Kinetics and action of N-methylthiotetrazole in volunteers and patients

Kinetics and Action of N-Methylthiotetrazole in Volunteers and Patients Population-Based Clinical Comparisons of Antibiotics With and Without This Moiety

Jerome J. Schentag, PharmD, Lynda S. Welage, PharmD, James S. Williams, MD, John H. Wilton, PhD, Martin H. Adelman, PhD, David Rigan, MD, and Thaddeus H. Grasela, PharmD, Buffalo, New York

Since the discovery of clinical bleeding in association with the N - m e t h y l t h i o t e t r a z o l e - ( N M T T ) containing antibiotic m o x a l a c t a m [1-3], there has been m u c h discussion regarding the risk of bleeding with other N M T T - c o n t a i n i n g antibiotics, such as cefoperazone, cefmetazole, cefamandole, and cefotetan [4]. Antibiotics with similar side chains, such as cefazolin, ceftriaxone, and even a z t r e o n a m have also been implicated in cases of clinical h y p o p r o t h rombinemia. It is controversial whether these episodes of h y p o p r o t h r o m b i n e m i a or bleeding are associated with patient factors [3,5] or whether the clotting cascade is somehow directly paralyzed by N M T T [6,7]. Over the past 5 years, there has been m u c h in vitro and a n i m a l m o d e l e v i d e n c e for N M T T - a s s o c i a t e d toxicity [6-8], b u t virtually no study of N M T T p h a r m a c o k i n e t i c s or its effects in either patients or volunteers. Only one previous s t u d y d e m o n s t r a t e d measurable N M T T concentrations in h u m a n subjects [9]. We therefore began a series of investigations to elucidate the pharmacologic i m p o r t a n c e Of N M T T in h u m a n subjects. We sought to d e t e r m i n e w h e t h e r N M T T in blood constitutes a major risk factor, a p o t e n t i a t o r of underlying clinical risk, or an interesting observation of i m p o r t a n c e only to animal systems or in vitro systems. H e r e i n we p r e s e n t an overview of our prelimin a r y find!ngs in volunteers and patients.

Methods Normal volunteers: Thirteen healthy volunteers as determined by history and physical and laboratory examinations were entered, of whom 11 completed the study. All subjects had baseline prothrombin and earlobe bleeding times within normal limits. Subjects provided written informed Consent in accordance with the protocol approved by the Millard Fillmore Hospital Human ReFrom the State University of New York at Buffalo School of Pharmacy, the Clinical Pharmacokinetics Laboratory, and the Department of Surgery, Millard Fillmore Hospital, Buffalo, New York. Requests for reprints should be addressed to Jerome J. Schentag, PharmD, Clinical Pharmacokinetics Laboratory, Millard Fillmore Hospital, 3 Gates Circ!e, Buffalo, New York 14209.

40

search Committee. In a three-way randomized crossover fashion, each volunteer was given 2 g of moxalactam, cefotetan, and cefoperazone. The dose was given over 30 minutes using an intravenous pump. Before the dosage, each had an intravenous catheter implanted for blood sampling. A baseline blood sample was obtained before the dose. Samples were then obtained at 0.25.0.5, 0.75, 1, 1.5, 2.5, 4.5, 6.5, 8.5, and 12.5 hours after dosing. Cumulative urine was collected from 0 to 24 and from 24 to 48 hours. Both urine and plasma specimens were assayed for NMTT and parent cephalosporin concentrations. In addition, an aliquot of each administered dose was assayed to determine the exact amount of parent compound and NMTT infused. Assays: Concentrations of cefoperazone and moxalactam in plasma and urine were determined using reversedphase high-pressure liquid chromatography. The methods were developed at the Clinical Pharmacokinetics Laboratory, and will be reported in greater detail in subsequent publications from this study. Within-day and between-day coefficients of variation were under 8.8 percent. Cefotetan was analyzed by an analytical contract group using a validated procedure. NMTT was analyzed at the Clinical Pharmacokinetics Laboratory using a Waters 6000 high-pressure liquid chromatography pump, a Waters 712B autosampler, a Krat0s 757 variable ultraviolet wavelength detector at 225 nm, and a Spectra-Physics 4270 integrator. Samples were chromatographed on a Beckman 5# C18 Ultrasphere immunoperoxidase column (4.6 mm by 25 cm) preceded by a Brownlee RP-8 guard column, using a 0.1 M phosphate buffer (pH 7.75) with methanol (80:20 ratio) and containing 0.005 M tetrabutylammonium hydroxide as a mobile phase at a flow rate of 1 ml/minute. Plasma (1 ml) and urine (1 ml of a 1:20 dilution) samples were prepared by cleanup over Analytichem SAX and C18 Bondelut solidphase extraction columns. Overall intraday and interday variability was 12.1 percent and 11.9 percent, respectively, for seeded plasma controls (25#1 injections), and 12.5 percent and 7.2 percent, respectively, for seeded urine controls (20 ~1 injections). Overall recovery in plasma and urine samples was 88 percent and 94 percent, respectively, for NMTT and 68 percent and 92 percent for 2-thiobarbituric acid, the internal standard. Pharmacokinetic analysis: Plasma concentrations of the parent compound and the N M T T side chain were

