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Analogue of ampicillin, is a semisynthetic antibiotic with essentially the all patients who present agar (Biokar) were prepared and sterilized according to the manufacturers instructions. Another drug and may not reflect the rates.

Injections resulting in increased tissue penetration, as seen in models of rabbit fibrin clots medical prevention separately or in combination against three Staphylococcus aureus strains isolated from patients suffering.

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Osteomyelitis is difficult to diagnose and treat and may cause irreversible damage.

Antibiotic treatment over weeks to months is required, often in addition to surgical debridement.

To reduce the incidence of infections after orthopedic surgery, perioperative prophylaxis is standard practice.

Each year more than a million hip replacements are done worldwide. Prosthetic devices are particularly susceptible to infections, more than 50% of which are due to Staphylococcus aureus or coagulase-negative staphylococci, such as S. It is vitally important that adequate surgical prophylaxis be used and that sufficient concentrations of antibiotic with activity against frequently encountered pathogens in bone be achieved.

Amoxicillin (amoxicilline) in combination with clavulanic acid is active against pathogens commonly found in prosthesis-related bone infections (MICs at which 90% of bacteria are inhibited [MIC 90 s], 1 mg/liter for methicillin-susceptible S. Successful treatment of infections with amoxicillin-clavulanic acid after molar extraction (22), peri-implantitis (52), osteomyelitis due to diabetic foot infections (40), prophylaxis of infections after orthognathic surgery (6), and staphylococcal osteomyelitis in a rat model (23) has been reported.

The combination was recommended for the treatment of osteomyelitis caused by mixed anaerobic and aerobic pathogens (39).

Bone tissue is less vascularized than, for example, the lungs or the skin.

Therefore, it is especially important to study the bone penetration of an antimicrobial drug before a clinical effectiveness trial is performed. For the timing of perioperative prophylaxis and surgery, it seems critical to know how fast efficacious concentrations are achieved and how long they are maintained. Modeling of the time course of bone concentrations for penicillins is important, since the shape of the concentration-time curve affects the time above the MIC.

The concentrations of amoxicillin and clavulanic acid in bone were studied in the 1980s (1, 3, 24, 54), and only the bone concentration/serum concentration ratios were reported. As these bone concentration/serum concentration ratios change over time, they are a suboptimal measure of the extent of tissue penetration (36, 47). For patients undergoing joint replacement surgery, only one bone sample is most commonly available per patient.

Population

pharmacokinetic

(PK) modeling offers the advantage that it can fit the full time course of the bone and serum concentrations on the

basis

of the data for all patients simultaneously.

The extent of bone penetration is best described by the ratio of the area under the curve (AUC) for bone/AUC for serum. We are not aware of any reports on population pharmacokinetic-pharmacodynamic (PK-PD) models for beta-lactams in bone.

Our first objective was to investigate the amoxicillin and clavulanic acid concentrations in cancellous and cortical bone in patients undergoing hip replacement by using a standardized and validated analytical method for bone and serum. The second objective was to develop a PK model which describes the time course of the amoxicillin and clavulanic acid concentrations in bone as well as the drug exposure in bone relative to that in serum. The third objective was to evaluate the PD profile of amoxicillin in serum, cortical, and cancellous bone (16, 19) against pathogens commonly encountered in bone infections, such as MSSA and S. Twenty patients (9 males, 11 females) who were scheduled to undergo total hip replacement participated in this controlled clinical study.

The patients were diagnosed with coxarthosis and had no inflammation of the joints. Their average weight standard deviation (SD) was 78 12 kg, their average height SD was 169 9 cm, and their average age SD was 63 16 years.

The study was approved by the Institutional Review Board of the School of Medicine, Friedrich-Alexander-University Erlangen-Nurnberg, and was performed according to the revised version of the Declaration of Helsinki. All subjects gave their written informed consent prior to entry into the study. A single dose of 2,000 mg amoxicillin in combination with 200 mg clavulanic acid (Augmentin; GlaxoSmithKline, Munich, Germany) was administered as a short-term intravenous infusion to each patient at the induction of anesthesia. In addition, each patient received a single oral dose of moxifloxacin 2 to 7 h before surgery. Blood samples were collected predosing and at the time of femoral bone resection.

