In Vitro In Vivo Extrapolation and Bioequivalence Prediction for Immediate-Release Capsules of Cefadroxil Based on a Physiologically-Based Pharmacokinetic ACAT Model.

Physiologically based pharmacokinetic (PBPK) modeling is a mechanistic concept, which helps to judge the effects of biopharmceutical properties of drug product such as in vitro dissolution on its pharmacokinetic and in vivo performance. With the application of virtual bioequivalence (VBE) study, the drug product development using model-based approach can help in evaluating the possibility of extending BCS-based biowaiver. Therefore, the current study was intended to develop PBPK model as well as in vitro in vivo extrapolation (IVIVE) for BCS class III drug i.e. cefadroxil. A PBPK model was created in GastroPlus™ 9.8.3 utilizing clinical data of immediate-release cefadroxil formulations. By the examination of simulated and observed plasma drug concentration profiles, the predictability of the proposed model was assessed for the prediction errors. Furthermore, mechanistic deconvolution was used to create IVIVE, and the plasma drug concentration profiles and pharmacokinetic parameters were predicted for different virtual formulations with variable cefadroxil in vitro release. Virtual bioequivalence study was also executed to assess the bioequivalence of the generic verses the reference drug product (Duricef®). The developed PBPK model satisfactorily predicted Cmax and AUC0-t after cefadroxil single and multiple oral dose administrations, with all individual prediction errors within the limits except in a few cases. Second order polynomial correlation function obtained accurately predict in vivo drug release and plasma concentration profile of cefadroxil test and reference (Duricef®) formulation. The VBE study also proved test formulation bioequivalent to reference formulation and the statistical analysis on pharmacokinetic parameters reported 90% confidence interval for Cmax and AUC0-t in the FDA acceptable limits. The analysis found that a validated and verified PBPK model with a mechanistic background is as a suitable approach to accelerate generic drug development.

Cefadroxil and cephalexin pharmacokinetics and pharmacodynamics in children with musculoskeletal infections.

Cephalexin, a first-generation cephalosporin, is the first-line oral therapy for children with musculoskeletal infections due to methicillin-susceptible Staphylococcus aureus (MSSA). Cefadroxil, a similar first-generation cephalosporin, is an attractive alternative to cephalexin given its longer half-life. In this study, we describe the comparative pharmacokinetics (PK) and pharmacodynamics (PD) of cephalexin and cefadroxil in children with musculoskeletal infections. Children aged 6 months to 18 years with a musculoskeletal infection were enrolled in a prospective, open-label, crossover PK study and given single oral doses of cefadroxil (50-75 mg/kg up to 2,000 mg) and cephalexin (50 mg/kg up to 1,375 mg). Population PK models were developed and used for dosing simulations. Our primary PD target was the achievement of free antibiotic concentrations above the minimum inhibitory concentration (fT >MIC) for 40% of the day for MICs ≤ 4 mg/L. PK of cephalexin (n = 15) and cefadroxil (n = 14) were best described using a one-compartment, first-order absorption model, with a lag time component for cefadroxil. PK parameters were notable for cefadroxil's longer half-life (1.61 h) than cephalexin's (1.10 h). For pediatric weight bands, our primary PD target was achieved by cephalexin 25 mg/kg/dose, maximum 750 mg/dose, administered three times daily and cefadroxil 40 mg/kg/dose, maximum 1,500 mg/dose, administered twice daily. More aggressive dosing was required to achieve higher PD targets. Among children with musculoskeletal infections, oral cephalexin and cefadroxil achieved PD targets for efficacy against MSSA. Given less frequent dosing, twice-daily cefadroxil should be further considered as an alternative to cephalexin for oral step-down therapy for serious infections due to MSSA.

A Critical Review of Cephalexin and Cefadroxil for the Treatment of Acute Uncomplicated Lower Urinary Tract Infection in the Era of "Bad Bugs, Few Drugs".

