Changes in Fecal Flora and Comparative Multiple-Dose Pharmacokinetics of Ceftibuten, Cefpodoxime Proxetil and Amoxycillin/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’, Carf 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, New 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 AUCtot of 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 AUCtot of 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

INTRODUCTION vitro activity against Gram-positive and Gram-negative bacteria [1,2]. Ceftibuten, cefpodoxime proxetil and Besides partially improved pharmacokinetic character- amoxycillin/clavulanate are widely used antibiotics for isitics, the main advantages of new oral cephalosporins upper and lower respiratory as well as urinary tract over older derivatives are their broader spectra of in infections. Ceftibuten is an orally active third-generation Corresponding author and reprint requests: non-ester cephalosporin. It has broad in vitro anti- H. Lode, Department of Pulmonary and Infectious Diseases, bacterial activity against the majority of Gram-negative City Hospital Berlin-ZehIendorf/Hleckeshorn, pathogens, including Klebsiella spp., good activity Zum Heckeshorn 80, D-14109 Berlin, Germany against streptococci [2-51, marginal activity against Tel: +49 30 8002 22 22 Fax: f49 30 8002 26 23 penicillin-susceptible Streptococcus pneumoniae [3,6-91, E-mail: [email protected] and poor activity against Staphylococcus aureus. It shows Accepted 13 January 1999 an absorption rate between 75% and 90% [4,10] and,

339 340 Clinical Microbiology and Infection, Volume 5 Number 6, June 1999 though resistant to most broad-spectrum 0-lactamases, MATERIALS AND METHODS including those of many Enterobcteriaceae, it is cleaved by a 0-lactamase produced by Bacteroides spp. so that Study design very little active substance is found in the colon [ll]. This open randomized triple crossover study was Cefpodoxime proxetil is a prodrug ester of a third- designed to examine changes in fecal flora and pharma- generation cephalosporin with similar in vitro anti- colnetics with the three oral antibiotics ceftibuten, cef- bacterial activity, except for better activity against podoxime proxetil and amoxycillin/clavulanate during Streptococcus pneumoniae and enhanced antistaphylo- and following 1 week of dosing in normal volunteers. It coccal activity, a similar long elimination half-life as was not blinded, for technical reasons such as amount of ceftibuten, but a rather low maximal serum concen- medication and bioequivalence of formulated blinded tration compared to other oral cephalosporins [2,9,12]. medcation, because of the principal aims of studying It has an absorption rate of about 50% [13]. Besides the fecal flora and pharmacokinetics, which are not fkaction of active drug entering the bowel, significant influenced by an unblinded design, and in order to amounts of the prodrug would be expected to reach the facilitate sampling for drug concentration. It was colon and change the fecal flora in case of activation, conducted from October 1992 to March 1993. The i.e. de-esterification by intestinal esterases. Neverthe- protocol was approved by the ethics committee of the less, in clinical trials the reported incidence of diarrhea Freie Universitat of Berlin, Germany. Medication intake with cefpodoxime proxetil is low [ 14-1 61, which is also was directly observed in each volunteer on day 1, and on the case for ceftibuten [17-191. the mornings of day 2 and day 7; all other medication The impact of amoxycillin/clavulanate, one of the had to be confirmed directly by telephone calls fkom most fkequently used broad-spectrum antimicrobial the volunteer to the responsible physician, and each agents, on the gastrointestinal tract is frequent, so that volunteer recorded a diary protocol with exact time of it was used in our study as 'positive control'. Clinical data each medication, possible adverse event, and stools, are inconsistent concerning the incidence of diarrhea including the quality of feces. All diaries were checked with amoxycillin/clavulanate. There are several studies for possible differences with the protocol made when reporting a much higher incidence of diarrhea in receiving the phone calls. In case of adverse events, the clinical trials with amoxycillin/clavulanate than with volunteers were asked to contact immediately the ceftibuten and ceeodoxime proxetil [20-221. Alterna- investigating physician, who performed a precise inter- tively, a few studies have shown a low incidence of loose view, and, if necessary, performed or organized consul- stools with amoxycdldclavulanate, or have not shown tations or further diagnostic or therapeutic procedures. any important difference [23,24]. Previous studies have shown that amoxycillin/clavulanate causes alterations Volunteers in the normal flora [25-281. Eighteen healthy volunteers aged between 18 and 50 This study was performed in an open randomized years-nine non-pregnant females with a proven barrier triple crossover fashion to examine the pharmaco- method of contraception, and nine males-were lnetics of ceftibuten, cefpodoxime proxetil and randomly assigned to the study. Written informed amoxyc&n/clavulanate following multiple dosing over consent was given after fdexplanation of the protocol. 7 days in 18 normal volunteers. Furthermore, this The mean age was 33.1 years (SD 5.7 years; range design was chosen for a novel statistical approach to 22-42 years). Mean height was 175 cm (SD 10 cm; analyzing changes in fecal flora. Previous evaluations of range 157-190 cm). Mean weight was 71.9 kg (SD 10.2 fecal flora have not addressed the problem of statistical kg; range 56-90 kg). Mean creatinine clearance was significance. For this study, the objective was not only 117 mL/min (SD 23 mL/min; range 82-171 d/min). the evaluation of the differences in fecal flora during Volunteers with a hypersensitivity or serum sickness treatment with a single antibacterial drug, but the in response to any 0-lactam drug were excluded, as statistically meaningful comparison of all three drugs in were volunteers taking dlicit drugs or any medication the same volunteers. With each volunteer serving as within 1 week of study entry or during the study, their own control for each microorgnism analyzed, volunteers who had used antimicrobial drugs within 1 and maintaining the comparison of effects within the month of study entry, volunteers with diarrhea, loose same person, variability was reduced to a minimum, as stools, or current or recent gastrointestinal disease, interindividual baseline differences were eliminated. By volunteers who had recently traveled to areas endemic calculation of an area under the curve (colony-forming for gastrointestinal parasites, or volunteers who had units/g feces versus time) for each microorganism, undergone recent hospitalization for any reason, in standard tests allowed a statistical comparison of fecal order to eliminate volunteers potentially colonized flora changes in this relatively small study population. with resistant organisms. Hamacher et al: Fecal flora and pharmacokinetics in three oral D-lactams 341

