
%
S-=
+It
II N
b
n
5
AZTREON A M
lK-Aztreonam, labelled as shown, has been preparedg.
9-43
N
\
H3cTc00H I
3.
Physical Properties
Infrared5 ectra Til efin 5rare mectrum of aztreonam in KBr/MeOH i s presented in Figure 2. Infrared spectra (KBr pellets) of the t w o pol morphic forms a and fl (see 3.1 1) are presented in Figures 3 an 410. 3.1
cy
NMR S ectra h z proton NMR spectrum of aztreonam in DMSO-db (Figure 5) is described in Table 1. The spectrum was obtained on a Varian XL-100-15 NMR spectrometer equipped with a Nicolet TT-100 data system. Instrumental settings: sweep width 1600Hz (quadrature detection); pulse width, 300; pulse delay, 2 sec.; data points, 8192; acquisition time, 2.56 sec.; and probe temperature, 300 C. The 2.6 Hz coupling constant between the protons of the beta-lactam ring confirms their relative (trans) stereochemistry. The chemical shift of the thiazole proton (6 = 6.82) confirms the 2-configuration for the oxime double bond1 1.
3.2
The proton decoupled 1 5 . 1 MHz carbon-13 NMR spectrum of aztreonam in DMSO-d6 (Figure 6) is assigned in Table 2, obtained on a JEOL FX-270 spectrometer using a Smm C/H dual probe. Spectral parameters; sweep width, 15,500 Hz; 500 pulses; 3.5 p sec. pulse (450); pulse delay, 1.5 sec.; bilevel decoupling; 16384 data pointsll.
6 -,
i'.. .
k FIGURE 2.
I.R. Spectrum of Aztreonam Research Standard AZ028. KBr/MeOH Instrument: PE983
I
8
8 ¶
8
L
a
I.R. Spectrum of Aztreonam Research Standard A2028 (p - Form). KBr Pellet Instrument: PE 983 Y
FIGURE 4.
I
8
8 ¶
8
L
a
I.R. Spectrum of Aztreonam Research Standard A2028 (p - Form). KBr Pellet Instrument: PE 983 Y
FIGURE 4.
W
FIGURE 5.
Proton NMR Spectrum of Aztreonam Research Standard A2028 Instrument: Varian XL-100-15
FIGURE 6.
Carbon-13 NMR Spectrum of Aztreonam Instrument: JEOL FX-270
in OMSO at 60° C.
11
AZTREONAM
TABLE 1
100 MHz Proton NMR of Aztreonam in DMSO-d6
Chemical Shift (ppm from TMS)
Number of Protons
Assignment
1.42 d (J = 6.2)
3
CH3CH
1.49 s
6
OC(CH3)2
3.72 d,q (J = 6.2,2.6)
1
C H3-CH-CH
4.38 d,d(J = 9.0,2.6)
1
NH-a-CH
6.86 s
1
Th iazole-H
9.33 d(J = 9.0)
1
NH-CH
-7.6 broad
>4
NH363, COOH, XH2O
_.
The proton and carbon-13 NMR spectra of aztreonam are consistent with the postulated structure! 1. 3.3
Mass5 ectra h g u r e 7) and negative (Figure 8) mass spectral& 13 were obtained on a double-focusing magnetic sector instrument Model ZAB-1 F, VG Analytical Ltd., Altrincham, U.K., equipped with a fast atom bombardment source using 4-8 kV xenon neutral atoms. Aztreonam gave a very prominent [M-HIion (base peak) in the negative ion detection mode and a significant MH + ion in the positive ion detection mode. Weak, but perceptible, dimeric ions were also observed.
KLAUS FLOREY
12
TABLE 2
Carbon-13 NMR Data for Aztreonam in DMSO at 600 C.
12
Chemical Shifta
Assiqnmentb
174.1
12
170.2 161.7
9 1
160.0 144.8 132.8 111.1
5 6 7 8 10 2 3 11,lla 4
82.6 60.6 56.9 23.7 17.9
a PPM from TMS with DMSO at 39.5 b Assignments based on long range C-H coupling constants.