First-generation oral cephalosporins (cephalexin and cefadroxil) have traditionally been considered second-line treatment options for uncomplicated lower urinary tract infections (uLUTIs).  However, in the current age of "bad bugs, few drugs", where there are increasingly limited oral options against resistant Enterobacteriaceae, there is an urgent need to rethink how best to utilize the available antibiotic armamentarium.  This review examines the historical clinical trials and experimental studies of cephalexin and cefadroxil, particularly through the modern lens of pharmacokinetics/pharmacodynamics (PK/PD), to better appreciate the efficacy of these drugs in uLUTIs.  Furthermore, newer cefazolin-cephalexin surrogate testing, as recommended by the Clinical and Laboratory Standards Institute (CLSI) and the United States Committee on Antimicrobial Susceptibility Testing (USCAST), has recategorized cephalexin in many instances from resistant to susceptible.  We conclude that cephalexin and cefadroxil have very good early bacteriological and clinical cures in uLUTIs due to non-extended-spectrum beta-lactamase-producing (ESBL) Enterobacteriaceae comparable to many traditionally first-line agents.  Cephalexin can be conveniently administered as 500 mg twice or thrice daily, similar to cefadroxil (500 mg twice daily); therefore, either agent may be used as a fluoroquinolone-sparing alternative. Cephalexin may be the more practical choice for many clinicians because reliable antimicrobial susceptibility test interpretative criteria (STIC) are provided by CLSI, USCAST, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), whereas direct cefadroxil STIC is offered only by EUCAST.

In Silico Prediction of the Absorption and Disposition of Cefadroxil in Humans using an Intestinal Permeability Method Scaled from Humanized PepT1 Mice.

It is difficult to predict the pharmacokinetics and plasma concentration-time profiles of new chemical entities in humans based on animal data. Some pharmacokinetic parameters, such as clearance and volume of distribution, can be scaled allometrically from rodents, mammals, and nonhuman primates with good success. However, it is far more challenging to predict the oral pharmacokinetics of experimental drug candidates. In the present study, we used in situ estimates of intestinal permeability, obtained in silico and from rat, wild-type (WT), and humanized PepT1 (huPepT1) mice, to predict the systemic exposure of cefadroxil, an orally administered model compound, under a variety of conditions. Using the GastroPlus simulation software program (Simulations Plus, Lancaster, CA), we found that the C max and area under the plasma concentration-time curve from time zero to the last measurable concentration of cefadroxil were better predicted using intestinal permeability estimates (both segmental and jejunal) from huPepT1 than from WT mice, and that intestinal permeabilities based on in silico and rat estimates gave worse predictions. We also observed that accurate predictions were possible for cefadroxil during oral dose escalation (i.e., 5, 15, and 30 mg/kg cefadroxil), a drug-drug interaction study (i.e., 5 mg/kg oral cefadroxil plus 45 mg/kg oral cephalexin), and an oral multiple dose study [i.e., 500 mg (6.7 mg/kg) cefadroxil every 6 hours]. Finally, the greatest amount of cefadroxil was absorbed in duodenal and jejunal segments of the small intestine after a 5 mg/kg oral dose. Thus, by combining a humanized mouse model and in silico software, the present study offers a novel strategy for better translating preclinical pharmacokinetic data to oral drug exposure during first-in-human studies.

Controlled release of organic-inorganic nanohybrid:cefadroxil intercalated Zn-Al-layered double hydroxide.