Antibiotic dosage and administration were counted, isolated in pure culture, and identified, The test subjects received each oral study drug for as were the different colonies appearing on the selective 7 days (ceftibuten 400 mg PO daily; amoxycillin/ media. All fecal samples were also assayed for yeasts,. clavulanate 500/125 mg every 8 h; cefpodoxime Clostridiuni dficile was searched for, and its toxins were proxetil 200 mg every 12 h), followed by a 'washout' determined by, a tissue-culture assay with human period of 4 weeks after each drug administration embryonic intestinal cells and a control with specific period. As the study was performed in a randomized neutralizing antiserum, a method described by triple crossover mode, the subjects were randomized to Aronsson et a1 [30]. Counts were given as log number the six possible sequences (permutations) of study drug of bacteria/g feces. order. After three administrations and two washout Samples were assayed for p-lactamase activity using periods, each subject had received the three different the chromogenic cephalosporin nitrocefin as substrate medications. for a spectrophotometric determination of activity, as previously described [31,32]. One unit of P-lactamase Sampling was defined as the amount which formed 1.0 pmol of Stool samples product per minute under given conditions. Fecal samples were collected twice per week before each of the three study drug administration periods, on Bioassay the fourth day of medication, on the seventh (last) day The concentrations of the four substances in the serum of medication, on the eighth day (the first day after end and urine samples were determined with an agar of medication), and on days 12, 21 and 28 in the diffusion bioassay (cup plate method), as modified by washout period. Samples of approximately 5 g each Reeves and Bywater [33] (Table 1). Serum and urine were collected in sterile plastic containers and were saniples were assayed in triplicate, and five standard frozen in a -70°C freezer until analysis was performed. concentrations (in triplicate) were used on each plate.

