clavulanate

ORIGINAL ARTICLE

Changes in fecal flora and comparative multiple-dose pharmacokinetics of ceftibuten, cefpodoxime proxetil and amoxycillin/clavulanate Clin Microbiol Infect 1999; 5 : 339-354

Jirg Hamaclierl, Joyg Luepke I , Bruce E. Reidetzbeyg’, C a r f Erik Nord 3 , Kfairs Borrzer4, Peter Koeppe 5 , David Bristol arid Hartmdt Lode

’

‘Department of Pulmonary and Infectious Diseases, City Hospital Berlin-Zehlendorf/Heckeshorn, affil. Freie Universitat Berlin, Berlin, Germany; 2Schering-Plough Research Institute, Kenilworth, N e w Jersey, USA; 3Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden; ‘Institute of Clinical Chemistry and 5Department of Medical Physics, Klinikum Benjamin Franklin (Steglitz), Freie Universitat Berlin, Germany Objective: To investigate changes in fecal flora and multiple-dose pharmacokinetics with the oral antibiotics ceftibuten 400 mg daily and cefpodoxime proxetil (CPX) 200 mg every 12 h, compared to amoxycillin/clavulanate 500/125 mg every 8 h during and following 1 week of medication. Methods: In an open randomized triple crossover design, 18 (nine female, nine male) healthy volunteers received each drug for 7 days, followed by a ‘washout’ period of 4 weeks. Serum and urine levels of the substances were determined by bioassay, and for ceftibuten isomers by high-pressure liquid chromatography. Statistical analysis of quantitative aerobic and anaerobic cultures; of feces was performed, and P-lactamase activity was determined. Results: Ceftibuten showed a mean C, of 18.9 (SD 3.0) mg/L, a terminal half-life of 2.89 h, and an AUCtotof 100 (21.8) mg.h/L; protein binding was 631.7 (5.1)%, and accumulation was marginal. Cefpodoxime proxetil had a C, of 1.92 (0.61) mg/L, a terminal half-life of 1.97 (0.42) h and an AUCtotof 10.8 (3.3) mg.h/L; no accumulation was seen. Amoxycillin and clavulanate had C,,, values of 7.15 (2.16) mg/L and 3.39 (1.31) mg/L, terminal half-life values of 1.03 (0.15) h and 0.93 (0.17) h, AUCtot values of 20.0 (4.2) mg.h/L and 8.87 (3.10) mg.h/L, and there was no accumulation. Statistical analysis for ech microorganism in fecal1 samples showed significant differences between amoxycillin/clavulanate and the two third-generation cephalosporiris, but virtually no differences between ceftibuten and cefpodoxime proxetil. Eleven of 12 volunteers reported loose stools (days 2-7, mean duration 4.4 (SD 2.7) days) with amoxycillin/clavulanate, but nobody during ceftibuten administration and one volunteer during cefpodoxime proxetil administration. Conclusions: Ceftibuten showed excellent and cefpodoxime favorable pharmacokinetic properties, with significantly less pronounced fecal flora changes and intestinal side effects compared to amoxycillin/clavulanate. The multiple crossover design allows powerful microbiological statistical analysis and pharmacokinetic parameter comparisons. Key words: Ceftibuten, cefpocloxime proxetil, amoxycillin/clavulanate, fecal flora, statistical analysis, pharmacokinetics, p-lactamase activity, triple crossover design

vitro activity against Gram-positive and Gram-negative bacteria [1,2]. Ceftibuten, cefpodoxime proxetil and amoxycillin/clavulanate are widely used antibiotics for upper and lower respiratory as well as urinary tract infections. Ceftibuten is an orally active third-generation non-ester cephalosporin. It has broad in vitro antibacterial activity against the majority of Gram-negative pathogens, including Klebsiella spp., good activity against streptococci [2-51, marginal activity against penicillin-susceptible Streptococcus pneumoniae [3,6-91, and poor activity against Staphylococcus aureus. It shows an absorption rate between 75% and 90% [4,10] and,

INTRODUCTION

Besides partially improved pharmacokinetic characterisitics, the main advantages of new oral cephalosporins over older derivatives are their broader spectra of in Corresponding author and reprint requests: H. Lode, Department of Pulmonary and Infectious Diseases, City Hospital Berlin-ZehIendorf/Hleckeshorn, Zum Heckeshorn 80, D-14109 Berlin, Germany

Tel: +49 30 8002 22 22 Fax: f 4 9 30 8002 26 23 E-mail: [email protected] Accepted 13 January 1999 339

