- Email: [email protected]
Maternal and fetal pharmacokinetics of moxalactam given intrapartum
Maternal and fetal pharmacokinetics of moxalactam given intrapartum ROGER
E.
F. GARY
PH.D.
CUNNINGHAM,
J. GERALD MICKI
BAWDON,
L.
M.D.
QUIRK, ROARK,
M.D.,
PH.D.
R.N.
Dallas. Texas Two grams of moxalactam was given intravenously to 28 women at high risk for infection following cesarean delivery. After a mean time of 48 minutes from infusion, maternal sera, cord sera, and uterine tissue obtained at delivery had concentrations of moxalactam of 82 @/ml, 22.2 pg/ml, and 9.8 cLg/gm, respectively. The maternal serum halfltime was calculated to be 2.1 hours. R and S epimeric distribution was determined in these sera and tissues, and the mean R/S ratios were 0.95, 0.93, and 1.22 for the three groups, respectively. The significance of these observations is discussed. A new method in which a high-pressure liquid chromatographic assay is used is described, and results are compared to those obtained with the microbiological assay. The high-pressure liquid chromatographic method was found to be quick, accurate, and reproducible. (AM. J. OBSTET. GYNECOL. 144:548, 1982.)
MOXALACTAM is a “third-generation” cephalosporin with a broad spectrum of antimicrobial activity. Like most fi-lactam antimicrobials, it has R and S epimers and it has been suggested that the R epimer has greater antimicrobial activity. ‘-’ Moxalactam is effective in vitro against many aerobic and anaerobic pathogens frequently isolated from women with polymicrobial pelvic infections, with the exceptions of strains of Sta@lococcus aureus and Bacteroides fragilis. 4-S The drug has minimal adverse effects and may be given by rapid infusion of doses up to 4 gm. These features, along with its serum half-life of greater than 2 hours, make moxalactam an attractive selection for perioperative treatment of women at high risk for infection following cesarean delivery.
From the Depatiment of Obstetrics and Gynecology, University of Texas Health Science Center, Southwestern Medical School. Supported in part by a grant from Indianapolis, Indiana.
Eli Lilly
f3 Co.,
Presented in part at the Twenty-ninth Annual Meeting of the Society for Gynecologk Investigation, Dallas, Texas, March 24-27, 1982. Reprint requests: Dr. F. Gary Cunningham, Department of Obstetrics and Gynecology, University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas 75235.
546
To evaluate its clinical efficacy, we gave three-dose perioperative moxalactam therapy to more than 100 women at risk for cesarean section-related infection. During this investigation we developed a practical high-pressure liquid chromatographic assay for moxalactam and determined its concentrations and epimeric distribution in maternal and cord sera and uterine tissue following a 2 gm intravenous infusion given preoperatively.
Material and methods Patients. Of 130 women given three-dose perioperative moxalactam because they were at high risk for infection following cesarean delivery, 28 participated in this investigation. We have previously described risk factors for this group of nulliparous women, which include cephalopelvic disproportion and 6 or more hours of membrane rupture.’ After a decision was made to effect delivery by cesarean section and informed consent was obtained, 2 gm of moxalactam was given intravenously in 150 ml of normal saline over 20 minutes, and the time of the infusion was recorded. At delivery, maternal and cord sera were collected simultaneously, and in 11 women myometrial tissue was obtained from the incision site. The specimens were frozen at - 20” C, and assays were performed within 1 month of coliection. The concentration of moxalactam in the speci0002-9378/82/210546+05$00.50/O
0
1982 The
C.V.
Mosby
Co.