Blood samples Assay of ceftibuten in serum and urine by high-performance These were taken repeatedly for serum drug level liquid chromatograph y (HPL C) determination on days 1 and 7 after 0, 30, 60, 120, 240, After addition of the internal standard toluenesulfonic and 360 min, the last sample being taken at 8 h (volun- acid, ceftibuten was extracted from serum with teers under amoxycillin/clavulanate) and 12 h (volun- acetonitrile. Acetonitrile was back-extracted from the teers under cefiibuten and cefpodoxime proxetil). liquid phase with dichloroniethane. The aqueous supernatant was chromatogaphed on a reversed-phase Urine column (Nucleosil 5C18, 125 mm (length), 4 mm This was analyzed on days 1 and 7. On both days, urine (diameter); Macherey & Negel, Diiren, Germany). samples were analyzed from the periods 0-6 h and The mobile phase was acetonitrile/methanol/water 6-12 h for ceftibuten and cefpodoxime, 12-24 h for (130:20:850) containing 3.4 g tetrabutylammonium ceftibuten and 0-8 h for amoxycillin/clavulanate. phosphate, adjusted to pH 6.95 with 2 M sodium hydroxide solution. The absorbance of the eluate was Methods monitored at 256 nm. Retention times were 8.5 min Analysis of the fecal flora (cis-ceftibuten), 10.2 min (trans-ceftibuten) and 17.7 Microbial identification and quantification was done min (internal standard). Urine was diluted with mobile according to colonial morphology, Gram stain, and phase before chromotography. Validation of the method various serologic and biochemical tests. A 1-g sample yielded the following results for serum (urine): detection of stool was homogenized in 9 mL of prereduced limit 0.1 mg/L (5.0 mg/L), lower limit of quantifi- peptone yeast extract medium. Tenfold serial dilutions cation 0.2 mg/L (10.0 mg/L, linear range 26.0 mg/L were made to a dilution of' lo-*. The samples were (1000 mg/L), coefficient of variance between series inoculated and a quantitative estimate of each microbial 4.4-9.5% (2.1-4.9%), and recovery of 97.3-98.3% species was made as described by Heimdahl and (107.3%). Nord [29]. All manipulations of the anaerobic bacteria were carried out in an anaerobic chamber (Forma, Statistical fecal flora analysis Philadelphia, PA, USA) under 10% (vol/vol) hydrogen Each volunteer acted as his or her own control. in nitrogen. After 24 h (aerobic agar plates) or 48 h Separate analyses were performed for each of the (anaerobic agar plates) of incubation at 37"C, total organisms. The cultures obtained at the latter of the colony counts were determined from aerobic and two screening visits were used as the baselines. Change anaerobic blood agar plates. Different colony types from baseline was analyzed to estimate differences in 342 Clinical Microbiology and Infection, Volume 5 Number 6, June 1999

Table 1 Characteristics of the bioassays Lower limit of Variation detection (mg/L) coefficient (%) Standard Bacterium Pool Pool Substance solution (test strain) Agar Buffer serum Buffer serum

Ceftibuten (lot no. Sorensen’s buffer Morganella morganii DST agar 0.058 0.116 3.90 4.57 28’173/001) at pH 7.2 IF0 3848 (DSM 30164)

Cefpodoxime Sorensen’s buffer Morganella morganii Antibiotic 0.058 0.116 6.27 5.40 (lot no. 18) at pH 7.2 IF0 3848 medium 2 (DSM 30164) Oxoid

Amoxycillin Citrate buffer Bacillus subh’lis Antibiotic 0.116 0.116 6.95 4.25 (batch no. 28) at pH 7.2 ATCC 6633 medmm 2 Micrococcus Oxoid 0.010 0.030 5.40 3.28 luteus ATCC 8340

Cladanate Sorensen’s buffer Klebsiella CLED Agar with 0.030 0.058 6.42 3.04 (batch no. 54) at pH 7.0 aerogenes Na-benzylpenicillin NCTC 11228 (Ch.B.OY 8007)

the effects between the three study medications at each Pharmacokinetic calculations time point (days 4, 7, 8, 12, 21 and 28) for each The main pharmacokinetic parameters were standard- organism. All statistical testing was performed at the ized to 70-kg body weight (area, C,,, volume of p level of 0.05. distribution in the steady state, divided by the oral As the subjects were randomized to one of the six bioavailability (L&/’)) or to 1.73-m2 body surface possible treatment sequences that can be followed with area; (Cltot/’ CIRn). They were calculated model three drugs, there were three subjects in each treatment free (AUDtot,mean residence time (MRT), E-MRT sequence. Since one subject discontinued prematurely, (percentage of eliminated substance until MRT), C,,, she was excluded from statistical fecal flora analysis and V,,/’ total and renal clearance (Cltot/’ Clm), as well as replaced by a female volunteer who finished the whole assuming an open one-compartment model (AUCr0, study and was therefore included in the analysis T,,, tsonp)). The calculations were performed by according to the protocol. In order to include the standard methods [36]. Differences in determined para- repeated measures of the microbe counts at the six time meters were analyzed for single variables by painvise points after baseline, the area under the curve (AUC) t-test, and for multiple variables by multiple painvise change from the baseline for every subject was comparisons according to Wilcoxon and Wilcox [37]. calculated, working with loglo (colony-forming units A p-value <0.05 was considered significant. microbedg feces) values. Using this transformed variable, differences from baseline were computed for each subject. Because the loglo transformations were RESULTS used, differences were used, as opposed to ratios. This was performed for each treatment, so that a subject had Tolerance three values for each time point concerning each One volunteer (female, 27 years) was replaced during microorganism. The AUC was computed for each the first study period because of fever, severe headache, treatment and each subject (so each subject had three leukocytosis (16.2 x 109/L with 4% eosinophils) and AUC values concerning each microorganism). These diarrhea after 5 days of medication with cefiibuten (no AUC values were used for analyses. The between- Clostridium d@ile or toxins were found). According to treatment Merences were tested using a 3 x 3 crossover the protocol, the data of that volunteer were excluded ANOVA model, with a p-value <0.05 being con- from the final pharmacokinetic and fecal flora statistical sidered as significant [34,35]. The ANOVA model evaluation. included effects for sequence, subjects nested in Further adverse events are listed in Table 2. sequence, period, treatment, and carryover. I D 3 D 0 5 m 1 2 'v n m n 'v Table 2 Adverse events under study drug medlcation and their occurrence in relation to study drug period m Number of volunteers D 3 per period number a Number of TI Adverse event Drug volunteers Severity Duration (mean) I I1 I11 Relationship 5 Loose stools AM/CL 11 4 x mild, 7 x moderate 4.4 (SD 2.7) days 5 3 3 11 x prob. CFB 1 Mild 2 days 1 prob. CPX 1 Mild 4 days 1 prob.