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though resistant to most broad-spectrum 0-lactamases, including those of many Enterobcteriaceae,it is cleaved by a 0-lactamase produced by Bacteroides spp. so that very little active substance is found in the colon [ll]. Cefpodoxime proxetil is a prodrug ester of a thirdgeneration cephalosporin with similar in vitro antibacterial activity, except for better activity against Streptococcus pneumoniae and enhanced antistaphylococcal activity, a similar long elimination half-life as ceftibuten, but a rather low maximal serum concentration compared to other oral cephalosporins [2,9,12]. It has an absorption rate of about 50% [13]. Besides the fkaction of active drug entering the bowel, significant amounts of the prodrug would be expected to reach the colon and change the fecal flora in case of activation, i.e. de-esterification by intestinal esterases. Nevertheless, in clinical trials the reported incidence of diarrhea with cefpodoxime proxetil is low [14-1 61, which is also the case for ceftibuten [17-191. The impact of amoxycillin/clavulanate, one of the most fkequently used broad-spectrum antimicrobial agents, on the gastrointestinal tract is frequent, so that it was used in our study as 'positive control'. Clinical data are inconsistent concerning the incidence of diarrhea with amoxycillin/clavulanate. There are several studies reporting a much higher incidence of diarrhea in clinical trials with amoxycillin/clavulanate than with ceftibuten and ceeodoxime proxetil [20-221. Alternatively, a few studies have shown a low incidence of loose stools with amoxycdldclavulanate, or have not shown any important difference [23,24]. Previous studies have shown that amoxycillin/clavulanate causes alterations in the normal flora [25-281. This study was performed in an open randomized triple crossover fashion to examine the pharmacolnetics of ceftibuten, cefpodoxime proxetil and amoxyc&n/clavulanate following multiple dosing over 7 days in 18 normal volunteers. Furthermore, this design was chosen for a novel statistical approach to analyzing changes in fecal flora. Previous evaluations of fecal flora have not addressed the problem of statistical significance. For this study, the objective was not only the evaluation of the differences in fecal flora during treatment with a single antibacterial drug, but the statisticallymeaningful comparison of all three drugs in the same volunteers. With each volunteer serving as their own control for each microorgnism analyzed, and maintaining the comparison of effects within the same person, variability was reduced to a minimum, as interindividualbaseline differences were eliminated. By calculation of an area under the curve (colony-forming units/g feces versus time) for each microorganism, standard tests allowed a statistical comparison of fecal flora changes in this relatively small study population.

MATERIALS AND METHODS Study design

This open randomized triple crossover study was designed to examine changes in fecal flora and pharmacolnetics with the three oral antibiotics ceftibuten, cefpodoxime proxetil and amoxycillin/clavulanate during and following 1 week of dosing in normal volunteers. It was not blinded, for technical reasons such as amount of medication and bioequivalence of formulated blinded medcation, because of the principal aims of studying fecal flora and pharmacokinetics, which are not influenced by an unblinded design, and in order to facilitate sampling for drug concentration. It was conducted from October 1992 to March 1993. The protocol was approved by the ethics committee of the Freie Universitat of Berlin, Germany. Medication intake was directly observed in each volunteer on day 1, and on the mornings of day 2 and day 7; all other medication had to be confirmed directly by telephone calls fkom the volunteer to the responsible physician, and each volunteer recorded a diary protocol with exact time of each medication, possible adverse event, and stools, including the quality of feces. All diaries were checked for possible differences with the protocol made when receiving the phone calls. In case of adverse events, the volunteers were asked to contact immediately the investigating physician, who performed a precise interview, and, if necessary, performed or organized consultations or further diagnostic or therapeutic procedures. Volunteers

Eighteen healthy volunteers aged between 18 and 50 years-nine non-pregnant females with a proven barrier method of contraception, and nine males-were randomly assigned to the study. Written informed consent was given after f d explanation of the protocol. The mean age was 33.1 years (SD 5.7 years; range 22-42 years). Mean height was 175 cm (SD 10 cm; range 157-190 cm). Mean weight was 71.9 kg (SD 10.2 kg; range 56-90 kg). Mean creatinine clearance was 117 mL/min (SD 23 mL/min; range 82-171 d / m i n ) . Volunteers with a hypersensitivity or serum sickness in response to any 0-lactam drug were excluded, as were volunteers taking dlicit drugs or any medication within 1 week of study entry or during the study, volunteers who had used antimicrobial drugs within 1 month of study entry, volunteers with diarrhea, loose stools, or current or recent gastrointestinal disease, volunteers who had recently traveled to areas endemic for gastrointestinal parasites, or volunteers who had undergone recent hospitalization for any reason, in order to eliminate volunteers potentially colonized with resistant organisms.

H a m a c h e r et a l : F e c a l flo ra a n d p h a r m a c o k i n e t i c s i n t hr ee o r a l D - l a c t a m s

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Sampling Stool samples

were counted, isolated in pure culture, and identified, as were the different colonies appearing on the selective media. All fecal samples were also assayed for yeasts,. Clostridiuni dficile was searched for, and its toxins were determined by, a tissue-culture assay with human embryonic intestinal cells and a control with specific neutralizing antiserum, a method described by Aronsson et a1 [30]. Counts were given as log number of bacteria/g feces. Samples were assayed for p-lactamase activity using the chromogenic cephalosporin nitrocefin as substrate for a spectrophotometric determination of activity, as previously described [31,32]. One unit of P-lactamase was defined as the amount which formed 1.0 pmol of product per minute under given conditions.

Fecal samples were collected twice per week before each of the three study drug administration periods, on the fourth day of medication, on the seventh (last) day of medication, on the eighth day (the first day after end of medication), and on days 12, 21 and 28 in the washout period. Samples of approximately 5 g each were collected in sterile plastic containers and were frozen in a -70°C freezer until analysis was performed.

The concentrations of the four substances in the serum and urine samples were determined with an agar diffusion bioassay (cup plate method), as modified by Reeves and Bywater [33] (Table 1). Serum and urine saniples were assayed in triplicate, and five standard concentrations (in triplicate) were used on each plate.