Volume Number
Pharmacokinetics
144 5
of moxalactam
547
130 110 $
go-
Y “, 70ii a3
50-
% 30 y =09463X
l
+ 3.17
r = 0.94 II 0
I
I
I
I
I
I
I
I
20 40 60 80 100 Microbiological Assay ( pg/ml I
I(
120
Fig. 2. Comparison of moxalactam serum levels with the use of the microbiological assay and the high-pressure liquid chromatographic assay. A: Represents the line of perfect correlation. B: Represents the correlation coefficient obtained by linear least-squares regression. I
4
6
8 10
Retention
Time
0
2
I
I
I
4
6
(minutes
1
I
I
I
,
8 10
Fig. 1. HPLC chromatograms of serum extracts. A: Normal serum.B: Normal serum containing 24 pg/ml of moxalactam. The R and S epimers of moxalactam are indicated. The retention times of the R and S epimers were 6.8 and 8.0 minutes, respectively. A. U.: Absorbance units. mens was determined by both the microbiological and the high-pressure liquid chromatographic assays. Microbiological assay. The concentration of moxalactam was measured in serum and tissue extracts by the agar diffusion bioassay technique.s The test organism was Escherichiu coli ATCC 10536, and all samples were assayed in two dilutions and in duplicate. Assay was performed on serum samples with no extraction. Tissue samples were thawed, rinsed briefly in O.lOM phosphate buffer with a pH of 6.0, diced with a scalpel, weighed, covered with 10 ml of the phosphate buffer, and homogenized with a Potter Elvehjem homogenizer in a water and ice bath. The homogenates were centrifuged for 10 minutes at 3,000 x g to remove cellular debris, and the microbiological assay was performed on the supernatant. Tissue concentrations of moxalactam were corrected for blood contamination by means of the cyanmethemoglobin calorimetric method.$ High-pressure liquid chromatographic assay. The
high-pressure liquid chromatographic system was an ALC/GPC Model 204 liquid chromatograph (Waters Associates, Milford, Massachusetts). All analyses were performed on a reverse phase column (p Bondapak Crs, Waters Associates, Milford, Massachusetts). The eluate was monitored at 254 nm with the use of the 440 absorbance units full scale. Peaks were recorded on a 10 mV chart recorder (Houston Instruments, Houston, Texas) at a chart speed of 0.5 cm/min. The mobile phase, consisting of 0.1 M sodium phosphate (84%) and methanol (16%) at a pH of 3.2, was pumped at a flow rate of 2.0 ml/min at ambient temperature. The buffer was degassed by filtration with a 0.45 rnp FHUP filter (Millipore Corp., Bedford, Massachusetts). A standard curve was prepared with the use of spiked serum or uterine tissue extracts from pregnant women not given antimicrobials. The curve had a range of 0 to 100 pglml of moxalactam. Tissue specimen supernatants and sera were the same ones used for the microbiological assay. Five hundred microliters of sera or tissue supernatant was extracted in 2.0 ml of ice-cold acetonitrile to precipitate protein (high-pressure liquid chromatographic grade reagents were used throughout), vortexed for 1 minute, then centrifuged for 5 minutes at 3,000 x g at 4” C. The supernatant was decanted into a screw-capped tube containing 4 ml of methylene chloride, vortexed for 30 seconds, and centrifuged for 5 minutes at 3,000 X g. The upper aque-
548
Ncwcmher 1, 1982 Am. J. Obstet. Gynecol.
Bawdon et al.
Table I. Precision studies of high-pressure chromatographic assay for moxalactam Moxalactam concentration (t-&4
No.
Betweesbatch 5.0 47.6
deteminutims: 7 8
Within-batch 5.0 47.6
determinations: 10
10
liquid
Average recovely C%J
Mean
SD
0
4.87 46.89
0.52 1.50
10.7 3.2
97.4 98.5
5.31 47.40
0.05 2.30
9.2 4.8
106.0 99.7
CV = Coefficient of variation.
I
’
t
10
11
I1
30
50 Time
I1
70 (minutes
11
1
90 1
’
110
1
I
130
Fig. 3. Semilogarithmic graph of moxalactam concentrations in maternal and cord sera and uterine tissue levels obtained simultaneously. Specimens were grouped in 5-minute intervals, and number in parentheses indicates the number used to obtain the mean value. The same numbers of cord and maternal serum specimens were collected. Values include standard deviations when applicable. The dotted line through the maternal levels represents the calculated half-time of moxalactam in these pregnant women. A, Maternal serum level. l , Cord serum level. 0, Uterine tissue level. ous layer was removed and maintained at 4” C until chromatography was performed. A 50 ~1 aliquot of the aqueous layer was injected with a microsyringe (No. 810, Hamilton Co., Reno, Nevada), for the high-pressure liquid chromatographic assay.‘O, I1 All specimens were quantified by measuring the peak heights and comparing these with the standard curve. Comparison of assay methods. Accuracy of the high-pressure liquid chromatographic and microbiological procedures was compared with the use of assays performed within 3 days of each other to avoid correction for moxalactam degradation. Precision of the high-pressure liquid chromatographic assay was estimated by multiple determinations of spiked standards containing 5.0 and 47.6 pg/ml of moxalactam. For between-batch reproducibility, the spiked sera were assayed eight times over a 3-week period. For withinbatch reproducibility, the spiked sera were assayed 10 times within the same run. Results Representative high-pressure liquid chromatograms of serum extracts are illustrated in Fig. 1. The left