BACKGROUND: The intercalation of an antibiotic drug, cefadroxil (CD), into the inter-gallery of Zn, Al nitrate-layered double hydroxide (LDH) was accomplished using a co-precipitation method. This formed a nanostructured organic-inorganic hybrid material that can be exploited for the preparation of a controlled release formulation. MATERIALS AND METHODS: The drug-LDH nanohybrid was characterized by using field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) thermogravimetric (TG) analysis, X-ray powder diffraction (XRD) and UV-visible (UV-vis) absorption spectroscopy, which confirmed the intercalation process. Release tests of nanohybrid in the presence or absence of NaCl or poly-acrylamide (PAM) were performed in vitro in gastric (pH 1.2), lysosomal (pH 4.0), intestinal (pH 6.8) and blood (pH 7.4) simulated fluid using UV-vis spectroscopy. RESULTS: At pH 1.2, LDH was dissolved and intercalated antibiotic released from ZnAl-CD in a molecular form, which led to a significant increase in the antibiotic's solubility. Results showed that the release of drug from nanohybrid at pH 4.0, 6.8 and 7.4 was a sustained process. CONCLUSION: This material might reduce side effects by the release of the drug in a controlled manner. However, it was found that the presence of Cl or PAM species in the release media has a negative impact on the release behavior. The weathering mechanism is responsible for the release of CD from the nanocomposite at pH 1.2, while the mechanism of anion exchange may be responsible for the release behavior at pH 4.0, 6.8 and 7.4. A number of kinetic models were chosen to gain more insights into the mechanisms of drug release. At pH 1.2, the zero-order model most satisfactorily explained the release kinetics of CD, while the release data of CD at pH 4.0, 6.8 and 7.4 were governed by Bhaskar kinetics.

Effect of Different Sampling Schedules on Results of Bioavailability and Bioequivalence Studies: Evaluation by Means of Monte Carlo Simulations.

Bioavailability and bioequivalence study is one of the most frequently performed investigations in clinical trials. Bioequivalence testing is based on the assumption that 2 drug products will be therapeutically equivalent when they are equivalent in the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed and becomes available at the site of drug action. In recent years there has been a significant growth in published papers that use in silico studies based on mathematical simulations to analyze pharmacokinetic and pharmacodynamic properties of drugs, including bioavailability and bioequivalence aspects. The goal of this study is to evaluate the usefulness of in silico studies as a tool in the planning of bioequivalence, bioavailability and other pharmacokinetic assays, e.g., to determine an appropriate sampling schedule. Monte Carlo simulations were used to define adequate blood sampling schedules for a bioequivalence assay comparing 2 different formulations of cefadroxil oral suspensions. In silico bioequivalence studies comparing different formulation of cefadroxil oral suspensions using various sampling schedules were performed using models. An in vivo study was conducted to confirm in silico results. The results of in silico and in vivo bioequivalence studies demonstrated that schedules with fewer sampling times are as efficient as schedules with larger numbers of sampling times in the assessment of bioequivalence, but only if Tmax is included as a sampling time. It was also concluded that in silico studies are useful tools in the planning of bioequivalence, bioavailability and other pharmacokinetic in vivo assays.

Tenapanor administration and the activity of the H+ -coupled transporter PepT1 in healthy volunteers.

AIM: Tenapanor (RDX5791/AZD1722), an inhibitor of gastrointestinal Na+ /H+ exchanger NHE3, is being evaluated for the treatment of patients with constipation-predominant irritable bowel syndrome and the treatment of hyperphosphataemia in patients with chronic kidney disease on dialysis. By reducing intestinal H+ secretion, inhibition of NHE3 by tenapanor could indirectly affect H+ -coupled transporter activity, leading to drug-drug interactions. We investigated the effect of tenapanor on the activity of the H+ -coupled peptide transporter PepT1 via assessment of the pharmacokinetics of cefadroxil - a compound transported by PepT1 - in healthy volunteers. METHODS: In this open-label, two-period crossover, phase 1 study (NCT02140281), 28 volunteers received in random order: a single dose of cefadroxil 500 mg for 1 day; and tenapanor 15 mg twice daily over 4 days followed by single doses of both cefadroxil 500 mg and tenapanor 15 mg on day 5. There was a 4-day washout between treatment periods. RESULTS: Cefadroxil exposure was similar when administered alone or in combination with tenapanor {geometric least-squares mean ratios [(cefadroxil + tenapanor)/cefadroxil] (90% confidence interval): area under the concentration-time curve 93.3 (90.6-96.0)%; maximum concentration in plasma 95.9 (89.8-103)%}. Tenapanor treatment caused a softening of stool consistency and an increase in stool frequency, consistent with its expected pharmacodynamic effect. No safety concerns were identified and tenapanor was not detected in plasma. CONCLUSIONS: These results suggest that tenapanor 15 mg twice daily does not have a clinically relevant impact on the activity of the H+ -coupled transporter PepT1 in humans. This may guide future research on drug-drug interactions involving NHE3 inhibitors.