Carbon numbering as shown above.
I26
AZTREONAM
I
i
POSITIVE
ION
FAB
I
210 142
209
I00
200
7
313 356
4 36
-
FIGURE 7.
.
I,.. .
.I
300
. L*
1.
:.
,
I.
I,.
. ” ..
,
,. . 400
Positive Ion FAB-MS Spectrum of Aztreonam. Instr u ment : ZAB-I F-VG An a Iy t icaI Ltd .
._
,
II :
,.
I.
.
.
.
. 500 m/z
-l
-
80
122 AZTREONAM NEGATIVE
-
332 J
300
FIGURE 8.
ION
FAB
96
348
\IL 400
Negative Ion FAB-BS Spectrum of Aztreonam. Instrument: ZAB-IF-VG Analytical Ltd.
~
.
,.
.
Y .
.
,
.
.
, 500 m/z
AZTREONAM
15
In the ne ative ion spectrum, the ions resulting from the direct N - 0 bon cleavage yields the [m/z 3321- ion and i t s [mlz 1031- ion complement. The principal high mass fragment ion in the positive ion mass spectrum results from the loss of sulfur trioxide from the MH + ion. Cleavage of the monobactam ring of I gives rise to the 313 + ion in the positive ion detection mode and its [m/z 1221- complement in the negative ion detection mode.
8
The fragmentation patterns for the positive and negative mode 13,14 have been schematized in Figures 9 and 15. Thermospray mass spectra of aztreonam have been produced 107, but due to extensive degradation, off-line HPLC, combined with FAB, has been found more usefull6.
+ H3N
(M + H)+ 436+ (M+Na)+458+ (M + H - S03)+ 356 +
2
313+ + O = C = N H 313+
+
\
CH3-CH=NH
fl
OH
209+ + H02C-C-CH3
FIGURE 9. Positive Ion FAB Spectra of Aztreonam
CH3
KLAUS FLOREY
16
Ii3r+
/
311'
-5
122-
(M-HI- 434(M-H-SO3)- 354-
.-+
311-
+
1 O = C = N H /or CH3-CH = NH
FIGURE 10. Negative Ion FAB Spectra of Aztreonam Ultraviolet Spectra The ultraviolet spectra of Research Standard Batch AZ028 in water (conc. 50.91 mg/100 x 4/100 (pH.4)) and in methanol (conc. 50.41 mg/100 x 6/100 ml) are presented in Figure 11 17. 3.4
The absorptivities are as follows:
Water max
E(1%;1 cm)
Methanol max
E(l%; 1 cm)
233
248
235
244
2 58
237
264
278
285
222
280
251
The ultraviolet characteristics of aztreonam are p H dependent18 (Figure 12).
FIGURE 11.
Ultraviolet Spectra of Aztreonam (Batch AZ028) in Water and Methanol.
KLAUS FLOREY
18
1.0
0.8
0.6 Y
V
z
C
m
E 0 v)
s0.4
0.2
FIGURE 12.
UV-Absorption Spectra of Aztreonam as a Function of pH. (250 C, 5.7 x 10-5 M,ionic strength 0.5 M
KLAUS FLOREY
20
f 3 BATCH #b: BETA
[
4.211 #I
1
360
FIGURE 13.
180
418
I50
460
200
DSC Curves of Alpha and Beta Forms of Aztreonam.
510
250
OK
't
AZTREONAM
21
These pK values are in good agreement with values obtained by potentiometric, spectrophotometric and kinetic methodsls. 3.10 Solubility In aaueous solution, aztreonam distdavs minimum solubility near itiisoelectricpHof2.25 (- 10mg/ml)jl.