panel (A) depicts an extract from control serum, whereas panel B depicts a serum extract that was spiked with moxalactam. The resolution of R and S epimers from serum and tissue specimens was equivalent. In the 28 serum samples, the concentration of moxalactam determined by the high-pressure liquid chromatographic assay was compared to the concentration of the drug determined by microbiological assay (Fig. 2). The correlation coefficient, calculated by linear least-square regression, was 0.94. Comparable results were obtained when tissue levels were assayed by both methods. The precision of the high-pressure liquid chromatographic assay was tested. Between-batch recoveries were 97.4% and 98.5% for the two control levels over a 3-week period, and within-day determinations had recoveries of 106% and 99.7% (Table I). The mean interval from the completion of the moxalactam infusion to delivery was 48 minutes and ranged from 5 to 110 minutes. The mean maternal serum level was 62 pgiml and the simultaneous mean cord level was 22.2 pg/ml. The mean level of drug in 11 uterine tissue samples was 9.6 kgigm. The mean R/S epimeric ratios for the three groups of specimens were 0.95, 0.93, and 1.22 for maternal and cord sera and uterine tissue, respectively. Maternal serum moxalactam concentrations decreased with time following infusion (Fig. 3), and the calculated half-time was 2.1 hours. Comment The calculated serum half-time of moxalactam in these pregnant women was less than previously reported for nonpregnant individuals.‘. ‘* Since the kidney is the principal route of clearance, this decreased half-time reflects the increased glomerular filtration that is characteristic of pregnancy. The mean cord serum level of moxalactam was 36% of the simultaneous maternal level, and the uterine tissue level was 15% of the maternal level. Reported ranges for other
Volume Number
Pharmacokinetics
144 5
Table II. Simultaneous maternal serum, 0 to 2 hours after infusion or injection
cord serum,
and uterine
Cephalospmin
Dose
Cephalothin Cephaloridine Cefuroxime Cefotaxime
1 gm IM 1 gm IV 750 mg IM 1 gm IV gm 22 gm IV IV
Cefazolin Moxalactam
1.8-32 20.0
&ml
uterus
% Maternul*
12-18
2.2-2.8 1-15 6.0
8.2
1.9
7 5 33 23
39.6 22.2
30 36
132 62
549
tissue levels of some cephalosporins
Cord Matmal (t&f4
of moxalactam
pglgm
tissue
% Maternal
*
Reference No.
1.5 -
12.5 -
13 14 15 16
9.6
15.4
:
IM = Intramuscularly; IV = intravenously. *Indicates minimum percentage of maternal serum level. ?Present study. cephalosporins in maternal and cord sera as well as in uterine tissue are summarized in Table II. The broad range of levels probably is reflective of the half-times of these derivatives. Uterine tissue levels of cefuroxime and moxalactam were 12.5% and 15.4% of the maternal blood levels, respectively. These determinations exceed the minimum inhibitory concentration of many pathogens, and it seems reasonable to emphasize tissue levels rather than serum levels when antimicrobial sensitivities are being considered. The concentration of moxalactam and the distribution of its R and S epimers in cord serum and uterine tissue has not been reported previously. Freshly prepared solutions of moxalactam contain the epimers in equal amount?; however, these equilibrate in serum incubated at 37” C, and within 1% hours the R and S epimeric ratio is 0.82.’ This change probably represents differential binding of epimers to serum proteins or conversion of R epimer to the S form.‘* * Luthy and associates2 determined the R/S ratios in sera from six nonpregnant volunteers over 12 hours, and after 2 hours this decreased from 0.82 to 0.58 and plateaued at 0.5 after 5 hours. However, in another study no change in epimeric distribution was found over 8 hours3 In the present investigation, the mean R/S ratios were 0.95 and 0.93 for maternal and cord sera 1 to 2 hours after infusion, values that are similar to those of the latter report3 In uterine tissues the R/S ratio was 1.22 (t0.05 = 2.33, test of differences) and leads us to speculate that the R epimer has a greater affinity for tissue. A twofold increase in antimicrobial activity of the R epimer over the S epimer of moxalac-
REFERENCES
1. Wise, R., Wills, P. J., and Bedford, K. A.: Epimers of moxalactam: In vitro comparison of activity and stability, Antimicrob. Agents Chemother. 40:30, 1981.