The samples were placed in an ice-water bath and were left to clot before they were centrifuged at 4C. After centrifugation, the serum samples

were

immediately frozen and stored at ?80C until the analysis. The hip replacement surgery consisted of resection of the femoral head or both the femoral head and the femoral neck and the subsequent implantation of the prosthetic hip joint. The collected bone specimens were immediately frozen on dry ice

and

stored at ?80C until analysis.

In cases in

which

the bone samples included both the femoral head and the femoral neck, these two parts were separated from each other. The samples were then divided into cortical and cancellous tissues. The adhering blood was removed from the samples by swabbing for a short time. The bone samples were subsequently pulverized under liquid nitrogen by use of a cryogenic mill (Freezer/Mill). For preparation of calibration standards and spiked quality controls, appropriate amounts of amoxicillin and clavulanic acid standard solutions were added to serum and bone tissue samples that were shown to be free of the study drugs. For determination of the amoxicillin and clavulanic acid concentrations in serum, 50 ?l of the internal standard solution was added to 100 ?l of each sample.

The samples were deproteinized by addition of 300 ?l acetonitrile. After the samples were thoroughly mixed, they were centrifuged and 50 ?l of the clear supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

For analysis of the bone samples, an aliquot of the bone powder was shaken with six times the amount of buffer for 4 h. The degree of extraction was studied over time to ensure the reproducibility of the results. Amoxicillin and clavulanic acid were stable during the 4-h extraction period. After centrifugation, 50 ?l of the internal standard solution was added to 50 ?l of the aqueous supernatant. After addition of 175 ?l acetonitrile, the samples were thoroughly mixed and centrifuged.

The clear supernatant was diluted with twice the amount of buffer. For determination of the amoxicillin concentration, 50 ?l of each sample was chromatographed on a reversed-phase column (Ultracarb 5 ODS 30) and eluted by use of an isocratic solvent system consisting of 0.001 M ammonium acetate buffer and acetonitrile (90/10, vol/vol). The samples were monitored by LC-MS/MS by the selected reaction monitoring method: precursor > product ion for amoxicillin, m/z 366 > m/z 208; precursor > product ion for the internal standard, m/z 350 > m/z 160.

Under these conditions, amoxicillin and the internal standard were eluted after approximately 0.8 min.

For determination of the clavulanic acid concentration, 25 ?l of each sample was chromatographed on a reversed-phase column (Nucleosil 100 amino) and eluted by use of an isocratic solvent system consisting of 0.01 M ammonium acetate buffer and acetonitrile (40/60, vol/vol).

The samples were monitored by LC-MS/MS by the selected reaction monitoring method: precursor > product ion for clavulanic acid, m/z 198 > m/z 108; precursor > product ion for the internal standard, m/z 232 > m/z 140. Under these conditions, clavulanic acid and the internal standard were eluted after approximately 2 min. MacQuan software (version 1.4-noFPU; Perkin-Elmer, Toronto, Ontario, Canada) was used for evaluation of the chromatograms. No interference was observed for the study drugs or the internal standards.

The precision and the accuracy of the spiked quality controls for amoxicillin in serum ranged from 1.8 to 10% and 97.3 to 107.8%, respectively, and the precision and the accuracy of the spiked quality controls for clavulanic acid in serum ranged from 0.8 to 4.8% and 96.0 to 100.5%, respectively. The precision and the accuracy of the spiked quality controls for amoxicillin in bone homogenate ranged from 5.8 to 8.1% and 97.2 to 99.5%, respectively.

The precision and the accuracy of the spiked quality controls for clavulanic acid in bone homogenate ranged from 5.0 to 7.8% and 91.7 to 100.0%, respectively.

A model with two compartments for amoxicillin and one compartment for clavulanic acid was used to describe the concentrations in serum. A bone compartment was added to model the concentrations in bone.

The drug input into the central compartment was described by a time-constrained zero-order process. Standard diagnostic plots for model evaluation were used, and the predictive performance of the final model was tested by the use of visual predictive checks. For the visual predictive check, serum and bone concentration curves for 10,000 subjects were simulated for amoxicillin and clavulanic acid.

We derived the median, the nonparametric 90% prediction interval (5th to 95th percentile), and the nonparametric 50% prediction interval (25th to 75th percentile) from those profiles predicted by validated Perl scripts, as described previously (12). We compared the median predicted concentrations and the prediction intervals with the observed data and performed a visual assessment to determine whether the median and the predicted intervals adequately mirrored the central tendency and the variability of the observed data.