Upper gastrointestinal AM/CL 5 2 X moderate, 3 x mild 2.3 (SD 2.1) days 1 1 3 2 x poss.; 1 x prob. upsetlnausea CFB 1 Moderate 3 days 1 poss. CPX 1 Mild 6h 1 poss.

5 Headache AM/CL 2 Moderate 10 (SD 2) h 2 1 x poss.; 1 x prob. -7 m CFB 4 1 x mild, 2 x moderate 4.8 (SD 8.1) days 2 2 3 x poss.; 1 x prob. m 1 X severe CPX 6 4 x moderate, 1 x mild, 1.2 (SD 1.0) days 1 3 2 6 X poss. 1 x severe

Pruritus AM/CL 3 2 X dd,1 x moderate 1.5 (SD 0.7) days 2 1 3 x poss. D CFX 1 Mild 14h 1 poss. 3 lil Vaginal mycosis CFB 3 2 X moderate, 1 X mild 26 (SD 23) days 3 prob.

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.

W P W 344 Clinical Microbiology and Infection, Volume 5 Number 6, June 1999 laboratory ehemicalleiinical parameters case for amoxycillin/clavulanate and cefpodoxime Aspartate aminotransferase (ASAT), alanine amino- proxetil on day 7 (p=0.008), but without significant transferase (ALAT), alkaline phosphatase, bilirubin, overall change as determined by the AUC (~'0.29 and lactate dehydrogenase (LDH), protein, urea, creatinine, 0.31). There was no significant difference between glucose, sodium, potassium, hemoglobin, hematocrit, ceftibuten and cefpodoxime proxetil concerning E. coli white blood cell count including differential, platelet changes. count, urinary glucose, protein, microscopic examina- Significantly higher numbers of Klebsiella spp. were tion and Coombs test were within normal limits on the found on days 7 and 8 under arnoxycdldclavulanate first and last day of each treatment period of all volun- than under ceftibuten as well as cefpodoxime proxetil teers except the volunteer excluded as described above. (pc0.05 each). Again, there was no difference between ceftibuten and cefpodoxime proxetil. Microbiological results Significantly higher numbers of enterobacteria Descriptive analysis of fecal flora (Enterobactericeae excluding E. cog were found with The overview of the data in Figure 1(A-C) represents amoxycillin/clavulanate than with ceftibuten on days 7 microorganism numbers per gram dry feces. and 8, as well as with cefpodoxime proxetil on day 8 During amoxycillin/clavulanate administration (pc0.05 each), again without a difference between (Figure lC), there was no significant change in the ceftibuten and cefpodoxime proxetil. aerobic fecal microflora. There was marked increase of There were significantly less enterococci found yeasts (lo2-lo4, nine volunteers) during treatment, with amoxycdin/clavulanate than with ceftibuten on with rapid normalization by day 12. In anaerobic days 4,7 and 8 and in the AUC (p~0.006,0.003,0.008 bacteria, a slight increase of clostridia on days 8 (lo5), and 0.02, respectively). There were also significantly 12 (lo6), and 21 (lo5) compared to premedication less enterococci found with amoxycillin/clavulanate number (lo4)was seen; Clostridium dficile was detected than with cefpodoxime proxetd on days 4, 7, and 8 in six volunteers, whereas its toxin was found in five of (p<0.05 each), but not in the AUC (p=0.09). Again, those six; all six had loose stools (but six of 11 volunteers no significant difference was found between ceftibuten had loose stools without having Clostridium dfiile or and cefpodoxime proxetil. toxins in their stool samples). Significantly higher Candida albicans fecal concen- During ceftibuten administration (Figure 1A), the trations were found with amoxycdldclavulanate than aerobic microflora showed a marked increase of entero- with ceftibuten on days 4, 7 and 8 and in the AUC cocci (2 