The American Journal of Surgery

Symposium on the New Cephalosporins

plotted on semilogarithmic graph paper and analyzed using noncompartmental methods [10]. The cumulative NMTT urinary recovery was determined from the two consecutive 24-hour urine collections. From this cumulative recovery, the amount of NMTT given was subtracted. This difference between urinary NMTT recovery and NMTT administration was the amount of NMTT produced in vivo. Patients: Patient studies were also conducted to determine relationships between NMTT and effects on clotting factors. Two patients given cefoperazone have been studied thus far. Each underwent measurement of baseline Factors II and VII and prothrombin times. They were then given cefoperazone 2 g every 12 hours. The NMTT concentrations, prothrombin times, and clotting factors were simultaneously measured during therapy. Demographic data and clotting activity results are shown in Table I. Patient 1 had no change in prothrombin time from the baseline measurement, whereas Patient 2 had a dramatic increase in prothrombin time. Population surveillance: The Clinical Pharmacokinetics Laboratory network of clinical pharmacists was employed to survey patient populations given cefazolin (prophylaxis only), cefoxitin and cefotetan for either prophylaxis or treatment, and aminoglycoside and antianaerobic mixtures (treatment only) [11]. This network specializes in prospective, concurrent review with the capability of obtaining follow-up prothrombin times on all patients. Both hypoprothrombinemia and clinically evident bleeding were targeted. Clinical pharmacists in 44 hospitals participated. Chi-square testing was used to determine the association between various patient factors and hypoprothrombinemia (defined as an increase of 5 seconds in the prothrombin time over the baseline measurement) and partial thromboplastin time (defined as an increase of 10 seconds in the partial thromboplastin time over the baseline measurement). Results

Figure I shows plasma N M T T concentrations from each of the three antibiotics plotted versus time after a 2 g dose. The amount of N M T T associated with moxalactam and cefotetan decreased proportionally to the concentrations of the parent compound, whereas the N M T T concentrations from cefoperazone demonstrated a delayed serum peak and were not parallel to cefoperazone serum concentrations after 1 hour. Data on N M T T urine recovery, administered amounts, and in vivo N M T T production are shown in Table II. T he cefoperazone dose yielded th e highest u r i n a r y e xc r et i on of N M T T , chiefly as a result of higher in vivo N M T T formation. Moxalactam yielded less N M T T than cefoperazone, and both were higher than cefotetan (p <0.05). Figure 2 shows the measured N M T T concentrations in Patients 1 and 2 correlated with their respective clotting factor activity. Patient 1 had normal c l o t t i n g f a c t o r a c t i v i t y at bas e l i ne , and demonstrated no changes despite higher serum N M T T co n cen tr a t i ons . P a t i e n t 2 had a lower N M T T serum concentration, but manifested a de-

Volume 155 (5A), May 31, 1988

TABLE I

Risk Factors and Demographic Data For Patients 1 and 2

Parameter Prothrombin time (s) Partial thromboplastin time (s) Factor II (%) Factor VII (%) Albumin (g/dl)