Influence of peptide transporter 2 (PEPT2) on the distribution of cefadroxil in mouse brain: A microdialysis study.

Peptide transporter 2 (PEPT2) is a high-affinity low-capacity transporter belonging to the proton-coupled oligopeptide transporter family. Although many aspects of PEPT2 structure-function are known, including its localization in choroid plexus and neurons, its regional activity in brain, especially extracellular fluid (ECF), is uncertain. In this study, the pharmacokinetics and regional brain distribution of cefadroxil, a ß-lactam antibiotic and PEPT2 substrate, were investigated in wildtype and Pept2 null mice using in vivo intracerebral microdialysis. Cefadroxil was infused intravenously over 4h at 0.15mg/min/kg, and samples obtained from plasma, brain ECF, cerebrospinal fluid (CSF) and brain tissue. A permeability-surface area experiment was also performed in which 0.15mg/min/kg cefadroxil was infused intravenously for 10min, and samples obtained from plasma and brain tissues. Our results showed that PEPT2 ablation significantly increased the brain ECF and CSF levels of cefadroxil (2- to 2.5-fold). In contrast, there were no significant differences between wildtype and Pept2 null mice in the amount of cefadroxil in brain cells. The unbound volume of distribution of cefadroxil in brain was 60% lower in Pept2 null mice indicating an uptake function for PEPT2 in brain cells. Finally, PEPT2 did not affect the influx clearance of cefadroxil, thereby, ruling out differences between the two genotypes in drug entry across the blood-brain barriers. These findings demonstrate, for the first time, the impact of PEPT2 on brain ECF as well as the known role of PEPT2 in removing peptide-like drugs, such as cefadroxil, from the CSF to blood.

Comparative bioavailability and pharmacokinetic study of Cefadroxil capsules in male healthy volunteers of Pakistan.

The current study was aimed to judge bioequivalence between two formulations of cefadroxil capsules as guided by FDA guidelines. Another objective was to conduct pharmacokinetic evaluation in Pakistani population. A single-dose, randomized, cross-over pharmacokinetic study was conducted during the month of May'2013 to August'2013. Washout period was one week. Fourteen healthy male adult volunteers were enrolled in the study, however twelve completed the study. Cefadroxil plasma concentration was analyzed by using validated HPLC method. Protein precipitation was achieved by the addition of 6% tri chloro acetic acid in 1:1 ratio and detection was done at 260 nm. Retention time was 7.792 min and correlation coefficient (R2) was 0.9953 showing linearity of the method. Blood sampling was carried out at different time intervals after administration of either test (TEST 500 mg) or reference (REF® 500 mg) formulation. Pharmacokinetic parameters (AUC0→ ∞, AUC0→ t, Cmax, Tmax, t1/2 and kel) were calculated using Kinetica® PK/PD software. The geometric mean ratios and 90% confidence interval (CI) of these pharmacokinetic parameters for cefadroxil (test and reference) formulations were 0.986 (90.83-106.98%) for AUC0→ t; 0.967 (89.13-104.92%) for AUC0→ ∞ and 0.999 (91.06-109.69%) for Cmax. The differences between Tmax of both formulations were not found to be statistically significant (p-value was more than 0.05). The 90% CI of the test/reference AUC and Cmax ratio of cefadroxil were within the FDA recommended range for bioequivalence. Maximum plasma concentration Cmax was 12.5 µg/ml for test and 12.47 µg/ml for reference formulations. Average time to reach Cmax for test and reference formulation was 1.54 and 1.5 hrs. The two formulations of cefadroxil studied during the above study were verified bioequivalent. Maximum plasma concentration of cefadroxil was lower than those mentioned in some previous studies, while Tmax and half-life were near to values reported in literature.

Species differences in the pharmacokinetics of cefadroxil as determined in wildtype and humanized PepT1 mice.