x lo5 to 5 x lo7) and a marked decrease of between ceftibuten and amoxycilldclavulanate (p<0.05 E. coli (lo5 to 4 x 10'). In the anaerobic spectrum, a each), whereas the differences between amoxycillin/ slight decrease of anaerobic cocci was observed. There clavulanate and cefpodoxime proxetil did not reach were three patients harboring yeasts in feces, all three significant levels. There was no significant hfference developing vaginitis beginning 1-2 days after termina- between ceftibuten and cefpodoxime proxetil. tion of study drug medication and requiring medical Significantly less Bacteroides spp. as well as treatment. Clostridium d@le was found in six volunteers Lactobacillus spp. were found only on day 7 during during treatment, and its toxin in three of them, but the amoxycillin/clavulanate study period, but not none developed loose stools. with either cephalosporin (p<0.05 each). Again, there Cefpodoxime proxetil (Figure 1B) also caused a were no merences between cefbbuten and ce$odoxime marked increase of enterococci (4 x lo4 to lo7), but proxetil. no change of E. coli. In the anaerobic spectrum, a slight No significant differences were found for Bijdo- decrease of anaerobic cocci was seen on day 4 only. bacterium spp., anaerobic cocci, aerobic Gram-positive Nine of the volunteers had Clostridium d@cile in their rods, Clostridium spp., Staphylococcus aureus, group A stools, five of them with toxin. None of them streptococci, group B streptococci, Eillonella spp., developed loose stools, but another volunteer did. Citrobacter spp., Proteus spp., Actinomyces spp., Staphylo- coccus epidermidis, Micrococcus spp., Clostridium perfringens, Statistical analysis of the differences between the three Fusobacterium spp., Eubacterium spp., Bacillus spp., aerobic antibiotics Gram-negative rods, and fungi. The ANOVA model did not show any effect by Apart from higher numbers of Clostridium spp. sequence, subjects nested in sequence, period, treat- on day 21 with amoxycillin/clavulanate than with ment, or carryover. ceftibuten (p=O.O2), significant differences were never E. coli levels were significantly higher with amoxy- found between the three antibiotics on days 12, 21 and cillin/clavulanate than with ceftibuten on days 7 and 8 28, even when significant results were obtained during (p=0.002 and 0.01, respectively), which was also the treatment in all the bacteria and yeasts analyzed. A

r I I I1 I I I 9 Streptococci 9 Escherichia coli Anaerobic-cocci - 9 Clostridia I -1 9-- 8 I 8 6 a'.: .. m 7 7 3 7- ; 1: 6 6, 6-. I 5 . 5. 4 4. 3 3 3. 52 s2 52. :;6!II LactobacVli Staphylococci Enterobacteria 9 @'.I. . 2 8- .- 0 4 . 1 .a W 7 I m I m 3 . CL

sz-I: I I. . I 0 0 Bifidobacteria Fusobacteria 77 . Enterococci - Yeasts 10 a:. . -----I .. -0 9- I .-

6- . rn 40 4- .I. I . I .- 3- .. I. 1- I I- I II I I I W W 4 -1 0 4 78 12 21 28 Bacilli -10 4 76 12 21 28 ~EubacteriaI ! - 9- 6- . Days Days 7- . 6- 5. 4- 3-

52-11 I I1 I I I- -.10 476 12 21 28 Days Days

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 0 P administration on days 4, 7, and 8 and during the 'washout periods' on days 12, 21 and 28 are shown. m m m m r

Shaa shea L m n E 3 z Ln m E a 3 : . : .I I.-*;5 - .:L&+ I : 0 . . ;. -5 1 . -9 E .-0 shaa -L c shea -0 0 -m !iwa -6 (3 12 21 or t 01- - oz I U 1. 1 E m z 2s cn C m6 -0 v 0 5 9 L - 0 m 6 .-0 .-c 1 25 5 C t 5 9 L e

1. I I!- - :. 1C . .. .I. co* a m Eubacteria m 9- Bacilli 0-. . I - Wrn . 8- wi 3 I 2 Days 7- =: Days 7- . . 6.:: 6.. . .: . .- 5. 5- I a. .