-
The pH-solubility profile shown i n Figure 14 demonstrates that the solubility of the zwitterion of aztreonam is limiting a t pH 1-3, but as ionization occurs, solubility increases dramatically a t pH > 3. Solubilities of 40-50% w/v may be achieved a t pH's as low as 4-5 and maintained even under refrigerator conditions. The curve in Figure 14 a t RT is described as follows: S = So'[ 1
+
10 (pH-pKa2')
+ 10 (2pH-pKa2'-pKa3')
+
10 (PKa3'-PH) ]
where 5 is the total solubility and So' is the apparent intrinsic solubility of the uncharged or zwitterionic form of aztreonarn (see also 3.9). Solubility of the P-form in organic solvents: Methanol - 0.35% w/v19 Ethanol - 0.02% wh19 Solubility according t o USP terminology:22 Methanol Ethanol DMF DMSO To1uene CHC13 EtOAc
-
slightly soluble very slightly soluble soluble soluble insoluble - insoluble - insoluble
3.1 1 Crystal Properties, Polymorphism (see also Sections 3.1, 3.7 and 5.1)
Aztreonam has been observed i n three distinct crystalline forms: a, and E. All are pseudopolymorphs. The aform is obtained from aqueous solutions and is not very stable. It contains about 10-14%water (by K.F.). The crystals are fluffy rods and needles. The powder x-ray diffraction pattern is presented in Figure 15 and Table 323. Small amounts (>11%) of a-form in pform can be detected semiquantitatively by x-ray powder diffraction24. For a DSC pattern, see Section 3.7.
0 D
18.39
10.64
<
<
5
8.04
5.60
<
9.20
5.43
FIGURE 15.
Powder X-Ray Diffraction Pattern of Aztreonam, a - Form.
KLAUS FLOREY
24
TABLE 3 Powder X-Ray Diffraction Pattern of Artreonam, a -Form
Deq. 20
I
d)(Peak Height) - (
1/10
4.8
18.39
63 (lo)
1.oo
6.5
13.80
3
0.05
8.3
10.64
11
0.17
9.6
9.20
8
0.13
11.0
8.04
2
0.03
12.8
6.91
25
0.40
13.2
6.70
1
0.02
15.3
5.43
9
0.14
15.7
5.60
1
0.02
16.7
5.30
11
0.17
17.7
5.01
12
0.19
18.9
4.69
21
0.33
19.5
4.55
8
0.13
20.0
4.44
2
0.03
20.4
4.33
2
0.03
21.2
4.19
5
0.08
22.4
3.97
4
0.06
22.8
3.90
8
0.13
23.9
3.72
10
0.16
24.4
3.64
7
0.11
26.1
3.41
26
0.41
26.9
3.31
8
0.13
27.3
3.26
11
0.17
3.20
4
0.06
3.20
25
AZTREONAM
The p-form, which is obtained from a-material by recrystallizationfrom ethanol, is very stable and contains about 12 % ethanol. The crystals are dense aggregates and clusters. The powder x-ray diffraction pattern is presented in Figure 16 and Table 423. For a DSC pattern, see Section 3.7. The &-form is an orthorhombic pseudopolymorph, consisting of a 1:1 solvate of aztreonam with dimethylacetamide. It is relatively stable but will not normally be encountered, since dimethylacetamide is not used for recrystallization. A single crystal x-ray analysis of the r-form has been made26. In this form, aztreonam is zwitterionic with a proton on the cyclic nitrogen atom of the aminothiazole ring. An intramolecular H-bond occurs between the amide proton and the carbonyl oxygen atom of the carboxylic acid. Each molecule of dimethylacetamide i s the receptor of an intermolecular H-bond from the carboxyl proton of aztreonam.
Crystal properties for the dimethylacetamide (1: 1) complex are as follows26: 0
a = 11.726 (5) A, a = 900,
0
b = 22,139(8) A,
= 900, y = 900,
0
c = 9.920(3) A 0 3
v = 2575(3) A
dobs = 1.32 g cm-3. Method or comments: Flotation in hexaneKCl4 dcalc. = 1.359 cm-3 for 2 = 4 and formula of asym. unit:
c17H26N609S2
Formula: C13H17N50852. C4HgNO Space Group: P212121
100
100
10
90
SO
110
w
70
i n
(D
&-
60
50
40
w
t deg20 W h)
N
h)Q
N N
N
A
A
m
-+ A Q
d
m
o
0 ~ - - ~ ~ ~ ~ - ~ - - - - ~ - ~ - - - = - ~ ~
FIGURE 16.
m
a
0
al
P
N
0)
N
Powder X-Ray Diffraction Pattern of Aztreonam, fi - Form, Batch AZ028.