tam has been observedle3; however, with the difficulty in ascertaining the minimum inhibitory concentration end point in agar dilution and with only one tube dilution difference, it is difficult to relate any clinical significance to these findings.‘, I7 The method for assaying moxalactam reported here offers distinct advantages over other systems. The procedure requires a simple extraction in acetonitrile and then methylene chloride. This extraction has been used effectively to determine the concentration of other /3-lactam antimicrobials and worked well in the present study. The chromatographic separation takes less than 10 minutes and effectively separates the R and S epimers.“’ I1 Furthermore, the epimers can be quantified over the range of moxalactam concentrations found following therapeutic doses. The HPLC assay was shown to be as accurate as the standard microbiological assay for moxalactam. Moreover, the simplicity of extraction and the speed with which the chromatographic separation can be performed allow generation of quantitative data on the day the specimen is received. In summary, a high-pressure liquid chromatographic assay for moxalactam was developed and its concentration was determined in maternal serum, cord serum, and uterine tissue. Moxalactam entered the fetus quite rapidly, presumably by simple diffusion since epimeric distribution in cord and maternal sera was similar. Clinical consequences of these observations are relevant in that the infants are born with significant levels of this antimicrobial which has a half-time of more than 6 hours in the neonate.‘*
2. Luthy, R., Blaser, J., Bonetti, A., et al.: Comparative multiple-dose pharmacokinetics of cefotaxime, moxalactam, and ceftazidime, Antimicrob. Agents Chemother. 20:567, 1981.
550
Bawdon et al.
3. Miner, D. J., Coleman, D. L., Shepard, A. M., et al.: Determination of moxalactam in body fluids, Antimicrob. Agents Chemother. 20~252, 1981. 4. Barza, M., Tally, F. P., Jacobus, N. V., et al.: In vitro activitv of LY 127935. Antimicrob. Agents Chemother. 16:287, 1979. 5. Gibbs, R. J., Blanco, J. D., Castaneda, Y. S., et al.: Therapy of obstetrical infections with moxalactam, Antimicrab. Agents Chemother. 17:1004, 1980. 6. Cunningham, F. G., Hemsell, D. L., dePalma, R. T., et al.: Moxalactam for obstetric and gynecologic infections, AM. 1. OBSTET. GYNECOL. 139:915, 1981. 7. dePalma, R. T., Cunningham, F. G., Leveno, K. J., et al.: Continuing investigation of women at high-risk for infection followyng cesarean birth, Obstet. Gynecol. In press. 8. Parsons, J. N., Romano, J. M., and Levison, M. E.: Pharmacology of a new l-oxa-plactam (LY 127935) in normal volunteers, Antimicrob. Agents Chemother. 17:226,1980. 9. Fairbanks, V. F.: Hemoglobin derivatives and myoglobin, in Teitz. N. W.. editor: Fundamentals of Clinical Chemistry, Philadelphia, 1976, W. B. Saunders Co., p. 401. J. T., and Bawdon, R. E.: Determination of 10. Rudrik, penicillinase-resistant penicillins in serum using high pressure liquid chromatography, J. Liquid Chromatogr. 4:1525, 1981. 11. Dajani, A. S., Thirumoothi, M. C., Bawdon, R. E., et al.: Pharmacokinetics of intramuscular-ceforanide in children, Antimicrob. Agents Chemother. 21:282, 1982.
November Am. J. Obstet.
12.
13.
14.
15.
16.
17.
18.
1, 1982 Gynecol.
Polk, R. E., Kline, B. J., and Markowirz, S. M.: Cefazolin and moxalactam pharmacokinetics after simultaneous intravenous infusion, Antimicrob. Agents Chemother. 20: 576, 1981. Sheng, K. T., Huang, N. N., and Promadhattavedi, V.: Serum concentrations of cephalothin in infants and children and placental transmission of the antibiotic, in Sylvester, J. C., editor: Proceedings of the Fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D. C., 1964, American Society for Microbiology, p. 200. Barr, W., and Graham, R. M.: Placental transmission of cephaloridine, J. Obstet. Gynaecol. Br. Commonw. 74: 739, 1967. Craft, I., Mullinger, B. M., and Kennedy, M. R. K.: Placental transfer of cefuroxime, Br. J. Obstet. Gynaecol. 88:141, 1981. Kafetzis, D. A., Lazarides, C. V., Sifas, C. A., et al.: Transfer of cefotaxime in human milk from mother to fetus, J. Antimicrob. Chemother. (Suppl. A) 6:135, 1980. Rolfe, R. D., and Finegold, S. M.: Comparative in vitro activity of new p-lactam antibiotics against anaerobic bacteria, Antimicrob. Agents Chemother. 20~600, 1981. Schaad, V. B., McCracken, G. H., Threlkeld, N., et al.: Clinical evaluation of a new broad-spectrum oxa-betalactam antibiotic, moxalactam, in neonates and infants, J. Pediatr. 98:129, 1981.