Days Days

0 P Figure 1C AmoxyciUin/clawlanate. Fecal microorganisms of the 18 volunteers, with median value of microorganisms per gram feces and regression (-) during the study .I 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. 348 Clinical Microbiology and Infection, Volume 5 Number 6, June 1999

The only significant difference found between and volunteer 3 under amoxycillin/clavulanate having ceftibuten and cefpodoxime proxetil was in Eubacterium yeasts in all stools). spp. on day 4 (p=0.016), with higher CFU/g feces under ceftibuten. Pharrnacokinetics The main pharmacokinetic parameters were standard- P-Lactamase activity in feces ized to 70 kg body weight. They are given in Table 4. P-Lactamase activity was found in baseline stools of Figure 2 shows the median and standard ranges of each treatment period in six, four, and eight of the 18 cis-ceftibuten and trans-ceftibuten, respectively, on day volunteers (Table 3). At the end of treatment (day 7/8), 1 after oral 400 mg ceftibuten every 24 h in 18 volun- P-lactamase was seen in two more volunteers under teers. The values are normalized to a body weight of ceftibuten, as it was at the end of the washout period 70 kg. (days 21-28). With cefpodoxime proxetil, the p-lactamase activity was seen in 10 more volunteers (14/18) 20 J- under treatment, reducing to nine volunteers after 10: 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 1: 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 0.1 period. In conclusion, and as best demonstrated in 1 Table 3, there seems to be a trend to more P-lactamase 0.03 1 . , . , , , . , . 0 2 4 6 8 10 12 activity at the end of amoxycillin/clavulanate treatment Time after oral application (h) and a more marked increase under cef$odoxime proxetil administration than under ceftibuten administration. Figure 2 Serum regression (-), median of cis-ceftibuten Loose stools were not always associated with p-lacta- and trans-ceftibuten (.) and standard range in serum after mase activity (volunteer 17 under cefpodoxime proxetil oral 400 mg ceftibuten every 24 h in 18 volunteers on day having neither p-lactamase activity, nor yeasts in stool, 1. Values are normalized to a body weight of 70 kg.

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

Cefpodoxime Amoxycillin/ Ceftibuten proxetil clavulanate

Days 7 Days Days 7 Days Days 7 Days Volunteer no. Day 0 and 8 21-28 Day0 and8 21-28 Day 0 and 8 21-28

~~ 1 0.336 0.855 1.881 0.057 0.741 0.348 0.946 2.394 2.337 2 0.570 3.135 0.251 0.698 2.508 1.505 1.870 4.788 2.850 3 0 0 0.040 0 2.138 0 0 0 0 4 0 0 0 0 0.186 0 0 1.208 0.200 5 0.262 0.938 0.182 0.308 1.744 0.673 0.148 1.653 0.299 6 0 0 0 0 0 0 0 0.074 0 7 0 0 0 0 0 0 1.083 0.239 0 8 1.615 1.243 1.425 0 0.399 0.245 0 0.755 1.254 9 0 0.014 0.150 0 0.314 1.299 0 1.520 0 10 0 0.171 0 0.105 0.162 0 0 0.091 0 11 0 0 0 0 4.560 0.577 0 4.275 1.226 12 0 0.120 0.703 0 0.715 0 0 1.505 0.274 13 0 0 0 0 0.504 0 0 0.088 0 14 0 0 0 0 0.684 0.182 0.160 0.257 0 15 1.71 0.137 3.192 0 0.969 0 0.855 1.026 1.550 16 0 0 0 0 0 0.128 0 0.177 0 17 0 0 0 0 0 0 0.108 6.555 0 18 0.735 0 0 0 0.148 0.673 0.099 0.122 0.276

’One unit is defined as the amount which formed 1.0 pmol of product per minute under the given conditions. Harnacher et al: Fecal flora and pharrnacokinetics in three oral p-lactarns 349

Table 4 Pharmacokinetic parameters

Cefpodoxime Amoxycillin Clavulanate (200 mg (500 mg (125 mg Ceftibuten (4(!0 mg cis-Ceftibuten trans-Ceftibuten every 12 h) every 8 h) every 8 h) Parameter every 24 h) (bioassay) (HPLC) (HPLC) (bioassay) (bioassay) (bioassay) c,,lax (mg/L) 18.9 (3.0) 18.1 (4.3) 3.39 (1.20) 1.92 (0.61) 7.15 (2.16) 3.38 (1.73) 21.2 (5.3)" Tmx (h) 2.07 (0.55) 2.20 (0.63) 2.26 (0.66) 2.23 (0.35) 1.35 (0.24) 1.32 (0.37) 1.60 (0.38)" 1.43 (0.34)" tmpih) 2.89 (0.61) 2.50 (0.62) 1.92 (0.30) 1.97 (0.42) 1.03 (0.15) 0.93 (0.17) 2.39 (0.30)" AUCm, (mg/h/L) 100.0 (21.8) 82.23 (18.48) 14.0 (4.0) 10.8 (3.3) 20.0 (4.2) 8.87 (3.10) 115.0 (26.4)b MRT (h) 4.22 (0.35) 4.02 (0.33) 3.93 (0.43) 3.89 (0.43) 2.34 (0.18) 2.25 (0.30) 2.77 (0.48)' L