Antibiotic dosage and administration

The test subjects received each oral study drug for 7 days (ceftibuten 400 mg P O daily; amoxycillin/ clavulanate 500/125 mg every 8 h; cefpodoxime proxetil 200 mg every 12 h), followed by a 'washout' period of 4 weeks after each drug administration period. As the study was performed in a randomized triple crossover mode, the subjects were randomized to the six possible sequences (permutations) of study drug order. After three administrations and two washout periods, each subject had received the three different medications.

Blood samples

These were taken repeatedly for serum drug level determination on days 1 and 7 after 0, 30, 60, 120, 240, and 360 min, the last sample being taken at 8 h (volunteers under amoxycillin/clavulanate) and 12 h (volunteers under cefiibuten and cefpodoxime proxetil). Urine

This was analyzed on days 1 and 7. On both days, urine samples were analyzed from the periods 0-6 h and 6-12 h for ceftibuten and cefpodoxime, 12-24 h for ceftibuten and 0-8 h for amoxycillin/clavulanate. Methods Analysis of the fecal flora

Microbial identification and quantification was done according to colonial morphology, Gram stain, and various serologic and biochemical tests. A 1-g sample of stool was homogenized in 9 mL of prereduced peptone yeast extract medium. Tenfold serial dilutions were made to a dilution of' lo-*. The samples were inoculated and a quantitative estimate of each microbial species was made as described by Heimdahl and Nord [29]. All manipulations of the anaerobic bacteria were carried out in an anaerobic chamber (Forma, Philadelphia, PA, USA) under 10% (vol/vol) hydrogen in nitrogen. After 24 h (aerobic agar plates) or 48 h (anaerobic agar plates) of incubation at 37"C, total colony counts were determined from aerobic and anaerobic blood agar plates. Different colony types

Bioassay

Assay of ceftibuten in serum and urine by high-performance liquid chromatography (HPLC)

After addition of the internal standard toluenesulfonic acid, ceftibuten was extracted from serum with acetonitrile. Acetonitrile was back-extracted from the liquid phase with dichloroniethane. The aqueous supernatant was chromatogaphed on a reversed-phase column (Nucleosil 5C18, 125 mm (length), 4 mm (diameter); Macherey & Negel, Diiren, Germany). The mobile phase was acetonitrile/methanol/water (130:20:850) containing 3.4 g tetrabutylammonium phosphate, adjusted to pH 6.95 with 2 M sodium hydroxide solution. The absorbance of the eluate was monitored at 256 nm. Retention times were 8.5 min (cis-ceftibuten), 10.2 min (trans-ceftibuten) and 17.7 min (internal standard).Urine was diluted with mobile phase before chromotography. Validation of the method yielded the following results for serum (urine): detection limit 0.1 mg/L (5.0 mg/L), lower limit of quantification 0.2 mg/L (10.0 mg/L, linear range 26.0 mg/L (1000 mg/L), coefficient of variance between series 4.4-9.5% (2.1-4.9%), and recovery of 97.3-98.3% (107.3%). Statistical fecal flora analysis

Each volunteer acted as his or her own control. Separate analyses were performed for each of the organisms. The cultures obtained at the latter of the two screening visits were used as the baselines. Change from baseline was analyzed to estimate differences in

Clinical M i c r o b i o l o g y and Infection, Volume 5 Number 6, June 1999

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Table 1 Characteristics of the bioassays Lower limit of detection (mg/L)

Substance

Standard solution

Ceftibuten (lot no. 28’173/001)

at pH 7.2

Cefpodoxime (lot no. 18)

at pH 7.2

Amoxycillin (batch no. 28)

Citrate buffer at pH 7.2

Cladanate (batch no. 54)

Sorensen’s buffer

Sorensen’s buffer

Sorensen’s buffer at pH 7.0

Bacterium (test strain)

Variation coefficient (%)

Agar

Buffer

Pool serum

Buffer

Pool serum

Morganella morganii I F 0 3848 (DSM 30164)

DST agar

0.058

0.116

3.90

4.57

Morganella morganii I F 0 3848 (DSM 30164)

Antibiotic medium 2 Oxoid

0.058

0.116

6.27

5.40

Bacillus subh’lis ATCC 6633 Micrococcus luteus ATCC 8340

Antibiotic medmm 2 Oxoid

0.116

0.116

6.95

4.25

0.010

0.030

5.40

3.28

Klebsiella aerogenes NCTC 11228

CLED Agar with Na-benzylpenicillin

0.030

0.058

6.42

3.04

the effects between the three study medications at each time point (days 4, 7, 8, 12, 21 and 28) for each organism. All statistical testing was performed at the p level of 0.05. As the subjects were randomized to one of the six possible treatment sequences that can be followed with three drugs,there were three subjects in each treatment sequence. Since one subject discontinued prematurely, she was excluded from statistical fecal flora analysis and replaced by a female volunteer who finished the whole study and was therefore included in the analysis according to the protocol. In order to include the repeated measures of the microbe counts at the six time points after baseline, the area under the curve (AUC) change from the baseline for every subject was calculated, working with loglo (colony-forming units microbedg feces) values. Using this transformed variable, differences from baseline were computed for each subject. Because the loglo transformations were used, differences were used, as opposed to ratios. This was performed for each treatment, so that a subject had three values for each time point concerning each microorganism. The AUC was computed for each treatment and each subject (so each subject had three AUC values concerning each microorganism). These AUC values were used for analyses. The betweentreatment Merences were tested using a 3 x 3 crossover ANOVA model, with a p-value <0.05 being considered as significant [34,35]. The ANOVA model included effects for sequence, subjects nested in sequence, period, treatment, and carryover.