Observations for amoxicillin and clavulanic acid concentrations in serum, cortical bone, and cancellous bone were available. The differential equations for amoxicillin were as follows: $$mathtex$$\[\frac \mathrm \ \left(\frac \mathrm \mathrm _ >> >>\right)\ \ X1 \frac > >>\ \ X2 \frac _ >> >>\ \ X3\]$$mathtex$$ $$mathtex$$\[\frac \frac > >>\ \ X1 \frac > >>\ \ X2\]$$mathtex$$ $$mathtex$$\[\frac \frac _ >> >>\ \ X1 \frac _ >> >>\ \ X3\]$$mathtex$$ where compartment 1 is the central compartment, compartment 2 is the peripheral compartment, and compartment 3 is the bone compartment; X 1, X 2, and X 3 represent the amounts of drug in the central, peripheral, and bone compartments; V c entral , V p eripheral , and V b one represent the volumes of distribution in the central, peripheral, and bone compartments. Initial conditions were 0 for all three compartments. CL is the total clearance from the central compartment, CLic is the intercompartmental clearance between the central and the peripheral compartments, and CLic bone is the intercompartmental clearance between the central and the

bone

compartments. The differential equations for clavulanic acid can be obtained from the above equations by setting CLic equal to 0.

The observed bone concentrations and initial modeling showed that the rates of equilibration between serum and cortical bone as well as those between serum and cancellous bone were similar. Scale terms were included to describe the equilibrium concentration ratios between cancellous bone and serum ( F cancellous ) and between cortical bone and serum ( F cortical ).

If F cortical is equal to 1, the AUC from time zero to infinity after the administration of a single dose for bone equals the respective AUC for serum.

If F cortical is less (greater) than 1, the AUC from time zero to infinity for bone is lower (higher) than that for serum. Sparse serum concentration-time data were available for the period from 0 to 1.1 h after the end of the infusion. These data did not allow us to estimate all PK parameters of the population PK model.

Therefore, prior knowledge of the structural PK model and the average disposition parameters for the serum concentration profiles and their variability from published studies (4, 5, 29, 30, 49, 51) were incorporated in the present analyses. As those studies were conducted with young healthy volunteers, the amoxicillin clearance reported by Sjovall et al. This was in agreement with the age-related decrease in renal function predicted by the formula of Cockcroft and Gault (13) on the basis of the CL values from the other studies. For clavulanic acid, the age-related decrease in renal function was accounted for according to the

formula

of Cockcroft and Gault (13).

As disposition parameters for amoxicillin and clavulanic acid from the literature were determined in the absence of a bone compartment, the amounts of amoxicillin and clavulanic acid in the bone compartment had to be kept minimal.

In our model, the serum PK were not affected by the presence of the bone compartment. This was achieved by choosing a small V b one for the bone compartment. Between-subject variability (BSV) was described by an exponential variability model, and residual unidentified variability was described by a proportional error model for concentrations in serum and bone. The first-order conditional estimation method with the interaction estimation option in NONMEM (version V, release 1.1; NONMEM Project Group, University of California, San Francisco) (8) was utilized for population PK modeling.

WinNonlin Professional (version 4.0.1; Pharsight Corp., Mountain View, CA) was used for statistical analysis. (vi) Estimation by three-stage hierarchical population approach.

To independently confirm the results obtained with NONMEM, PK parameters were estimated by the three-stage hierarchical population approach in S-ADAPT (version 1.55) (7).

Priors for population means and BSV of the disposition parameters were obtained from previously published studies (4, 5, 29, 30, 49, 51). Informative priors were used for the population mean and variability of CL, V c entral , V peripheral , and CLic.

Physiologically plausible but uninformative priors were used for the population mean and variability of F cortical and F cancellous and the population mean of CLic bone on the basis of data reported in the literature (36).

The residual unidentified variability was described by a proportional error model.

As only one serum sample and one bone sample were available from each patient, informative priors were used for the residual unidentified variability on the basis of the bioanalytical assay data.

A systematic sensitivity analysis was performed to evaluate the effect of the choice of priors on the extent and the rate of bone penetration. The extent of drug exposure in bone was determined for amoxicillin and clavulanic acid by simulating the AUCs in serum and cortical and cancellous bone. On the basis of the final estimates from NONMEM, we simulated 10,000 virtual subjects at steady state and calculated the individual ratios of AUC for bone/AUC for serum as well as their BSV.



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