PepT1 (SLC15A1) is a high-capacity low-affinity transporter that is important in the absorption of digested di/tripeptides from dietary protein in the small intestine. PepT1 is also crucial for the intestinal uptake and absorption of therapeutic agents such as the ß-lactam aminocephalosporins and antiviral prodrugs. Species differences, however, have been observed in PepT1-mediated intestinal absorption and pharmacokinetics, thereby, making it more difficult to predict systemic drug exposure. In the present study, we evaluated the in situ intestinal permeability of the PepT1 substrate cefadroxil in wildtype and humanized PepT1 (huPepT1) mice, and the in vivo absorption and disposition of drug after escalating oral doses. The in situ perfusions indicated that cefadroxil had a twofold higher affinity (i.e., twofold lower Km) for jejunal PepT1 in huPepT1 mice, lower but substantial permeability in all regions of the small intestine, and low but measureable permeability in the colon as compared to wildtype animals. The in vivo experiments indicated almost superimposable pharmacokinetic profiles between the two genotypes after intravenous bolus dosing of cefadroxil. In contrast, after oral dose escalation, the systemic exposure of cefadroxil was reduced in huPepT1 mice as compared to wildtype animals. Moreover, the AUC and Cmax versus dose relationships were nonlinear for huPepT1 but not wildtype mice, and similar to that observed from human subjects. In conclusion, our findings indicate that huPepT1 mice may provide a valuable tool in the drug discovery process by better predicting the oral pharmacokinetic profiles of PepT1 substrates in humans.

Population pharmacokinetic modeling of cefadroxil renal transport in wild-type and Pept2 knockout mice.

1. Cefadroxil is a broad-spectrum ß-lactam antibiotic that is widely used in the treatment of various infectious diseases. Currently, poor understanding of the drug's pharmacokinetic profiles and disposition mechanism(s) prevents determining optimal dosage regimens and achieving ideal antibacterial responses in patients. In the present retrospective study, we developed a population pharmacokinetic model of cefadroxil in wild-type and Pept2 knockout mice using the nonlinear mixed effect modeling (NONMEM) approach. 2. Cefadroxil pharmacokinetics were best described by a two-compartment model, with both saturable and nonsaturable elimination processes to/from the central compartment. Through this modeling approach, pharmacokinetic parameters in wild-type and Pept2 knockout mice were well estimated, respectively, as follows: volume of central compartment V1 (3.43 versus 4.23 mL), volume of peripheral compartment V2 (5.98 versus 8.61 mL), intercompartment clearance Q (0.599 versus 0.586 mL/min) and linear elimination rate constant K10 (0.111 versus 0.070 min(-1)). Moreover, the secretion kinetics (i.e. V(m1) = 17.6 nmoL/min and K(m1) = 37.1 µM) and reabsorption kinetics (i.e. V(m2) = 15.0 nmoL/min and K(m2) = 27.1 µM) of cefadroxil were quantified in kidney, for the first time, under in vivo conditions. 3. Our model provides a unique tool to quantitatively predict the dose-dependent nonlinear disposition of cefadroxil, as well as the potential for transporter-mediated drug interactions.

Effects of 1α,25-dihydroxyvitamin D3 , the natural vitamin D receptor ligand, on the pharmacokinetics of cefdinir and cefadroxil, organic anion transporter substrates, in rat.

Evidence in the literature suggests that 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], the vitamin D receptor ligand, down-regulated the expression of the rat renal organic anion (renal organic anion transporter, rOAT) and oligopeptide (rPEPT) transporters, but increased intestinal rPEPT1 expression. We investigated, in rats, the intravenous and oral pharmacokinetics of 2 mg/kg cefdinir and cefadroxil, two cephalosporins that are eliminated via renal OAT1/OAT3 and are substrates of PEPT1/PEPT2, with and without 1,25(OH)2 D3 treatment. The area under the plasma concentration-time curve (AUC) of cefdinir or cefadroxil after 1,25(OH)2 D3 treatment was increased significantly because of decreased clearance (CL). Both kidney uptake and cumulative urinary recovery were significantly decreased, whereas liver uptake and fecal recovery remained unchanged in 1,25(OH)2 D3 -treated rats. Similar changes in AUC and CL were observed for both drugs upon coadministration of probenecid, the OAT inhibitor. Oral availability of cefdinir and cefadroxil remained unchanged with 1,25(OH)2 D3 treatment, suggesting lack of a role for intestinal rPEPT1. Rather, reduction of rOAT1/rOAT3 mRNA expression in kidney with 1,25(OH)2 D3 -treatment was observed, confirmed by decreased function in MDCKII cells overexpressing human OAT1 and OAT3. These composite results suggest that 1,25(OH)2 D3 treatment reduces cefdinir and cefadroxil clearances by diminution of renal OAT1/OAT3 expression, implicating a role for 1,25(OH)2 D3 in eliciting transporter-based drug interactions.