(Ch.B.OY 8007)

Pharmacokinetic calculations

The main pharmacokinetic parameters were standardized to 70-kg body weight (area, C,,, volume of distribution in the steady state, divided by the oral bioavailability (L&/’) or to 1.73-m2 body surface area; (Cltot/’ CIRn). They were calculated model free (AUDtot,mean residence time (MRT), E-MRT (percentage of eliminated substance until MRT), C,,, V,,/’ total and renal clearance (Cltot/’ Clm), as well as assuming an open one-compartment model (AUCr0, T, tsonp)). The calculations were performed by standard methods [36]. Differences in determined parameters were analyzed for single variables by painvise t-test, and for multiple variables by multiple painvise comparisons according to Wilcoxon and Wilcox [37]. A p-value <0.05 was considered significant.

RESULTS Tolerance

One volunteer (female, 27 years) was replaced during the first study period because of fever, severe headache, leukocytosis (16.2 x 109/L with 4% eosinophils) and diarrhea after 5 days of medication with cefiibuten (no Clostridium d@ile or toxins were found). According to the protocol, the data of that volunteer were excluded from the final pharmacokinetic and fecal flora statistical evaluation. Further adverse events are listed in Table 2.

1.5 (SD 0.7) days 14h 26 (SD 23) days

2 X d d , 1 x moderate Mild 2 X moderate, 1 X mild

4 6

3 1 3

CPX

AM/CL CFX

CFB

Pruritus

Vaginal mycosis

1

1

1

5 1 1

I

2

3

2 2

1 1

3

I1

3

1 1

2

2

3

3

I11

Number of volunteers per period number

prob.

3 x poss. poss.

6 X poss.

1 x poss.; 1 x prob. 3 x poss.; 1 x prob.

2 x poss.; 1 x prob. poss. poss.

11 x prob. prob. prob.

Relationship

CFB, ceftibuten 400 mg daily; CPX, cefpodoxime proxetil200 mg every 12 h; Ah4/CL, amoxycillin/clavulanate 500/125 mg every 8 h; vol, volunteer; SD, standard deviation; poss, possibly related to study drug mehcation; prob., probably related to study drug medication; rel., definitely related to study drug medication.

1.2 (SD 1.0) days

10 (SD 2) h 4.8 (SD 8.1) days

Moderate 1 x mild, 2 x moderate 1 X severe 4 x moderate, 1 x mild, 1 x severe

2

AM/CL CFB

Headache

2.3 (SD 2.1) days 3 days 6h

2 X moderate, 3 x mild Moderate Mild

5 1 1

AM/CL CFB CPX

Upper gastrointestinal upsetlnausea

4.4 (SD 2.7) days 2 days 4 days

Mild Mild

4 x mild, 7 x moderate

1 1

11

AM/CL CFB CPX

Loose stools

Duration (mean)

Severity

Drug

Adverse event

Number of volunteers

Table 2 Adverse events under study drug medlcation and their occurrence in relation to study d r u g period

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laboratory ehemicalleiinical parameters

Aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), protein, urea, creatinine, glucose, sodium, potassium, hemoglobin, hematocrit, white blood cell count including differential, platelet count, urinary glucose, protein, microscopic examination and Coombs test were within normal limits on the first and last day of each treatment period of all volunteers except the volunteer excluded as described above. Microbiological results Descriptive analysis of fecal flora

The overview of the data in Figure 1(A-C) represents microorganism numbers per gram dry feces. During amoxycillin/clavulanate administration (Figure lC), there was no significant change in the aerobic fecal microflora. There was marked increase of yeasts (lo2-lo4, nine volunteers) during treatment, with rapid normalization by day 12. In anaerobic bacteria, a slight increase of clostridia on days 8 (lo5), 12 (lo6), and 21 (lo5) compared to premedication number (lo4)was seen; Clostridium dficile was detected in six volunteers, whereas its toxin was found in five of those six; all six had loose stools (but six of 11 volunteers had loose stools without having Clostridium d f i i l e or toxins in their stool samples). During ceftibuten administration (Figure 1A), the aerobic microflora showed a marked increase of enterococci (2 x lo5 to 5 x lo7) and a marked decrease of E. coli (lo5 to 4 x 10'). In the anaerobic spectrum, a slight decrease of anaerobic cocci was observed. There were three patients harboring yeasts in feces, all three developing vaginitis beginning 1-2 days after termination of study drug medication and requiring medical treatment. Clostridium d@le was found in six volunteers during treatment, and its toxin in three of them, but none developed loose stools. Cefpodoxime proxetil (Figure 1B) also caused a marked increase of enterococci (4 x lo4 to lo7), but no change of E. coli. In the anaerobic spectrum, a slight decrease of anaerobic cocci was seen on day 4 only. Nine of the volunteers had Clostridium d@cile in their stools, five of them with toxin. None of them developed loose stools, but another volunteer did. Statistical analysis of the differences between the three antibiotics