21
AZTREONAM
TABLE 4 Powder X-Ray Diffraction Pattern of Aztreonam, BATCH #A2028
Deq. 20
rnl
I
[Peak Heiaht)
fl -Form
1/10 [Relative Peak Heiqht)
27.8
3.21
13
.17
27.0
3.30
7
.09
26.8
3.32
7
.09
26.0
3.42
8
.11
25.5
3.49
1
.01
24.7
3.60
16
.21
23.9
3.72
14
.18
23.5
3.78
18
.24
23.0
3.95
2
.03
21.8
4.07
76
1.00
21.2
4.19
53
.70
20.2
4.39
4
.05
18.9
4.69
55
.72
18.2
4.87
9
.12
17.8
4.98
17
.22
16.0
5.21
46
.61
15.8
5.60
8
.11
15.4
5.75
6
.08
14.0
6.32
3
.04
11.5
7.69
58
.76
9.6
9.20
6
.08
8.9
9.93
14
.18
lo
KLAUS FU)REY
28
4.
Methods of Analysis 4.1
Elemental Analysis
Calculatedfor CI~HI~NSO~S~
%
Found for Batch #A2028 (Res. Standard) [After Drying1
C
35.8
35.77
H
3.9
4.07
N
16.1
15.82
5
14.7
14.58
Microbioloqical Assay The basic microbiological agar diffusion assay method for aztreonam uses E. coli S.C. #12155 as organism, U.S.P. agar Medium #1, and phosphate buffer pH 6 (U.S.P. #6) as diluent27. U.S.P. Medium #2 hasalso been employed28. 4.2
The assay can be used f o r c o n f i r m a t i o n o f chromatographic assays of bulk and formulation. It has found its greatest use in bod fluid assays when high sensitivity (0.06 mcg/ml) is required 2 ,28.
7
lodometric Analysis The well-known iodometric analvsis for O-lactam was tried unsuccessfully for aztreonam29. 4.3
Ultraviolet Analysis Ultraviolet absorbance a t 310 nm has been used t o follow the dissolution of attreonam capsules in 0.1M HCW. 4.4
Colorimetric Analysis The alkaIine hydroxyla mine-ferr ic nitrate a ut o mated method for P-lactams has been adapted t o aztreonam t o assay powders and solutions. I t was shown t o be linear over a concentration of 377 to 1887 mg/m131. 4.5
29
AZTREONAM
4.6
Chromatoqraphic Analysis
4.61 Thin-La er &stems t o detect aztreonam are shown in Table 5. Aztreonam can be detected under short wave U.V. light.
Hiqh Performance Liquid Several svstems have been developed f o r aztreonam. They are basedbn: 4.62
1) Reversed phase columns (CIS) with a mobile phase consisting of a mixture of low pH (mostly pH3) phosphate buffer containing tetrabutyl ammonium hydrogen sulfate (TBAHS) with acetonitrile in approximately 80:20 ratio 27,35,54. A c8 column has also been used36, and so has been methanol instead of acetonitrile in the mobile phase 37. 2) A reversed phase column (c18) with a mobile phase consisting of acetonitrile, ammonium acetate and tetrabutylammonium bromide (TBAB) at a ratio o f 33:10:5 at a pH of 738. 3) A normal phase (silica) column, using 0.1% orthophosphoric acid and 3% acetonitrile in water39A0. This sytem has also been used preparativelyls.