Patient 1" Patient 21 Maximum Maximum Baseline Effect Baseline Effect 12.4

12.6

13.8

22.3

23.1

25.8

25.2

69.9

>100 >100 3.9

94 >100 4.1

40 32 2.6

16 8.5 1.6

* Patient 1 was a 56 year old female with a chronic leg ulcer growing pseudomonas. She was in otherwise good health. Baseline serum albumin level was normal. Treatment with cefoperazone 2 g every 12 hours. Patient 2 was a 51 year old female with severe peripheral vascular disease and an infected amputation stump. Preantibiotic baseline clotting factor activity was normal. Treatment with cefoperazone 2 g every 12 hours.

IO-

NMTT FEASMA OON~ENT~ATE)N (/zg/ml)

TIME(hours)

Figure 1. Mean N-methylthiofetrazole ( N M T D concentrations versus time (n = 11) are shown for moxalactam (asterisked line), cefotetan (circles), and cefoperazone ( crossed line). The moxalactam-and cefotetan-derived NMTT concentrations were parallel to serum concentrations of the parent cephalosporln. Cefoperazone concentrations were atypical, reflecting pronounced differences in Its disposition.

crease in the activity of clotting factors II and VII. Thus, there was no correlation between N M T T concentration and change in clotting factor activity in these two patients. Population surveillance of 484 patients (Table III) revealed an overall incidence of hypoprothrombinemia of less than 10 percent. These data are given in Table IV. There were no statistical differences between antibiotics in the incidence of hypoprothrombinemia. Two clinical factors significantly correlated with prolongation of the partial thromboplastin time: nasogastric suction and t r e a t m e n t as opposed to prophylactic t herapy (p <0.05). Clinical bleeding was rare (4 cases in 484 patients). Other adverse effects are listed in Table V.

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Schentag et al

TABLE II

N-Methylthiotetrazole (NMTT) Recovery, Administration, and in Vivo Production (values expressed as the mean 4- standard deviation)

Parameter

Cefoperazone

Moxalactam

Cefotetan

NMTT urine recovery (mg) NMTT given (mg) In vivo NMTT production (mg)

73.6 4- 44.3*

57.4 4- 26.2

29.7 4- 22.9

0

75-

CLOTrlNG FACTOR ACTIVITY

15.2 4- 0.9 t 25.8 4- 1.4~ 22.1 4- 3.0 57.0 4- 45.9tw 33.8 4- 21,2t 9.0 4- 10.9 n

* p <0.05 versus cefotetan. p <0.01 versus moxalactam and versus cefotetan. p <0.01 versus cefotetan. w p <0.058 (cefoperazone versus maxalactam).

T A B L E III

50-

(%)

Drug Surveillance Network AntibioticAssociated Coagulopathy: Demographic Data*

Total no. of subjects No. of males Mean weight (Ib) Mean serum creatinine level (mg/dl) Mean serum albumin level (g/dl) Mean age (yr)

484 187 166,5 4- 48.6 1.2 4- 1.2 3.8 4- 0.7 52.6 4- 19.2

* Statistical values indicate the mean 4- standard deviation.

0

I 1

I I 2 3 NMTr CONCENTRATION (#glml)

Figure 2. Correlation between N-methylthiotetrazole (NMTT) concentrations in Patients I and 2 and corresponding assays of clotting factor II (circles) and VII (asterisks) activity. There was no apparent relationship, as higher NMTT concentrations were not associated with a decline in clotting factor activity. Both patients received the same dose of cefoperazone, 2 g every 12 hours.