The ANOVA model did not show any effect by sequence, subjects nested in sequence, period, treatment, or carryover. E. coli levels were significantly higher with amoxycillin/clavulanate than with ceftibuten on days 7 and 8 (p=0.002 and 0.01, respectively), which was also the

case for amoxycillin/clavulanate and cefpodoxime proxetil on day 7 (p=0.008), but without significant overall change as determined by the AUC (~'0.29 and 0.31). There was no significant difference between ceftibuten and cefpodoxime proxetil concerning E. coli changes. Significantly higher numbers of Klebsiella spp. were found on days 7 and 8 under arnoxycdldclavulanate than under ceftibuten as well as cefpodoxime proxetil ( p c 0 . 0 5 each). Again, there was no difference between ceftibuten and cefpodoxime proxetil. Significantly higher numbers of enterobacteria (Enterobactericeae excluding E. c o g were found with amoxycillin/clavulanate than with ceftibuten on days 7 and 8, as well as with cefpodoxime proxetil on day 8 ( p c 0 . 0 5 each), again without a difference between ceftibuten and cefpodoxime proxetil. There were significantly less enterococci found with amoxycdin/clavulanate than with ceftibuten on days 4 , 7 and 8 and in the AUC (p~0.006,0.003,0.008 and 0.02, respectively). There were also significantly less enterococci found with amoxycillin/clavulanate than with cefpodoxime proxetd on days 4, 7, and 8 (p<0.05 each), but not in the AUC (p=0.09). Again, no significant difference was found between ceftibuten and cefpodoxime proxetil. Significantly higher Candida albicans fecal concentrations were found with amoxycdldclavulanate than with ceftibuten on days 4, 7 and 8 and in the AUC between ceftibuten and amoxycilldclavulanate (p<0.05 each), whereas the differences between amoxycillin/ clavulanate and cefpodoxime proxetil did not reach significant levels. There was no significant hfference between ceftibuten and cefpodoxime proxetil. Significantly less Bacteroides spp. as well as Lactobacillus spp. were found only on day 7 during the amoxycillin/clavulanate study period, but not with either cephalosporin (p<0.05 each). Again, there were no merences between cefbbuten and ce$odoxime proxetil. No significant differences were found for Bijdobacterium spp., anaerobic cocci, aerobic Gram-positive rods, Clostridium spp., Staphylococcus aureus, group A streptococci, group B streptococci, Eillonella spp., Citrobacter spp., Proteus spp., Actinomyces spp., Staphylococcus epidermidis, Micrococcus spp., Clostridium perfringens, Fusobacterium spp., Eubacterium spp., Bacillus spp., aerobic Gram-negative rods, and fungi. Apart from higher numbers of Clostridium spp. on day 21 with amoxycillin/clavulanate than with ceftibuten (p=O.O2), significant differences were never found between the three antibiotics on days 12, 21 and 28, even when significant results were obtained during treatment in all the bacteria and yeasts analyzed.

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Figure 1A Ceftibuten. Fecal microorganisms of the 18 volunteers, with median value of microorganisms per gram feces and regression (-) during the study periods. The study drug administration was from day 0 to day 7. The results of two stool samples collected in the week before each study drug administration as well as of samples during study drug administration on days 4, 7, and 8 and during the 'washout periods' on days 12, 21 and 28 are shown.

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Figure 1C AmoxyciUin/clawlanate. Fecal microorganisms of the 18 volunteers, with median value of microorganisms per gram feces and regression (-) during the study periods. The study drug administration was from day 0 to day 7. The results of two stool samples collected in the week before each study drug administration as well as of samples during study drug administration on days 4, 7, and 8 and during the 'washout periods' on days 12, 21 and 28 are shown.

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The only significant difference found between ceftibuten and cefpodoxime proxetil was in Eubacterium spp. on day 4 (p=0.016), with higher CFU/g feces under ceftibuten. P-Lactamase activity in feces P-Lactamase activity was found in baseline stools of each treatment period in six, four, and eight of the 18 volunteers (Table 3). At the end of treatment (day 7/8), P-lactamase was seen in two more volunteers under ceftibuten, as it was at the end of the washout period (days 21-28). With cefpodoxime proxetil, the p-lactamase activity was seen in 10 more volunteers (14/18) under treatment, reducing to nine volunteers after 21-28 days. After amoxyciUin/clavulanate medication, there was an increase of p-lactamase activity fiom eight to 17 of 18 volunteers, and a reduction to eight of 18 volunteers in the samples of days 21-28 (five being other volunteers than those with initial activity). In this group, one volunteer had loose stools but never had P-lactamase activity demonstrated in this medication period. In conclusion, and as best demonstrated in Table 3, there seems to be a trend to more P-lactamase activity at the end of amoxycillin/clavulanate treatment and a more marked increase under cef$odoxime proxetil administration than under ceftibuten administration. Loose stools were not always associated with p-lactamase activity (volunteer 17 under cefpodoxime proxetil having neither p-lactamase activity, nor yeasts in stool,

and volunteer 3 under amoxycillin/clavulanate having yeasts in all stools). Pharrnacokinetics The main pharmacokinetic parameters were standardized to 70 kg body weight. They are given in Table 4. Figure 2 shows the median and standard ranges of cis-ceftibuten and trans-ceftibuten, respectively, on day 1 after oral 400 mg ceftibuten every 24 h in 18 volunteers. The values are normalized to a body weight of 70 kg. 20J10:

1:

0.1

1 .