Determination of cefadroxil in rat plasma and urine using LC-MS/MS and its application to pharmacokinetic and urinary excretion studies.

A simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of cefadroxil, a first-generation cephalosporin, in rat plasma and urine. Rat samples were deproteinized with methanol, and then injected into the LC-MS/MS system (electro-spray ionization, positive mode) for quantification. Drugs were separated on a Synergi™ 4 µm Polar-RP 80A column (150 mm × 2.0 mm, 4 µm) with a mixture of 0.1% formic acid and methanol (62:38, v/v) as the mobile phase at 0.2 mL/min. Detection was performed using multiple reaction-monitoring modes at m/z 364.1→208.1 (for cefadroxil) and m/z 368.1→174.2 (for cefaclor, the internal standard). Method was specific and linear over the concentration range of 10-10,000 ng/mL. Validation parameters for cefadroxil, including accuracy, precision, absolute matrix effect, and stability in rat plasma and urine, were acceptable according to the biological method validation guidelines of the FDA (2001) [16]. Cefadroxil levels in plasma up to 1440 min or 480 min and urine up to 96 h were quantifiable following oral and intravenous cefadroxil administrations to rats at a dose of 2mg/kg, each, suggesting that the method is appropriate for routine pharmacokinetic studies including urinary recovery in rats.

Impact of lipopolysaccharide-induced inflammation on the disposition of the aminocephalosporin cefadroxil.

The purpose of this study was to determine if the disposition of cefadroxil, an α-amino-containing ß-lactam antibiotic, changes during lipopolysaccharide (LPS)-induced acute inflammation. Six hours after LPS or saline treatment, mice received 1 nmol/g cefadroxil intravenously along with inulin for glomerular filtration rate (GFR) determination. Serial blood samples, along with tissue and urine samples, were collected at predetermined time points. In order to determine inflammation-induced changes in GFR, renal tubular secretion, and reabsorption, it was necessary to coadminister 70 mg/kg probenecid. Changes in the expression of the mRNA of transporters involved in cefadroxil disposition in the kidneys and choroid plexus were also investigated 6 h after LPS treatment. The results demonstrated marked increases in blood, cerebrospinal fluid, and tissue cefadroxil concentrations with LPS treatment. Tissue-to-blood concentration ratios were decreased by 4.6-fold in the choroid plexus and by 2.5-fold in the kidneys during LPS-induced inflammation. Renal, but not choroid plexus, mRNA expression of peptide transporter 2, organic-anion transporter 1 (OAT1), OAT3, and multidrug resistance-associated protein 4 was mildly reduced in LPS-treated mice. The renal clearance of cefadroxil was substantially decreased by LPS treatment (3-fold). GFR was also reduced by 3-fold in LPS-treated mice, but no significant differences in the fractional reabsorption of cefadroxil and renal secretion once normalized by GFR were observed. These findings demonstrated that LPS-induced inflammation has a dramatic effect on the renal excretion of cefadroxil. It appears that changes in transporter expression played a minor role during LPS treatment but that renal dysfunction, associated with GFR reduction, was responsible for the substantial increase in plasma cefadroxil concentration-time profiles.

In vivo absorption and disposition of cefadroxil after escalating oral doses in wild-type and PepT1 knockout mice.