TABLE IV

Drug Surveillance Network AntibioticAssociated Coagulopathy: Data on Patients With Elevated Prothrombin Times (EPT) and Elevated Partial Thromboplastin Times (EPTT)*

Treatment Group

Comments

It was somewhat surprising to find greater NMTT production from cefoperazone than moxalactam in view of the greater incidence of clinical bleeding associated with moxalactam [4-6,12,13]. This may be the first evidence for lesser importance of NMTT, at least in relation to other clinical risk factors. The data from two cefoperazone-treated patients also point to the importance of clinical risk factors. Preliminary patient data clearly illustrated that adequate baseline vitamin K activity can offset the effects of higher in vivo NMTT concentrations. Our population surveillance studies of 484 patients in U.S. hospitals found a rather low incidence of hypoprothrombinemia and even less overt bleeding in the first 484 patients. There was no apparent correlation between hypoprothrombinemia and bleeding, and no antibiotic revealed a higher overall incidence of either hypoprothrombinemia or bleeding. In view of the low potency of NMTT in the presence of adequate baseline amounts of vitamin K, few patients are actually at risk for hypoprothrombinemia. These studies are continuing. Taken together, our clinical and volunteer studies fail to provide major support for the NMTT-hypoprothrombinemia hypothesis [6,7]. Specifically, antibiotics like cefoperazone, with a lower incidence of clinical bleeding, produced higher amounts of NMTT. In patients, higher NMTT concentrations

42

I 4

Prophylaxis Cefazolin Cefoxitin Cefotetan Treatment Aminoglycoside-antianaerobic Cefoxitin Cefotetan

n

EPT

EPTT

89 135 26

0 2 ~1.5) 1 (3.8)

1 (1.1) 1 2 (1.5) 1 (3,8)

81 48 18

1 (1.2) 0 1 (5.6)

4 (4.9) 1 (2.1) 1 (5.6)

* An elevated prothrombin time is one more than 5 seconds longer than the baseline measurement; an elevated partial thromboplastin time is one more than 10 seconds longer than the baseline measurement. 1"Values in parentheses are percentages.

did not necessarily lead to more antagonism of clotting factor activity. In surveillance studies, there was no greater incidence of hypoprothrombinemia or bleeding in the NMTT-containing antibiotic cefotetan than encountered in similar patients given antibiotics without NMTT. The clinical studies reported here require continued examination and expansion to include more patients. However, they are consistent with earlier findings published before the NMTT hypothesis was advanced. Specifically, it has been known for over 15 years that vitamin K depletion alone is sufficient to produce clinical hypoprothrombinemia and occasionally clinical bleeding [14]. Secondly, The American Journal of Surgery

S y m p o s i u m on the N e w C e p h a l o s p o r i n s

TABLE V

Drug Surveillance Network Antibiotic-Associated Coagulopathy: Adverse Reactions and Vitamin K Therapy Drug

Bleeding

Elevated SC Level

Elevated LFT Result

Loose Stools

Vitamin K for EPT

Cefazolin Cefoxitin Cefotetan Aminoglycosideantianaerobic

0 1 0

1 1 1

0 4 0

2 3 2

0 2 2

2

2

4

3

0

EPT = elevated prothrombin time; LFT = liver function tests; SC = serum creatinine.

antibiotics without the NMTT group appear quite capable of producing hypoprothrombinemia in seriously ill patients [3,15,16]. This includes cefoxitin and biologically stable antibiotics like the aminoglycosides. Hypoprothrombinemia secondary to cefoxitin and aminoglycosides is also responsive to vitamin K, indicating its common etiology with vitamin K deficiency. Finally, surgical patients who are unable to ingest vitamin K have always been at great risk for hypoprothrombinemia, and, in some cases, bleeding [14]. Any antibiotic might cause hypoprothrombinemia in a vitamin K-depleted patient, but the various cephalosporins are given to patients who clearly differ in the various baseline risks due to differences in baseline illness, in this setting, some cephalosporins might have an apparently higher incidence of hypoprothrombinemia due to more frequent administration to higher-risk subgroups. These lines of argument (which favor patient variables as the major risk factor) are balanced by the greater ability of some antibiotics to produce hypoprothrombinemia in vitamin K-depleted rats [17]. These studies identify one specific NMTTcontaining antibiotic (moxalactam) as a higher risk than antibiotics lacking this moiety. What animal models do not clarify is the comparative incidence of hypoprothrombinemia among the various NMTT-containing cephalosporins, and the correlations between the degree of hypoprothrombinemia and the measured NMTT concentrations. Furthermore, they do not establish a correlation between NMTT concentration and bleeding in vivo. Presumably, it would be useful to measure comparative concentrations of free NMTT side chains in rats and then attempt correlation between the free NMTT concentrations and the relative degrees of hypoprothrombinemia or bleeding. Since hypoprothrombinemia may also be aggravated by the eradication of gastrointestinal flora, it would be essential to determine whether antibiotics of equal antianaerobic and antiaerobic potency differ in the onset of hypoprothrombinemia in the rat model. Until animal studies are conducted to establish firm correlations between NMTT concentrations and effects on clotting factor activity, it is an open question whether NMTT is important in patients.