Retention times in these systems vary from 1 to 10 minutes. Detection by U.V. absorption has been carried out at 210,220,254,280 and 293 nm. Detection limits of 0.1 p/ml have been achieved. The various systems have been used t o determine s t a b i l i t y o f b u l k d r u g and dosage form, as w e l l as pharmacokinetics in biological fluids. 4.63
Electrophoretic
Three electrophoretic systems were used to determine aztreonam33:
TABLE 5
Rf Values of SQ 26,776 after Thin-LayerChromatoaraphy in Different Solvent Systems Solvent System
Rf Value ---
Ref
1 0.25mm Silica Gel GF (Analtech)
n-Propanol/acetic acid/water/ethyl acetate (70: 2:35: 60)
0.68
32
2 0.25mm Silica Gel GF (Analtech)
n-Propanol/acetic acid (9: 1)
0.53
32
3 0.25mm Silica Gel GF (Analtech)
Chloroform/methanol/arnmonia (50: 50: 5)
0.38 (very broad)
32
4 0.25mm Silica Gel GF (Analtech)
Chloroform/methanol/acetic acid/ethyl acetate (50:50:2: 50)
0.26
32
5 0.25mm Silica Gel (Analtech)
Chloroform/methanol/methylisobutyl ketone (1 :1:1)
0.21 (very broad)
32
6 0.25mm SilicaGel GF (Analtech)
Ethyl acetate/methanol/acetic acid/methyl isobutyl ketone (50:50:2:50)
0.46
32
7 0.25mm Silica Gel GF (Analtech)
b-ButanoVethyl acetate/water/acetic acid (1 :1:1: 1)
0.62
33
8 0.25mm Silica Gel GF (Analtech)
n-Butanol/acetic acidlwater (3:1:1)
0.50
32
9 0.25mm Silica Gel 60 F254(Merck)
n-PropanoVacetic acid/water/ethyl acetate (70:2:35:60)
0.32
34
10 0.25mm Silica Gel 60 F254 (Merck)
n-ButanoVethyl acetate/water/acetic acid (1 :1: 1:1)
0.50
33
11 0.25mm Silica Gel G (Analtech) impregnated with tetradecane
Mcllvaine's buffer pH 6.Yacetone (200:3)
-0.5
34
12 0.25mm Silica Gel (Merck)
n-Propanol-ethyl acetate-pH 7.0 phosphate buffer (70:60 :35)
-0.50
34
13 Analtech RPS
Water-acetonitrile-sodi um perchlorate (995: 5: 0.7)
-0.80
33
Plate
AZTREONAM
Svstern
5.
31
SUDDO~~
BufferhH
Voltslcrn Minutes
1
Celldose (Eastman)
Pyridine acetate 0.05M. pH 4.0
20
60
2
CelIdose (Eastman)
2M Phosphate
10
45,81
3
Celldose (Eastman)
Phosphate 0.05M. pH 6.9
20
60
+
Stability - Deqradation
5.1 Solid Stabilit T e sta ility of a and fl crystalline forms has been studied (see 3.1 1 Crystal Properties). 5.1 1 a-Form
T h e - f o r m (crystallization f r o m aqueous ethanol or methanol) is not very stable. A 1% loss at RT and an 80% loss at 800 C. after a one-week stora e has been reported (Figure 17)41. An energy of activation of 25 cal/mole, assuming a first-order model and 20 kcal/mole, assuming a zero-model was calculated. Both energies o f activation were in the range indicative of hydrolysis reactions.
1
w
5.12 T e -form (recrvstallized from anhvdrous ethanol) is stable. Aft&' a 12-monthastorageat -200, + 5%: + 330, + 400, + 220, 80% RH and 400 C./75% RH batches s t i l l pass specifications at all temperatures and humidity stations42. Even under the most rigorous storage conditions (40% C./75% RH), the samples were found to have undergone only a slight increase (<2% respectively by TLC) in impurities and a small drop (3.0to 3.51% by HPLC) in potency.
Solution Stabilit In aaueous solttion, the most imDortant source of instability of azireonam over the whole pH range is hydrolysis o f the beta-lactam ring19. 5.2
In weakly acid solutions (pH 2 t o 5), hydrolysis i s preceded by isomerization of the side chain as shown in Figure 18 (conversion of Z (syn) t o E (anti) isomer)1619. Care has t o be exercised that E-isomer formation does not occur as an artifact
KLAUS FJBREY
32
0 1 2 3 . ( 5 6 7 8 9 1 O I I I 2 l 3
TI ME
FIGURE 17.