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 Ceftibuten shows a high protein binding rate of standard ranges of serum concentrations of the four about 63%, which is higher than that of other new oral study drugs in the 18 volunteers, standardized to a body cephalosporins [2,10], high serum levels, and a longer weight of 70 kg. half-life, resulting in the largest AUC. This is also the On comparison of the results of ceftibuten based case for the whole group of new oral cephalosporins on HPLC with those determined by bioassay, no sig- after correction for dose [5]. This pharmacokinetic nificant differences in the pharmacokinetic parameters profile allows twice-daily or even once-daily oral were found (two-sided Wilcoxon-Wilcox test [37]). administration for treatment of several types of infections [4,38]. The significant 12% rise of C,, DISCUSSION found after multiple dosage is consistent with the 13% rise described by Barr et a1 [39]. Accumulation reached Despite the many similar indications for oral amoxy- significance only in model-derived statistics, not in cillin/clavulanate, ceftibuten. and cefpodoxime proxetil, model-free calculations, despite our rather high numbers there are important differences in their pharmaco- of subjects tested. The levels of the eightfold less kinetic profiles as well as in their impacts on fecal flora. antibacterially active trans-ceftibuten isomer [39] in Compared with amoxycillin, the half-life of ceftibuten serum were about two to three times higher (15.1% of was about 2.8 times longer, and that of cefpodoxime total cefiibuten, or 17.8% of cis-ceftibuten) than two times longer (1.03 h), and the areas under the described in plasma [39-411; this might be partly due curve were five times larger with ceftibuten, and about to cis-trans interconversion during sample processing to 50% smaller with cefpodoxirne. More meaningfully the serum. ratio of AUC and dose per 24 h for ceftibuten was Cefpodoxime is characterized by low serum levels, about 19 times higher, and in cefpodoxime about two a moderately long elimination half-life and late T,,,, times higher, than for amoxycillin. All three p-lactams allowing twice-daily dosing 1421, and a much lower were well absorbed, as evidenced by their intermediate protein binding of 18-23% compared to ceftibuten to high urinary recovery. [43]. There is no evidence of accumulation [43-451. 350 Clinical Microbiology and Infection, Volume 5 Number 6, June 1999

cis-ceftibuten T = 24 h trans-ceftibuten = 24 h 20 10 ...... ::.::...... ::. :,.:":::::.::.:': :.::. I ......

wE C .-0 c.2 cc 8 0.1 sc ......

...... 0.002 0 1 6 0 1 6 7 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,jI,:I~ ......