,

0

2

0.03

.

,

,

4

,

. ,

6

8

.

1

12

10

Time after oral application (h)

Figure 2 Serum regression (-), median of cis-ceftibuten and trans-ceftibuten (.) and standard range in serum after oral 400 mg ceftibuten every 24 h in 18 volunteers on day 1. Values are normalized to a body weight of 70 kg.

Table 3 P-Lactamase activity in U/g feces” Cefpodoxime proxetil

Ceftibuten

Volunteer no.

Day 0

1 2 3 4 5 6 7 8 9 10

0.336 0.570 0 0 0.262 0 0 1.615 0 0 0 0 0 0 1.71 0 0 0.735

Amoxycillin/ clavulanate

Days 7 and 8

Days 21-28

Day0

Days 7 and8

Days 21-28

Day 0

Days 7 and 8

0.855 3.135 0 0 0.938 0 0 1.243 0.014 0.171

1.881 0.251 0.040 0 0.182 0 0 1.425 0.150 0 0 0.703 0 0 3.192 0 0 0

0.057 0.698 0 0 0.308 0 0 0 0 0.105 0 0 0 0 0 0 0 0

0.741 2.508 2.138 0.186 1.744 0 0 0.399 0.314 0.162 4.560 0.715 0.504 0.684 0.969 0 0 0.148

0.348 1.505 0 0 0.673 0 0 0.245 1.299 0 0.577 0 0 0.182 0 0.128 0 0.673

0.946 1.870 0 0 0.148 0 1.083 0 0 0 0 0 0 0.160 0.855 0 0.108 0.099

2.394 4.788 0 1.208 1.653 0.074 0.239 0.755 1.520 0.091 4.275 1.505 0.088 0.257 1.026 0.177 6.555 0.122

Days 21-28 ~~

11

12 13 14 15 16 17 18

0

0.120 0 0 0.137 0 0 0

’One unit is defined as the amount which formed 1.0 pmol of product per minute under the given conditions.

2.337 2.850 0 0.200 0.299 0 0 1.254 0 0 1.226 0.274 0 0 1.550 0 0 0.276

Harnacher e t al: Fecal f l o r a a n d pharrnacokinetics i n t h r e e oral p-lactarns

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Table 4 Pharmacokinetic parameters

Parameter c,,lax

Ceftibuten (4(!0 mg every 24 h) (bioassay)

(mg/L)

18.9 (3.0) 21.2 (5.3)"

cis-Ceftibuten (HPLC)

Cefpodoxime (200 mg every 12 h) (bioassay)

Amoxycillin (500 mg every 8 h) (bioassay)

Clavulanate (125 mg every 8 h) (bioassay)

3.39 (1.20)

1.92 (0.61)

7.15 (2.16)

3.38 (1.73)

trans-Ceftibuten (HPLC)

18.1 (4.3)

Tmx (h)

2.07 (0.55)

2.20 (0.63)

2.26 (0.66)

2.23 (0.35)

1.35 (0.24) 1.60 (0.38)"

1.32 (0.37) 1.43 (0.34)"

ih)

2.89 (0.61)

2.50 (0.62)

1.92 (0.30) 2.39 (0.30)"

1.97 (0.42)

1.03 (0.15)

0.93 (0.17)

t m p

AUCm, (mg/h/L)

MRT (h) L
Ur.f

(1/70 kg)

(Yo)

CL"t if (mL/min/1.73 m')

C1,n (mL/min/l.73 m2)

100.0 (21.8) 115.0 (26.4)b 4.22 (0.35)

82.23 (18.48)

14.0 (4.0)

4.02 (0.33)

3.93 (0.43)

10.8 (3.3) 3.89 (0.43)

20.0 (4.2) 2.34 (0.18) 2.77 (0.48)'

8.87 (3.10) 2.25 (0.30)

11.5 (1.6) 9.6 (3.9)"

13.55 (2.68) 17.48 (7.09)

64.9 (24.9)

48.4 (15.3)

39.6 (11.2) 47.9 (11.1)6

22.2 (7.0)

60.8 (19.3)

55.91 (17.45)

13.6 (6.9)

46.3 (16.5)

48.8 (1 1.0)

20.0 (10.5)

66.9 (17.5)

85.39 (23.81)

527 (179)

331 (128)

388 (98)

39.1 (10.6)

46.91 (17.44) 37.92 (8.92)"

69 (34)

139 (36)

187 (57)

240 (77) 45.5 (23.7) 61.4 (39.6)"

Mean (standard deviation) for day I , and for day 7 only when significantly different (p<0.05; Wilcoxon or Wilcoxon-Wilcox, respectively; marked as "p<0.05; bp
Figures 3-5 show the corresponding median and standard ranges of serum concentrations of the four study drugs in the 18 volunteers, standardized to a body weight of 70 kg. On comparison of the results of ceftibuten based on HPLC with those determined by bioassay, no significant differences in the pharmacokinetic parameters were found (two-sided Wilcoxon-Wilcox test [37]). DISCUSSION