PURPOSE: To determine the effect of PepT1 on the absorption and disposition of cefadroxil, including the potential for saturable intestinal uptake, after escalating oral doses of drug. METHODS: The absorption and disposition kinetics of [3H]cefadroxil were determined in wild-type and PepT1 knockout mice after 44.5, 89.1, 178, and 356 nmol/g oral doses of drug. The pharmacokinetics of [3H]cefadroxil were also determined in both genotypes after 44.5 nmol/g intravenous bolus doses. RESULTS: PepT1 deletion reduced the area under the plasma concentration-time profile (AUC0-120) of cefadroxil by 10-fold, the maximum plasma concentration (Cmax) by 17.5-fold, and increased the time to reach a maximum plasma concentration (Tmax) by 3-fold. There was no evidence of nonlinear intestinal absorption since AUC0-120 and Cmax values changed in a dose-proportional manner. Moreover, the pharmacokinetics of cefadroxil were not different between genotypes after intravenous bolus doses, indicating that PepT1 did not affect drug disposition. Finally, no differences were observed in the peripheral tissue distribution of cefadroxil (i.e., outside gastrointestinal tract) once these tissues were corrected for differences in perfusing blood concentrations. CONCLUSIONS: The findings demonstrate convincingly the critical role of intestinal PepT1 in both the rate and extent of oral administration for cefadroxil and potentially other aminocephalosporin drugs.

Relevance of PepT1 in the intestinal permeability and oral absorption of cefadroxil.

PURPOSE: To determine the contribution of intestinal PepT1 on the permeability and oral absorption of the ß-lactam antibiotic drug cefadroxil. METHODS: The effective permeability (P eff ) of cefadroxil was evaluated in wild-type and PepT1 knockout mice following in situ single-pass intestinal perfusions. The plasma concentration-time profiles of cefadroxil were also examined after oral gavage. RESULTS: The P eff (cm/s) of cefadroxil in wild-type mice was 0.49 × 10(-4) in duodenum, 0.80 × 10(-4) in jejunum, 0.88 × 10(-4) in ileum and 0.064 × 10(-4) in colon. The P eff (cm/s) in PepT1 knockout mice was significantly reduced in small intestine, but not in colon, as shown by values of 0.003 × 10(-4), 0.090 × 10(-4), 0.042 × 10(-4) and 0.032 × 10(-4), respectively. Jejunal uptake of cefadroxil was saturable (Km = 2-4 mM) and significantly attenuated by the sodium-proton exchange inhibitor 5-(N,N-dimethyl)amiloride. Jejunal permeability of cefadroxil was not affected by L-histidine, glycine, cephalothin, p-aminohippurate or N-methylnicotinamide. In contrast, cefadroxil permeability was significantly reduced by glycylproline, glycylsarcosine, or cephalexin. Finally, PepT1 ablation resulted in 23-fold reductions in peak plasma concentrations and 14-fold reductions in systemic exposure of cefadroxil after oral dosing. CONCLUSIONS: The findings are definitive in demonstrating that PepT1 is the major transporter responsible for the small intestinal permeability of cefadroxil as well as its enhanced oral drug performance.

Oral availability of cefadroxil depends on ABCC3 and ABCC4.