(weeks)
Aztreonam Alpha Form: Solid-state Stability: Temperature Profile
I m 0 In
d N
1.:
4,
m
X
V
X-Z
N X
5: N
L? z
I
N
W
I N
\ /
X
/
a
KLAUS FLOREY
34
9
durin chromatographic separation43. The pH-rate profile for the hydro ysis of aztreonam at 350 C. and constant ionic strength (l.~ = 0.5M KCI) is presented in Figure 1919. It shows that aztreonam is most stable in the pH 5-7 range. The Arrhenius plot for aztreonam degradation is presented in Figure 2019. It has also been shown41 that in aqueous, buffer-free solution, aztreonam is about 5 times more stable in the pH 4-7 range than most penicillins and cephalosporins, including ampicillin and cephradine. At pH 5-7, aztreonam shows 10% de radation in 300-500 hours. Phosphate, tris, borate and car onate buffers accelerate degradation .
%
3
Other de radation products that have been observed are the desulfonate productsu:
H '.R
Lf3
and
0
H
R\'
NrB"3 NB2
0-C,
ow
Degradation of aztreonam t o dimers and trimers in aqueous solution has also been describedss. Li ht Stabilit M e x o o s e d one week t o 400 and 900 footcandles of fludrestent illumination and 33-35% C. temperature yellowed and showed a 6% conversion t o the 'E' isomer. Samples stored in the dark a t 330 C. or exposed t o room light and temperature f o r one week showed n o y e l l o w i n g or chromatographic evidence of significant '2' t o 'E' isomerizationl9. 5.3
Pink Discoloration After long standing at a pH of <5, a pink discoloration of aztreonam has been observed. Solids exposed t o moisture may also exhibit this phenomenonlg. 5.4
PH
FIGURE 19: AZTREONAM. pH Rate Profile for Degradation of Aztreonam at 3 5 O C. an p = 0.5M. The rate constants have been extrapolated to zero buffer concentration.
1o'lr
FIGURE 20: AZTREONAM. Arrhenius Plots for Aztreonam De radation. Key: [51 pH 3.66;A pH 5.40; pH 7.92
0
KLAUS FLOREY
36
5.5 Stability in Bioloqical Fluids
Aztreonam is stable in human serum and urine for at least 10 months when kept a t -780 C.46. However, aztreonam is unstable in human serum even at -200 C. where a loss of 5% was observed after 7 days. However, immediate dilution (1120)in pH 6.0 phosphate buffer great1 increased stability a t -200 C. t o a loss of
i
Aztreonam is more stabile in urine. It loses about 2.5%/24 hours at 250 C., 1.5%124 hours a t 50 C. and only 0.515%12 weeks a t -200 C. It, therefore, should be stored a t -200 C. and assayed within 4-6 weeks47. Aztreonam was found to be stable in hemodialysis and peritoneal dialysis fluids for at least four days a t room temperature to -200 C.43. There was no loss of activity in waste dialysate buffered to pH 6 after storage of -200 C. for seven weeks. However an activity lossof -9% occurred in the unbuffered dialysate after two weeks' storage46. 6.
Drua Metabolism, Pharmacokinetics
Metabolism and pharmacokinetics of aztreonam have been studied with 14C labeled aztreonam in the rat, dog and monkeya as well as in human volunteers48A9. Distribution in male and female ratsso, in rat tissue51 and fetuses and milk of rats52 has also been described. In human volunteers, after intravenous administration, the pharmacokinetic properties of aztreonam in serum were described by an open, linear, two-compartment model. After intramuscular administration of aztreonam t o human volunteers, the serum concentration vs. time data was described by a biex ponent ia I equat ion w h 3 represented a one-compartme nt model with first-order absorption and elimination. Bioavailability after an intramuscular dose was 100%. After either intravenous or intramuscular administration, aztreonam was eliminated primarily by urinary excretion of unchanged aztreonam (about 66% of dose); only 1% of the dose was found as unchanged aztreonam in the feces, presumably as a result of bilia secretion. The values determined for the average elimination alf-life of aztreonam were 1.6 and 1.7h, respectively, after intravenous and intramuscular administration. Aztreonam did n o t undergo extensive metabolism; the most prominent biotransformation roduct of aztreonam was the derivative resulting from the lydrolytic opening of the beta-lactam ring. Excretion o f this compound in urine and feces accounted for 7 and 3% of the
7l
AZTREONAM
37
administered dose, respectively. It was eliminated a t a considerably slower rate than was aztreonam53. Other metabolites, accounting for 4 t o 5% of the radioactivity in urine, have not yet been identified53. 7. 1.