Volume 155 (5A), May 31, 1988

Clearly, our studies and numerous other studies establish baseline vitamin K depletion as the major variable for prediction of antibiotic-associated coagulopathy [13-15,18]. A low serum albumin level is an important clue to a vitamin K depletion syndrome (Table I). In such patients, most antibiotics can cause hypoprothrombinemia. High-risk patients should be given vitamin K to reverse their deficiency, and this may lower or remove their risk of bleeding abnormalities from drug-associated factors. Once vitamin K deficiency is corrected, no studies, including our previous ones with moxalactam [3], have proved that the vitamin K-replenished patient is at greater risk for either hypoprothrombinemia or clinical bleeding. If patients with adequate vitamin K do have more frequent bleeding with moxalactam, it may be caused by platelet defects rather than hypoprothrombinemia [19]. Indeed, impairment of platelet function could explain why moxalactam, with intermediate amounts of NMTT production, has a greater incidence of clinical bleeding than cefoperazone. Therefore, although antibiotics containing NMTT (but devoid of platelet defects) such as cefoperazone, cefmetazole, cefamandole, and cefotetan deserve continued clinical surveillance, studies to date indicate these antibiotics present a lower risk to patients without vitamin K deficiency or to those prophylactically given vitamin K. In low-risk patients, our studies advance the hypothesis that the risk of clinical bleeding with these agents is unlikely to be greater than with antibiotics lacking this side chain.

Summary Normal volunteers and patients were studied to determine the relative importance of NMTT and patient risk factors in the production of hypoprothrombinemia. The normal volunteers demonstrated in vivo NMTT production, but the order of magnitude (cefoperazone, moxalactam, and cefotetan in descending order) was different from the usual order of clinical risk. In patients, there was not a NMTT-concentration-versus-effect relationship. Patients who were vitamin K deficient were more sensitive to lower NMTT concentrations than those

43

Schentag et al

with n o r m a l v i t a m i n K status. In surveillance studies, N M T T - c o n t a i n i n g antibiotics were no m o r e freq u e n t l y associated with h y p o p r o t h r o m b i n e m i a or bleeding t h a n antibiotics t h a t lack this moiety.

10.

11.

References 1. Joehl RJ,'Rasback DA, Ballard JO, Weitekamp MR, Sattler FR. Moxalactam: evaluation of clinical bleeding in patients with abdominal infection. Arch Surg 1983; 118: 1259-61. 2. Pakter RL, Russell TR, Mielke CH, West D. Coagulopathy associated with the use of moxalactam. JAMA 1982; 248: 1100-2. 3. Baxter JG, Marble DA, Whitfield LR, Wels PB, Walczak P, Schentag JJ. Clinical risk factors for prolonged PT/PTT in abdominal sepsis patients treated with moxalactam or tobramycin plus clindamycin. Ann Surg 1985; 201: 96-102. 4. Schentag JJ, Welage LS, Grasela TH, Adelman MH. Determinants of antibiotic-associated hypoprothrombinemia. Pharmacotherapy 1987; 7: 80-6. 5. Sattler FR, Weitekamp MR, Ballard JO. Potential for bleeding with the new beta-lactam antibiotics. Ann Intern Med 1986; 105: 924-31. 6. Lipsky JJ, N-methyl-thio-tetrazole inhibition of the gamma carboxylation of glutamic acid: possible mechanism for antibiotic-associated hypoprothrombinemia. Lancet 1983; 2: 192-3. 7. Kerremans AL, Lipsky JJ, Van Loon J, Gallego MO, Weinshilbourn RM. Cephalosporin-induced hypoprothrombinemia: possible role for thiol methylation of 1-methyltetrazole-5thiol and 2-methyl-l,3,4-thiadiazole-5-thiol. J Pharmacol Exp Ther 1985; 235: 382-8. 8. Uotila L, Suttie JW. Inhibition of vitamin K-dependent carboxylase in vitro by cefamandole and its structural analogs. J Infect Dis 1983; 150: 571-8. 9. Aronoff GR, Wolen RL, Obermeyer BD, Black HR. Pharmacokinetics and protein binding of cefamandole and its 1-

12. 13.