Despite the many similar indications for oral amoxycillin/clavulanate, ceftibuten. and cefpodoxime proxetil, there are important differences in their pharmacokinetic profiles as well as in their impacts on fecal flora. Compared with amoxycillin, the half-life of ceftibuten was about 2.8 times longer, and that of cefpodoxime two times longer (1.03 h), and the areas under the curve were five times larger with ceftibuten, and about 50% smaller with cefpodoxirne. More meaningfully the ratio of AUC and dose per 24 h for ceftibuten was about 19 times higher, and in cefpodoxime about two times higher, than for amoxycillin. All three p-lactams were well absorbed, as evidenced by their intermediate to high urinary recovery.

Ceftibuten shows a high protein binding rate of about 63%, which is higher than that of other new oral cephalosporins [2,10], high serum levels, and a longer half-life, resulting in the largest AUC. This is also the case for the whole group of new oral cephalosporins after correction for dose [5]. This pharmacokinetic profile allows twice-daily or even once-daily oral administration for treatment of several types of infections [4,38]. The significant 12% rise of C,, found after multiple dosage is consistent with the 13% rise described by Barr et a1 [39]. Accumulation reached significance only in model-derived statistics, not in model-free calculations, despite our rather high numbers of subjects tested. The levels of the eightfold less antibacterially active trans-ceftibuten isomer [39] in serum were about two to three times higher (15.1% of total cefiibuten, or 17.8% of cis-ceftibuten) than described in plasma [39-411; this might be partly due to cis-trans interconversion during sample processing to serum. Cefpodoxime is characterized by low serum levels, a moderately long elimination half-life and late T,,,, allowing twice-daily dosing 1421, and a much lower protein binding of 18-23% compared to ceftibuten [43]. There is no evidence of accumulation [43-451.

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trans-ceftibuten = 24 h

cis-ceftibuten T = 24 h 20

10

2 E w

................................................

I

0

7

:,.:":::::.::.:': :.:. . . .. ... .. ..... ..... .. ... .. ... .. ..... ..... .. ... .. ... .. ..... .. ... .. ... .. ... .. ..

::.:: : : .

1

C

0 .c.

2

c

c

8c

0.1

s

............................ ............................................

...............................

0.002 0

6

1

1

6

Time after first oral dose (days)

Time after first oral dose (days)

Figure 3 Serum concentrations (median and standard ranges) on day 1 and day 7; regression and simulation based on day 1 data with oral 400 mg ceftibuten every 24 h in 18 volunteers. Values are normalized to a body weight of 70 kg. ?=time between two drug administrations.

Cefpodoxime proxetil; T = 12 h

5

,

j

I

,

:

I

~

.............................................

0.05

1

0

E 2

4

6

8

Time after oral application(h)

Figure 5 Serum regression (-) of amoxycillin 500 mg every 8 h and clavulanate 125 mg every 8 h (median and standard ranges) in 18 volunteers on day 1. Values are standardized to a body weight of 70 kg.

Simulation, based on day 1 data Data: day 1 (medianzstd range) Data: day 7 (medianzstd range)

0.01

0

1

6

7

Time after first oral dose (days) Figure 4 Serum concentrations (median and standard ranges) on day 1 and day 7, regression and simulation based on day 1 data with oral 400 mg cefpodozime proxetil every 12 h in 18 volunteers. Values are normalized to a body weight of 70 kg. time between two drug administrations.

Compared to published data from Saathoff et al [46] with 10 male volunteers and those of Trembley et al [47] with 12 volunteers, our data showed identical T-, but lower ,,C , (1.92 versus 2.54 mg/L), and a shorter elimination half-life (1.97 versus 2.87 h), resulting in a smaller AUCtOt(10.8 versus 14.0mg, h/L), which might be explained by differences in gender. As food intake augments the bioavailability of cephalo-

Harnacher e t a l : Fecal f l o r a a n d p h a r m a c o k i n e t i c s i n t h r e e o r a l P-lactarns