E.
F. GARY
PH.D.
CUNNINGHAM,
J. GERALD MICKI
BAWDON,
L.
M.D.
QUIRK, ROARK,
M.D.,
PH.D.
R.N.
Dallas. Texas Two grams of moxalactam was given intravenously to 28 women at high risk for infection following cesarean delivery. After a mean time of 48 minutes from infusion, maternal sera, cord sera, and uterine tissue obtained at delivery had concentrations of moxalactam of 82 @/ml, 22.2 pg/ml, and 9.8 cLg/gm, respectively. The maternal serum halfltime was calculated to be 2.1 hours. R and S epimeric distribution was determined in these sera and tissues, and the mean R/S ratios were 0.95, 0.93, and 1.22 for the three groups, respectively. The significance of these observations is discussed. A new method in which a high-pressure liquid chromatographic assay is used is described, and results are compared to those obtained with the microbiological assay. The high-pressure liquid chromatographic method was found to be quick, accurate, and reproducible. (AM. J. OBSTET. GYNECOL. 144:548, 1982.)
MOXALACTAM is a “third-generation” cephalosporin with a broad spectrum of antimicrobial activity. Like most fi-lactam antimicrobials, it has R and S epimers and it has been suggested that the R epimer has greater antimicrobial activity. ‘-’ Moxalactam is effective in vitro against many aerobic and anaerobic pathogens frequently isolated from women with polymicrobial pelvic infections, with the exceptions of strains of Sta@lococcus aureus and Bacteroides fragilis. 4-S The drug has minimal adverse effects and may be given by rapid infusion of doses up to 4 gm. These features, along with its serum half-life of greater than 2 hours, make moxalactam an attractive selection for perioperative treatment of women at high risk for infection following cesarean delivery.
From the Depatiment of Obstetrics and Gynecology, University of Texas Health Science Center, Southwestern Medical School. Supported in part by a grant from Indianapolis, Indiana.
Eli Lilly
f3 Co.,
Presented in part at the Twenty-ninth Annual Meeting of the Society for Gynecologk Investigation, Dallas, Texas, March 24-27, 1982. Reprint requests: Dr. F. Gary Cunningham, Department of Obstetrics and Gynecology, University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas 75235.
546
To evaluate its clinical efficacy, we gave three-dose perioperative moxalactam therapy to more than 100 women at risk for cesarean section-related infection. During this investigation we developed a practical high-pressure liquid chromatographic assay for moxalactam and determined its concentrations and epimeric distribution in maternal and cord sera and uterine tissue following a 2 gm intravenous infusion given preoperatively.
Material and methods Patients. Of 130 women given three-dose perioperative moxalactam because they were at high risk for infection following cesarean delivery, 28 participated in this investigation. We have previously described risk factors for this group of nulliparous women, which include cephalopelvic disproportion and 6 or more hours of membrane rupture.’ After a decision was made to effect delivery by cesarean section and informed consent was obtained, 2 gm of moxalactam was given intravenously in 150 ml of normal saline over 20 minutes, and the time of the infusion was recorded. At delivery, maternal and cord sera were collected simultaneously, and in 11 women myometrial tissue was obtained from the incision site. The specimens were frozen at - 20” C, and assays were performed within 1 month of coliection. The concentration of moxalactam in the speci0002-9378/82/210546+05$00.50/O
0
1982 The
C.V.
Mosby
Co.