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methyl H-tetrazole-5 thiol side chain in subjects with normal and impaired renal function. J Infect Dis 1986; 153: 1069-74. Jusko WJ. Guidelines for collection and analysis of pharmacokinetic data. In: Evans W, Schentag J, Jusko W, eds. Applied pharmacokinetics. Spokane, WA: Applied Therapeutics, Inc., 1986: 9-84. Grasela TH, Schentag JJ. A clinical pharmacy oriented drug surveillance network. I: Program description. Drug Intell Clin Pharm 1987; 21: 902-8. Barza M, Furie B, Brown AE, Furie BC. Defects in vitamin Kdependent carboxylation associated with moxalactam treatment. J Infect Dis 1986; 153:1166-9. Morris DL, Fabricius PJ, Ambrose NS, Scammell B, Burdon DW, Keighley MR. A high incidence of bleeding is observed in a trial to determine whether addition of metronidazole is needed with latamoxef for prophylaxis in colorectal surgery. J Hosp Infect 1984; 5: 398-408. Ham FM. Hypoprothrombinemia in patients undergoing prolonged intensive care. Med J Aust 1971; 2: 716-8. Schentag JJ, Vari AJ, Winslade NE, et al. Treatment with aztreonam or tobramycin in critical care patients with nosocomial gram-negative pneumonia. Am J Med 1985; 78 (suppl 2A): 34-42. Brown RB, Klar J, Lemeshow S, Teres D, Pastides H, Sand M. Enhanced bleeding with cefoxitin and moxalactam. Statistical analysis within a defined population of 1493 patients. Arch Intern Med 1986; 146: 2159-64. Lipsky JJ, Lewis JC, Novick WJ. Production of hypoprothrombinemia by moxalactam and 1-methyl-5-thiotetrazole in rats. Antimicrob Agents Chemother 1984; 25: 380-1. Mackie IJ, Walshe K, Cohen H, et al. Effects of N-methyl thiotetrazole cephalosporin on haemostasis in patients with reduced serum vitamin K ~concentrations. J Clin Pathol 1986; 39: 1245-9. Weitekamp MR, Caputo GM, AI-Mondhiry HAB, Aber RC. The effects of latamoxef, cefotaxime, and cefoperazone on platelet function and coagulation in normal volunteers. J Antimicrob Chemother 1985; 16: 95-101.

FOCUS

Analysis of Prothrombin Time Prolongation in North American Cefotetan Clinical Trials: Questions and Answers

Neil H. Goldstein, M D , Wilmington, Delaware

Q: What has Stuart's experience been with hypoprothrombinemia associated with cefotetan? A: We undertook a retrospective analysis of prothrombin time prolongation in the North American cefotetan therapeutic clinical trials. All patients enrolled in these trials From the Division of Clinical and Medical Affairs, Stuart Pharmaceuticals, Wilmington, Delaware. Requests for reprints should be addressedto Neil H. Goldstein, MD, 'Division of Clinical and Medical Affairs, Stuart Pharmaceuticals,New Murphy Roadand Concord Pike, Wilmington,Delaware 19897.

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who had a pretherapy and end or post-therapy prothrombin time were included in this analysis. Table I shows demographics for the cefotetan and comparator drug patients. The types of infections treated are shown for each drug. The cefotetan trials included patients with intraabdominal, gynecologic, skin and soft tissue, and gonococcal infections. Cefoxitin was used in all but the gonococcal trials. Moxalactam was one of our early comparators and only appears in the early intraabdomi-

The American Journal of Surgery