Some cephalosporins, such as cefadroxil, are orally available. H(+)-coupled peptide transporter 1 mediates the transport of cephalosporins across the apical membrane of enterocytes. It is not known which mechanism(s) is responsible for the subsequent transport of cephalosporins across the basolateral membrane toward the circulation. We have tested whether ATP-binding cassette (ABC) transporters ABCC3 and/or ABCC4 are involved in the latter process. Transport experiments with plasma membrane vesicles expressing these transporters were used to determine whether ABCC3 and ABCC4 can transport cephalosporins in vitro. The involvement of Abcc3 and Abcc4 in the transport of cefadroxil from enterocytes was subsequently studied using intestinal explants from wild-type, Abcc3(-/-), Abcc4(-/-), and Abcc3(-/-)/Abcc4(-/-) mice in an Ussing chamber setup. Finally, appearance of cefadroxil in portal blood was investigated in vivo after intrajejunal administration of cefadroxil in wild-type, Abcc3(-/-), Abcc4(-/-), and Abcc3(-/-)/Abcc4(-/-) mice. ABCC3- and ABCC4-mediated transport of estradiol-17ß-glucuronide was dose-dependently inhibited by cephalosporins in vesicular transport experiments. Furthermore, transport of cefadroxil by ABCC3 and ABCC4 was saturable with K(m) values of 2.5 ± 0.7 and 0.25 ± 0.07 mM, respectively. Transport of cefadroxil from the apical to the basolateral side of jejunal tissue explants was unchanged in Abcc3(-/-) but significantly reduced (approximately 2-fold) in Abcc4(-/-) and Abcc3(-/-)/Abcc4(-/-) when compared with wild-type tissue. Upon instillation of cefadroxil in the jejunum, portal and peripheral blood concentrations were similar in Abcc3(-/-) and Abcc4(-/-) but approximately 2-fold reduced in Abcc3(-/-)/Abcc4(-/-) compared with wild-type mice. Our data demonstrate that intestinal absorption of cefadroxil depends partly on ABCC3 and ABCC4.

Distribution of glycylsarcosine and cefadroxil among cerebrospinal fluid, choroid plexus, and brain parenchyma after intracerebroventricular injection is markedly different between wild-type and Pept2 null mice.

The purpose of this study was to define the cerebrospinal fluid (CSF) clearance kinetics, choroid plexus uptake, and parenchymal penetration of PEPT2 substrates in different regions of the brain after intracerebroventricular administration. To accomplish these objectives, we performed biodistribution studies using [(14)C]glycylsarcosine (GlySar) and [(3)H]cefadroxil, along with quantitative autoradiography of [(14)C]GlySar, in wild-type and Pept2 null mice. We found that PEPT2 deletion markedly reduced the uptake of GlySar and cefadroxil in choroid plexuses at 60 mins by 94% and 82% (P<0.001), respectively, and lowered their CSF clearances by about fourfold. Autoradiography showed that GlySar concentrations in the lateral, third, and fourth ventricle choroid plexuses were higher in wild-type as compared with Pept2 null mice (P<0.01). Uptake of GlySar by the ependymal-subependymal layer and septal region was higher in wild-type than in null mice, but the half-distance of penetration into parenchyma was significantly less in wild-type mice. The latter is probably because of the clearance of GlySar from interstitial fluid by brain cells expressing PEPT2, which stops further penetration. These studies show that PEPT2 knockout can significantly modify the spatial distribution of GlySar and cefadroxil (and presumably other peptides/mimetics and peptide-like drugs) in brain.

Role and relevance of PEPT2 in drug disposition, dynamics, and toxicity.

Pept2 knockout mice are an important tool to evaluate the evolving role and relevance of this proton-coupled oligopeptide transporter beyond drug disposition, where the transporter also modulates the pharmacodynamic and toxicodynamic effects of drug substrates. Our in vivo studies with glycylsarcosine in Pept2 knockout mice have established "proof of concept" that PEPT2 can have a significant effect on dipeptide disposition. Subsequent studies with the aminocephalosporin antibiotic cefadroxil have shown relevance to pharmacology and infectious disease. Finally, studies with the endogenous peptidomimetic 5-aminolevulinic acid have demonstrated relevance to toxicology in the framework of porphyria- and lead-induced neurotoxicity. These studies have consistently demonstrated the dual action of PEPT2 with respect to its apical localization in choroid plexus epithelium and kidney in: 1) effluxing substrates from CSF into choroid plexus, thereby affecting regional pharmacokinetics in brain; and 2) reabsorbing substrates from renal tubular fluid into proximal tubules, thereby affecting systemic pharmacokinetics and exposure. Moreover, these studies have shown that the regional effect of PEPT2 in limiting substrate concentrations in the CSF is more dramatic than its effect in increasing systemic exposure. In the case of 5-aminolevulinic acid, such regional modulation of drug disposition translates directly into significant changes in neurotoxicity.