2. 3.
4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21.
References C.M. Cimarusti, R.B. Sykes, H.A. Applegate, D.P. Bonner, H. Breuer, H.W. Chang, T. Denzel, D.M. Floyd, A.W. Fritz, W.H. Koster, W.C. Liu, W.L. Parker, M.L. Rathnum, W.A. Slusarchyk, U.D. Treuner, M.G. Young, Am. College Clin. Pharm., 1,35 (1981). R.B. Syk& and I.Phillips (eds.), J. Antimicrob. Chemother. 8 (Supplement E), s(1981). R.B. Sykes, D.P. Bonner, K. Bush and N.H. Georgopapadakou, Antimicrob. Agents and Chemother., a , 8 5 (1982). C.M. Cimarusti and R.B. Sykes, Chem. in Brit., 19,302 (1983). C.M. Cimarusti, Trends Pharmacol. Sci., 4,468fi983). C.M. Cimarusti and R.B. Sykes, Med. Res. Rev., 4,l (1984). D.M. Floyd, A.W. Fritz and C.M. Cimarusti, J. Org. Chem., 47, 176 (1982). R.B. Sykes, W.L. Parker, C.M. Cimarusti, W.H. Koster, W.A. Slusarchyk, A.W. Fritz, D.M. Flo d, U.K. Patent Application GB2071650A, published Septem er 29,1981; also EP048953 (1982). P. Egli and L. Gnacek, Personal Communication. M. Paslawsky, The Squibb Institute, Personal Communication (1982). M. Porubcan, The Sauibb Institute. Personal Communication (1983). A.I. Cohen, P.T. Funke, B.N. Green, J. Pharm. Sci., 7 l , 1065 (1982). S.E. Unger and T.J. McCormick, Trends Anal. Chem., 4, 178 (1985). S.E. Unger, Int. J. Mass Spectrom. Ion Process.& 195 (1985). S.E. Unger and B.M. Warrack, Spectroscopy, 1 , s (1986). J. Adamovics and 5. Unger, J. Liquid Chromatogr., 9, 141 (1986). B. Kline and 5 . Willis, The Squibb Institute, Personal Commun ication. J.D. Pipkin and M. Davidovich, The Squibb Institute, Personal Communication (1983). J.D. Pipkin, Chemical Solubility of Pharmaceuticals, 2nd ed., p. 250, K.A. Connors, et. al. (eds.), John Wiley, New York (1986). J.DeVincentis, The Squib b l n s t i t Ute, Personal Communication. J.D. Pipkin, The Squibb Institute, Personal Communication (1981).