Volume Number
Pharmacokinetics
144 5
of moxalactam
547
130 110 $
go-
Y “, 70ii a3
50-
% 30 y =09463X
l
+ 3.17
r = 0.94 II 0
I
I
I
I
I
I
I
I
20 40 60 80 100 Microbiological Assay ( pg/ml I
I(
120
Fig. 2. Comparison of moxalactam serum levels with the use of the microbiological assay and the high-pressure liquid chromatographic assay. A: Represents the line of perfect correlation. B: Represents the correlation coefficient obtained by linear least-squares regression. I
4
6
8 10
Retention
Time
0
2
I
I
I
4
6
(minutes
1
I
I
I
,
8 10
Fig. 1. HPLC chromatograms of serum extracts. A: Normal serum.B: Normal serum containing 24 pg/ml of moxalactam. The R and S epimers of moxalactam are indicated. The retention times of the R and S epimers were 6.8 and 8.0 minutes, respectively. A. U.: Absorbance units. mens was determined by both the microbiological and the high-pressure liquid chromatographic assays. Microbiological assay. The concentration of moxalactam was measured in serum and tissue extracts by the agar diffusion bioassay technique.s The test organism was Escherichiu coli ATCC 10536, and all samples were assayed in two dilutions and in duplicate. Assay was performed on serum samples with no extraction. Tissue samples were thawed, rinsed briefly in O.lOM phosphate buffer with a pH of 6.0, diced with a scalpel, weighed, covered with 10 ml of the phosphate buffer, and homogenized with a Potter Elvehjem homogenizer in a water and ice bath. The homogenates were centrifuged for 10 minutes at 3,000 x g to remove cellular debris, and the microbiological assay was performed on the supernatant. Tissue concentrations of moxalactam were corrected for blood contamination by means of the cyanmethemoglobin calorimetric method.$ High-pressure liquid chromatographic assay. The
high-pressure liquid chromatographic system was an ALC/GPC Model 204 liquid chromatograph (Waters Associates, Milford, Massachusetts). All analyses were performed on a reverse phase column (p Bondapak Crs, Waters Associates, Milford, Massachusetts). The eluate was monitored at 254 nm with the use of the 440 absorbance units full scale. Peaks were recorded on a 10 mV chart recorder (Houston Instruments, Houston, Texas) at a chart speed of 0.5 cm/min. The mobile phase, consisting of 0.1 M sodium phosphate (84%) and methanol (16%) at a pH of 3.2, was pumped at a flow rate of 2.0 ml/min at ambient temperature. The buffer was degassed by filtration with a 0.45 rnp FHUP filter (Millipore Corp., Bedford, Massachusetts). A standard curve was prepared with the use of spiked serum or uterine tissue extracts from pregnant women not given antimicrobials. The curve had a range of 0 to 100 pglml of moxalactam. Tissue specimen supernatants and sera were the same ones used for the microbiological assay. Five hundred microliters of sera or tissue supernatant was extracted in 2.0 ml of ice-cold acetonitrile to precipitate protein (high-pressure liquid chromatographic grade reagents were used throughout), vortexed for 1 minute, then centrifuged for 5 minutes at 3,000 x g at 4” C. The supernatant was decanted into a screw-capped tube containing 4 ml of methylene chloride, vortexed for 30 seconds, and centrifuged for 5 minutes at 3,000 X g. The upper aque-
548
Ncwcmher 1, 1982 Am. J. Obstet. Gynecol.
Bawdon et al.
Table I. Precision studies of high-pressure chromatographic assay for moxalactam Moxalactam concentration (t-&4
No.
Betweesbatch 5.0 47.6
deteminutims: 7 8
Within-batch 5.0 47.6
determinations: 10
10
liquid
Average recovely C%J
Mean
SD
0
4.87 46.89
0.52 1.50
10.7 3.2
97.4 98.5
5.31 47.40
0.05 2.30
9.2 4.8
106.0 99.7
CV = Coefficient of variation.
I
’
t
10
11
I1
30
50 Time
I1
70 (minutes
11
1
90 1
’
110
1
I
130
Fig. 3. Semilogarithmic graph of moxalactam concentrations in maternal and cord sera and uterine tissue levels obtained simultaneously. Specimens were grouped in 5-minute intervals, and number in parentheses indicates the number used to obtain the mean value. The same numbers of cord and maternal serum specimens were collected. Values include standard deviations when applicable. The dotted line through the maternal levels represents the calculated half-time of moxalactam in these pregnant women. A, Maternal serum level. l , Cord serum level. 0, Uterine tissue level. ous layer was removed and maintained at 4” C until chromatography was performed. A 50 ~1 aliquot of the aqueous layer was injected with a microsyringe (No. 810, Hamilton Co., Reno, Nevada), for the high-pressure liquid chromatographic assay.‘O, I1 All specimens were quantified by measuring the peak heights and comparing these with the standard curve. Comparison of assay methods. Accuracy of the high-pressure liquid chromatographic and microbiological procedures was compared with the use of assays performed within 3 days of each other to avoid correction for moxalactam degradation. Precision of the high-pressure liquid chromatographic assay was estimated by multiple determinations of spiked standards containing 5.0 and 47.6 pg/ml of moxalactam. For between-batch reproducibility, the spiked sera were assayed eight times over a 3-week period. For withinbatch reproducibility, the spiked sera were assayed 10 times within the same run. Results Representative high-pressure liquid chromatograms of serum extracts are illustrated in Fig. 1. The left