0.05 1 E 0 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 Compared to published data from Saathoff et al [46] 0 1 6 7 with 10 male volunteers and those of Trembley et al Time after first oral dose (days) [47] with 12 volunteers, our data showed identical T-, but lower C,,, (1.92 versus 2.54 mg/L), and a Figure 4 Serum concentrations (median and standard shorter elimination half-life (1.97 versus 2.87 h), result- ranges) on day 1 and day 7, regression and simulation based ing in a smaller AUCtOt(10.8 versus 14.0mg, h/L), on day 1 data with oral 400 mg cefpodozime proxetil every 12 h in 18 volunteers. Values are normalized to a body weight which might be explained by differences in gender. of 70 kg. time between two drug administrations. As food intake augments the bioavailability of cephalo- Harnacher et al: Fecal flora and pharmacokinetics in three oral P-lactarns 351 sporin esters [5,48,49] and decreases that of ceftibuten significantly more suppression of Bacteroides spp., [39], the studies were performed after overnight fasting. lactobacilli and enterococci was found during amoxy- Amoxycillin had ratheir high serum levels and cillin/clavulanate treatment than with the new clavulanic acid had intermediate serum levels and cephalosporins; on the other hand, the numbers of their short T,, and half-life values were sirmlar 150-521. enterobacteria, including E. coli, Klebsiella spp., and Neither substance accumulated [53]. The urinary Candida albicans, were higher. P-Lactamase activity recovery of 48.8% of amoxycillin was similar to that of either on day 7 or day 8 was found in eight volunteers cefpodoxime and other p-lactams. We found a lower with ceftibuten, in 14 with cefpodoxime proxetil, and than reported urinary recovlery of 20.0% (SD 10.5%) in virtually all (17/18) with amoxycillin/clavulanic of clavulanic acid [50,51,53,54], which might be acid, despite its P-lactamase inhibitor component, influenced by its temperature- and pH-dependent whereas 0-lactamase was found in four to eight degradation [55-571. volunteers in the fecal samples before study drug The major advantage of our triple crossover design administration. using standard oral dosages and treatment durations was Our data differ from those of Todd and Benfield the ability to demonstrate drug-dependent fecal flora concerning the high prevalence of Clostridium d&cile alterations with statistical means rather than by descrip- [64] found in one-third to one-half of our volunteers: tive analysis. The crossover design was also aimed at in six under ceftibuten and amoxycillin/clavulanate circumventing interpersonal variations of the gastro- administration and in nine under ce@odoxime proxetil intestinal microflora, by being based on the intra- administration, about half of them being toxigenic. Our personal fecal flora, which is assumed to be remarkably results confirm others showing that neither Clostridium stable [58,59]. Changes of the ecosystem due to an d@cile and its toxins, nor yeasts, are closely associated antimicrobial agent depend on its antibiotic spectrum, with loose stools [60,65-67], which is also the case with route of administration, dose, duration, and pharmaco- p-lactamase activity. They underline the more complex kinetic properties such as incomplete absorption, pathogenesis of antibiotic-associated diarrhea. secretion in bile or through intestinal mucosa, activ- In our unblinded study, 11 of 18 volunteers (61%) ation of prodrugs, or inactivation, e.g. with microbial under amoxycillin/clavulanate reported a mean of 11 and sometimes pre-existing P-lactamases [l 1,59-611, loose stools over 4.4 days. The episode started in nine or by drug binding to fecal macromolecules [62]. of the 11 volunteers between day 1 and day 4, suggest- Nearly 40% of orally administered radiomarked ing very early causative changes that might therefore cefiibuten is found in feces [39]. In spite of this appar- not be avoidable by shortening the time of treatment. ently important intestinal exposure, very low bioassay- The very high incidence of loose stools with amoxy- determined fecal concentrations were described, as cillin/clavulanate contrasted with only one volunteer detectable levels were found in only two of 14 (5.6%) having loose stools under cefpodoxime proxetil, volunteers, neither having any P-lactamase activity and with none under cefiibuten. under 400 mg/24 h during 10 days in the study of In terms of symptoms, our 61% incidence of loose Brismar et al [ll]. Of the prodrug ester cefpodoxime stools contrasted also with the approximately 3.5% proxetil, about half of the d.ose normally stays in the diarrhea reported in more than 32 000 patients studied intestine. The other half gets absorbed and activated with amoxycillin/clavulanate by Neu et a1 [24] and the with de-esterification by non-specific esterases of the 4.1% of 9700 patients reviewed by Croydon [68]. They intestinal epithelium, and not in the lumen [13,63]. reported a maximal incidence of about 6% if no Although cleavage of the ester might take place in the specified gastrointestinal events were included, and this intestine, there seems to be an important degradation is in the range reported for cefpodoxime proxetil of the compound to <0.5% of the administered dose [14-161 and ceftibuten [17]. This raises the question of [63]. However, fecal antibiotic activity seems to differ why the reported incidence of loose stools or diarrhea considerably, as Edlund et a1 1[60] found measurable and under amoxycillin/clavulanate varies between extremes quite high cefpodoxime amoiints with a bioassay in only in the literature, reaching 34% in one large children’s three of 10 volunteers. Fecal amoxycillidclavulanate acute otitis media study [20]. Besides the hampering was below the limit of detection in a study from lack of a simple and clearcut definition of diarrhea, the Brumfitt et a1 [28]. Amoxycillin/clavulanate, used as a different dosing and treatment schedules and applic- ‘positive control’ concerning fecal flora effects in our ation modes, the heterogeneity of the treated patient study, induced significantly more pronounced changes groups, and the clinicians and investigators focusing on of the fecal flora, and caused considerably more other findings than loose stools, might be the most gastrointestinal side effects, mainly in terms of reported important biases in studies not primarily addressing this loose stools. According to the antibacterial spectrum, question. One drawback of our study focused on stool 352 Clinical Microbiology and Infection, Volume 5 Number 6, June 1999

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