1
KLAUS FLQREY
38
22. A. Pudzianowski, The Squibb Institute, Personal Communication (1983). 23. C. Sachs, The Squibb Institute, Personal Communication. 24. A. Pudzianowski, C. Sachs, The Squibb Institute, Personal Communication (1982). 25. DeIeted . 26. M. Malley and J. Gou outas, The Squibb Institute, Personal Communication (19847. 27. E.A. Swabb, M.A. Leitz, F.G. Pilkiewicz and A.A. Sugerman, J. Antimicrob. Chemotherap. 8 (Sup I. E), 131 (1981). 28. W. Ehret, H. Probst, G. Ruck eschel, J. Antimicrobial Chemother., 19,541(1987). 29. J. Gentile, C. Fassbender, H. Weisblatt, The Squibb Institute, Personal Communication (1981). 30. D. Both, The Squibb Institute, Personal Communication (1981). 31. C. Sherman, The Squibb Institute, Personal Communication. 32. M.S. Bathala and S.H. Weinstein, The Squibb Institute, Personal Communication (1981). 33. 0. Kocy, The Squibb Institute, Personal Communication (1984). 34. H.R. Roberts, The Squibb Institute, Personal Communication (1982). 35. F.G. Pilkiewicz, B.J. Remsburg, S.M. Fisher and R.B. Sykes, Antimicrobiol. Agents Chemother., 3,852(1983). 36. D.W. Marble, J.A. Bosso and R.J. Townsend, Am. J. Hosp. Pharm., 43,1732(1986). 37. M.J. Jamzand C. Riley, Am. 1. Hosp. Pharm., 42,1095. 38. F. Hehl, P. Birckel and H. Monteil, 1. Chromatogr., 413, 109 (1987). 39. J. Adamovics, Liq. Chromatogr. Mag.,& 393 (1984). 40- M.J. James and C.M. Riley, Am. J. Hosp. Pharm., 42, 1984 (1985). 41. J.D. Pipkin and E.P. Barry, The Squibb Institute, Personal Communication (1981). 42. A.R. Restivo and M.C. Blaich, Personal Communication (1983). 43- J.M. Joseph, The Squibb Institute, Personal Communication (1983). 44. S.E. Un er, T.J. McCormick, Analytical R&D Report dated Septem er 30,1983. 45. J.D. Pipkin, S.A. Ranadive, N.H. Chang, S.E. Unger, T.J.
8
%
McCormick, M. Porubcan, E.P. Barr , S.A. Varia and J. Adamovics, Am. Pharm. Assoc. Acad. PYl arm. Sci. Abst., l5(2)
135 (1985). 46. M.A. Leitz, 5 . Wind, The Squibb Institute, Personal Communication (1983).
AZTREONAM
47. 48.
49. 50.
51. 52. 53.
54.
8.
39
J.A. Manning, M.A. Leitz, T.B. Platt, The Squibb Institute , Personal Communication (1982). K.J. Kripalani, S.M. Singhvi, S.H. Weinstein, D.W. Everett, M.S. Bathala, A.V. Dean, C.E. Ita, L. Lawrence, F.S. Meeker, J.M. Shaw, B.D. Walker and B.H. Migdalof, Antimicrob. Agents Chemother., 26,119 (1984). E.A. Swabb, A.A. Sugerman, T.B. Platt, F. Pilkiewicz and M. Frantz, Antimicrob. Agents and Chemother., 21,944 (1982). S.M. Singhvi, C.E. Ita, J.M. Shaw and B.H. Migdalof, Pharmacologist, 2 5 , 1 1 5 (1983).
3
S.M. Singhvi, C.E. Ita, J.M. Shaw and B.H. M i dalof, Antimicrob. Agents and Chemotherap., 26,127 (1984 . S.M. Singhvi, C.E. Ita, J.M. Shaw, G.R. Keim and B.H. Migdalof, Antimicrob. Agents Chemother., 26,132 (1984). €.A. Swabb, S.M. Singhvi, M.A. Leitz, M. Frantz and A. Sugerman, Antimicrob. Agents Chemother., 24,394 (1983). A. Menlemans, J. Mohler, D. Vitteco , G. Haroche, M.A. Rosset, M. Vulpillat and J. Modai, J. C romatogr., 377, 466 (1986).
i?
Acknowledaement
For contributions t o the orofile, 1 am indebted t o G.A. Brewer, C. Cimarusti, F. Dondzija, P. Funke, J.Z. Gougoutas, J. Kirschbaum, 0. Kocy, B. Migdalof, M. Paslawsky, J.D. Pipkin, T. Platt, M. Porubcan, A. Pudzianowski, A. Restivo, H. Roberts and 5. Unger. I also would like to thank Marie Bruno for competent and patient secretarial assistance. .