panel (A) depicts an extract from control serum, whereas panel B depicts a serum extract that was spiked with moxalactam. The resolution of R and S epimers from serum and tissue specimens was equivalent. In the 28 serum samples, the concentration of moxalactam determined by the high-pressure liquid chromatographic assay was compared to the concentration of the drug determined by microbiological assay (Fig. 2). The correlation coefficient, calculated by linear least-square regression, was 0.94. Comparable results were obtained when tissue levels were assayed by both methods. The precision of the high-pressure liquid chromatographic assay was tested. Between-batch recoveries were 97.4% and 98.5% for the two control levels over a 3-week period, and within-day determinations had recoveries of 106% and 99.7% (Table I). The mean interval from the completion of the moxalactam infusion to delivery was 48 minutes and ranged from 5 to 110 minutes. The mean maternal serum level was 62 pgiml and the simultaneous mean cord level was 22.2 pg/ml. The mean level of drug in 11 uterine tissue samples was 9.6 kgigm. The mean R/S epimeric ratios for the three groups of specimens were 0.95, 0.93, and 1.22 for maternal and cord sera and uterine tissue, respectively. Maternal serum moxalactam concentrations decreased with time following infusion (Fig. 3), and the calculated half-time was 2.1 hours. Comment The calculated serum half-time of moxalactam in these pregnant women was less than previously reported for nonpregnant individuals.‘. ‘* Since the kidney is the principal route of clearance, this decreased half-time reflects the increased glomerular filtration that is characteristic of pregnancy. The mean cord serum level of moxalactam was 36% of the simultaneous maternal level, and the uterine tissue level was 15% of the maternal level. Reported ranges for other
Volume Number
Pharmacokinetics
144 5
Table II. Simultaneous maternal serum, 0 to 2 hours after infusion or injection
cord serum,
and uterine
Cephalospmin
Dose
Cephalothin Cephaloridine Cefuroxime Cefotaxime
1 gm IM 1 gm IV 750 mg IM 1 gm IV gm 22 gm IV IV
Cefazolin Moxalactam
1.8-32 20.0
&ml
uterus
% Maternul*
12-18
2.2-2.8 1-15 6.0
8.2
1.9
7 5 33 23
39.6 22.2
30 36
132 62
549
tissue levels of some cephalosporins
Cord Matmal (t&f4
of moxalactam
pglgm
tissue
% Maternal
*
Reference No.
1.5 -
12.5 -
13 14 15 16
9.6
15.4
:
IM = Intramuscularly; IV = intravenously. *Indicates minimum percentage of maternal serum level. ?Present study. cephalosporins in maternal and cord sera as well as in uterine tissue are summarized in Table II. The broad range of levels probably is reflective of the half-times of these derivatives. Uterine tissue levels of cefuroxime and moxalactam were 12.5% and 15.4% of the maternal blood levels, respectively. These determinations exceed the minimum inhibitory concentration of many pathogens, and it seems reasonable to emphasize tissue levels rather than serum levels when antimicrobial sensitivities are being considered. The concentration of moxalactam and the distribution of its R and S epimers in cord serum and uterine tissue has not been reported previously. Freshly prepared solutions of moxalactam contain the epimers in equal amount?; however, these equilibrate in serum incubated at 37” C, and within 1% hours the R and S epimeric ratio is 0.82.’ This change probably represents differential binding of epimers to serum proteins or conversion of R epimer to the S form.‘* * Luthy and associates2 determined the R/S ratios in sera from six nonpregnant volunteers over 12 hours, and after 2 hours this decreased from 0.82 to 0.58 and plateaued at 0.5 after 5 hours. However, in another study no change in epimeric distribution was found over 8 hours3 In the present investigation, the mean R/S ratios were 0.95 and 0.93 for maternal and cord sera 1 to 2 hours after infusion, values that are similar to those of the latter report3 In uterine tissues the R/S ratio was 1.22 (t0.05 = 2.33, test of differences) and leads us to speculate that the R epimer has a greater affinity for tissue. A twofold increase in antimicrobial activity of the R epimer over the S epimer of moxalac-
REFERENCES
1. Wise, R., Wills, P. J., and Bedford, K. A.: Epimers of moxalactam: In vitro comparison of activity and stability, Antimicrob. Agents Chemother. 40:30, 1981.