sporin esters [5,48,49] and decreases that of ceftibuten [39], the studies were performed after overnight fasting. Amoxycillin had ratheir high serum levels and clavulanic acid had intermediate serum levels and their short T, and half-life values were sirmlar 150-521. Neither substance accumulated [53]. The urinary recovery of 48.8% of amoxycillin was similar to that of cefpodoxime and other p-lactams. We found a lower than reported urinary recovlery of 20.0% (SD 10.5%) of clavulanic acid [50,51,53,54], which might be influenced by its temperature- and pH-dependent degradation [55-571. The major advantage of our triple crossover design using standard oral dosages and treatment durations was the ability to demonstrate drug-dependent fecal flora alterations with statistical means rather than by descriptive analysis. The crossover design was also aimed at circumventing interpersonal variations of the gastrointestinal microflora, by being based on the intrapersonal fecal flora, which is assumed to be remarkably stable [58,59]. Changes of the ecosystem due to an antimicrobial agent depend on its antibiotic spectrum, route of administration, dose, duration, and pharmacokinetic properties such as incomplete absorption, secretion in bile or through intestinal mucosa, activation of prodrugs, or inactivation, e.g. with microbial and sometimes pre-existing P-lactamases [l 1,59-611, or by drug binding to fecal macromolecules [62]. Nearly 40% of orally administered radiomarked cefiibuten is found in feces [39]. In spite of this apparently important intestinal exposure, very low bioassaydetermined fecal concentrations were described, as detectable levels were found in only two of 14 volunteers, neither having any P-lactamase activity under 400 mg/24 h during 10 days in the study of Brismar et al [ll]. Of the prodrug ester cefpodoxime proxetil, about half of the d.ose normally stays in the intestine. The other half gets absorbed and activated with de-esterification by non-specific esterases of the intestinal epithelium, and not in the lumen [13,63]. Although cleavage of the ester might take place in the intestine, there seems to be an important degradation of the compound to <0.5% of the administered dose [63]. However, fecal antibiotic activity seems to differ considerably, as Edlund et a1 1[60]found measurable and quite high cefpodoxime amoiints with a bioassay in only three of 10 volunteers. Fecal amoxycillidclavulanate was below the limit of detection in a study from Brumfitt et a1 [28]. Amoxycillin/clavulanate, used as a ‘positive control’ concerning fecal flora effects in our study, induced significantly more pronounced changes of the fecal flora, and caused considerably more gastrointestinal side effects, mainly in terms of reported loose stools. According to the antibacterial spectrum,

351

significantly more suppression of Bacteroides spp., lactobacilli and enterococci was found during amoxycillin/clavulanate treatment than with the new cephalosporins; on the other hand, the numbers of enterobacteria, including E. coli, Klebsiella spp., and Candida albicans, were higher. P-Lactamase activity either on day 7 or day 8 was found in eight volunteers with ceftibuten, in 14 with cefpodoxime proxetil, and in virtually all (17/18) with amoxycillin/clavulanic acid, despite its P-lactamase inhibitor component, whereas 0-lactamase was found in four to eight volunteers in the fecal samples before study drug administration. Our data differ from those of Todd and Benfield concerning the high prevalence of Clostridium d&cile [64] found in one-third to one-half of our volunteers: in six under ceftibuten and amoxycillin/clavulanate administration and in nine under ce@odoximeproxetil administration, about half of them being toxigenic. Our results confirm others showing that neither Clostridium d@cile and its toxins, nor yeasts, are closely associated with loose stools [60,65-67], which is also the case with p-lactamase activity. They underline the more complex pathogenesis of antibiotic-associated diarrhea. In our unblinded study, 11 of 18 volunteers (61%) under amoxycillin/clavulanate reported a mean of 11 loose stools over 4.4 days. The episode started in nine of the 11 volunteers between day 1 and day 4, suggesting very early causative changes that might therefore not be avoidable by shortening the time of treatment. The very high incidence of loose stools with amoxycillin/clavulanate contrasted with only one volunteer (5.6%) having loose stools under cefpodoxime proxetil, and with none under cefiibuten. In terms of symptoms, our 61% incidence of loose stools contrasted also with the approximately 3.5% diarrhea reported in more than 32 000 patients studied with amoxycillin/clavulanate by Neu et a1 [24] and the 4.1% of 9700 patients reviewed by Croydon [68]. They reported a maximal incidence of about 6% if no specified gastrointestinal events were included, and this is in the range reported for cefpodoxime proxetil [14-161 and ceftibuten [17]. This raises the question of why the reported incidence of loose stools or diarrhea under amoxycillin/clavulanate varies between extremes in the literature, reaching 34% in one large children’s acute otitis media study [20]. Besides the hampering lack of a simple and clearcut definition of diarrhea, the different dosing and treatment schedules and application modes, the heterogeneity of the treated patient groups, and the clinicians and investigators focusing on other findings than loose stools, might be the most important biases in studies not primarily addressing this question. One drawback of our study focused on stool

352

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changes is its unblinded design, with the stool changes data being based on volunteers’ reports. Despite the fact that more volunteers had yeasts in their stools under amoxycillidclavulanate than under the two cephalosporins, all three probable candida vaginitis episodes occurred 1-2 days after ceftibuten medication, a side effect also described in one patient in the study of Stein et al [17]. This side effect occurred after the third investigation period, suggesting that a prolonged and more complex disruption of the vaginal flora may have occurred. In conclusion, ceftibuten shows comparatively favorable, and cefpodoxime proxetil good, pharmacokinetic properties after oral administration, allowing longer dosing intervals than with amoxycillin/ clavulanate. The statistically confirmed more discrete ecological disturbances of the fecal flora by ceftibuten and cefpodoxime may be due to low concentrations of the substances [63] or to the substances being found only in a few persons in feces [11,60], whereas amoxycillin/clavulanate is found in considerable amounts in feces [26,59]. Their narrower antimicrobial spectrum compared to amoxycillin/clavulanate may contribute to less pronounced flora changes. These pharmacokinetic and microbiological observations are consistent with the clinical observations of much less frequent intestinal side effects with the dosages used [20-22,691. The multiple crossover design, calculating an area under the time curve for the log count of each microorganism, allows microbiological changes not only to be described, but also to be statistically analyzed, thus extending the utility of study results.

Ackowledgments

This work was supported by Schering Plough International, Inc., Kenilworth, New Jersey, USA, and was presented in part at the Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans 1993.

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