tam has been observedle3; however, with the difficulty in ascertaining the minimum inhibitory concentration end point in agar dilution and with only one tube dilution difference, it is difficult to relate any clinical significance to these findings.‘, I7 The method for assaying moxalactam reported here offers distinct advantages over other systems. The procedure requires a simple extraction in acetonitrile and then methylene chloride. This extraction has been used effectively to determine the concentration of other /3-lactam antimicrobials and worked well in the present study. The chromatographic separation takes less than 10 minutes and effectively separates the R and S epimers.“’ I1 Furthermore, the epimers can be quantified over the range of moxalactam concentrations found following therapeutic doses. The HPLC assay was shown to be as accurate as the standard microbiological assay for moxalactam. Moreover, the simplicity of extraction and the speed with which the chromatographic separation can be performed allow generation of quantitative data on the day the specimen is received. In summary, a high-pressure liquid chromatographic assay for moxalactam was developed and its concentration was determined in maternal serum, cord serum, and uterine tissue. Moxalactam entered the fetus quite rapidly, presumably by simple diffusion since epimeric distribution in cord and maternal sera was similar. Clinical consequences of these observations are relevant in that the infants are born with significant levels of this antimicrobial which has a half-time of more than 6 hours in the neonate.‘*
2. Luthy, R., Blaser, J., Bonetti, A., et al.: Comparative multiple-dose pharmacokinetics of cefotaxime, moxalactam, and ceftazidime, Antimicrob. Agents Chemother. 20:567, 1981.
550
Bawdon et al.
3. Miner, D. J., Coleman, D. L., Shepard, A. M., et al.: Determination of moxalactam in body fluids, Antimicrob. Agents Chemother. 20~252, 1981. 4. Barza, M., Tally, F. P., Jacobus, N. V., et al.: In vitro activitv of LY 127935. Antimicrob. Agents Chemother. 16:287, 1979. 5. Gibbs, R. J., Blanco, J. D., Castaneda, Y. S., et al.: Therapy of obstetrical infections with moxalactam, Antimicrab. Agents Chemother. 17:1004, 1980. 6. Cunningham, F. G., Hemsell, D. L., dePalma, R. T., et al.: Moxalactam for obstetric and gynecologic infections, AM. 1. OBSTET. GYNECOL. 139:915, 1981. 7. dePalma, R. T., Cunningham, F. G., Leveno, K. J., et al.: Continuing investigation of women at high-risk for infection followyng cesarean birth, Obstet. Gynecol. In press. 8. Parsons, J. N., Romano, J. M., and Levison, M. E.: Pharmacology of a new l-oxa-plactam (LY 127935) in normal volunteers, Antimicrob. Agents Chemother. 17:226,1980. 9. Fairbanks, V. F.: Hemoglobin derivatives and myoglobin, in Teitz. N. W.. editor: Fundamentals of Clinical Chemistry, Philadelphia, 1976, W. B. Saunders Co., p. 401. J. T., and Bawdon, R. E.: Determination of 10. Rudrik, penicillinase-resistant penicillins in serum using high pressure liquid chromatography, J. Liquid Chromatogr. 4:1525, 1981. 11. Dajani, A. S., Thirumoothi, M. C., Bawdon, R. E., et al.: Pharmacokinetics of intramuscular-ceforanide in children, Antimicrob. Agents Chemother. 21:282, 1982.
November Am. J. Obstet.
12.
13.
14.
15.
16.
17.
18.
1, 1982 Gynecol.
Polk, R. E., Kline, B. J., and Markowirz, S. M.: Cefazolin and moxalactam pharmacokinetics after simultaneous intravenous infusion, Antimicrob. Agents Chemother. 20: 576, 1981. Sheng, K. T., Huang, N. N., and Promadhattavedi, V.: Serum concentrations of cephalothin in infants and children and placental transmission of the antibiotic, in Sylvester, J. C., editor: Proceedings of the Fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D. C., 1964, American Society for Microbiology, p. 200. Barr, W., and Graham, R. M.: Placental transmission of cephaloridine, J. Obstet. Gynaecol. Br. Commonw. 74: 739, 1967. Craft, I., Mullinger, B. M., and Kennedy, M. R. K.: Placental transfer of cefuroxime, Br. J. Obstet. Gynaecol. 88:141, 1981. Kafetzis, D. A., Lazarides, C. V., Sifas, C. A., et al.: Transfer of cefotaxime in human milk from mother to fetus, J. Antimicrob. Chemother. (Suppl. A) 6:135, 1980. Rolfe, R. D., and Finegold, S. M.: Comparative in vitro activity of new p-lactam antibiotics against anaerobic bacteria, Antimicrob. Agents Chemother. 20~600, 1981. Schaad, V. B., McCracken, G. H., Threlkeld, N., et al.: Clinical evaluation of a new broad-spectrum oxa-betalactam antibiotic, moxalactam, in neonates and infants, J. Pediatr. 98:129, 1981.