Irinotecan-induced gastrointestinal damage alters the expression of peptide transporter 1 and absorption of cephalexin in rats.

Irinotecan causes severe gastrointestinal damage, which may affect the expression of intestinal transporters. However, neither the expression of peptide transporter 1 (Pept1) nor the pharmacokinetics of Pept1 substrate drugs has been investigated under irinotecan-induced gastrointestinal damage. Therefore, the present study quantitatively investigated the effects of irinotecan-induced gastrointestinal damage on the intestinal expression of Pept1 and absorption of cephalexin (CEX), a typical Pept1 substrate, in rats. Irinotecan was administered intravenously to rats for 4 days to induce gastrointestinal damage. The expression of Pept1 mRNA and the Pept1 protein in the upper, middle, and lower segments of the small intestine of irinotecan-treated rats was assessed by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. The pharmacokinetic profile of CEX was examined after its oral or intravenous administration (10 mg/kg). In irinotecan-treated rats, ∼2-fold increases in Pept1 protein levels were observed in all three segments, whereas mRNA levels remained unchanged. The oral bioavailability of CEX significantly decreased to 76% of that in control rats. The decrease in passive diffusion caused by intestinal damage may have overcome the increase in Pept1-mediated uptake. In conclusion, irinotecan may decrease the intestinal absorption of Pept1 substrate drugs; however, it increased the expression of intestinal Pept1.

The synergistic mechanism of ß-lactam antibiotic removal between ammonia-oxidizing microorganisms and heterotrophs.

Nitrifying system is an effective strategy to remove numerous antibiotics, however, the contribution of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and heterotrophs for antibiotic removal are still unclear. In this study, the mechanism of ß-lactam antibiotic (cefalexin, CFX) removal was studied in a nitrifying sludge system. Results showed that CFX was synergistically removed by AOB (Nitrosomonas, played a major role) and AOA (Candidatus_Nitrososphaera) through ammonia monooxygenase-mediated co-metabolism, and by heterotrophs (Pseudofulvimonas, Hydrogenophaga, RB41, Thauera, UTCFX1, Plasticicumulans, Phaeodactylibacter) through antibiotic resistance genes (ARGs)-encoded ß-lactamases-mediated hydrolysis. Regardless of increased archaeal and heterotrophic CFX removal with the upregulation of amoA in AOA and ARGs, the system exhibited poorer CFX removal performance at 10 mg/L, mainly due to the inhibition of AOB. This study provides new reference for the important roles of heterotrophs and ARGs, opening the possibilities for the application of ARGs in antibiotic biodegradation.

Drug in adhesive transdermal patch containing antibiotic-loaded solid lipid nanoparticles.

The structure of the skin only allows those hydrophobic elements to penetrate through the depth of the skin with low molecular weight (less than 500 Da) and low daily dose (less than 100 mg/day). Skin penetration of many drugs such as antibiotics at a high daily dose remains an unresolved challenge. In this study a transdermal patch using cephalexin as an antibiotic drug model was developed. Cephalexin was loaded into α-tocopherol succinate-based solid lipid nanoparticles (SLNs). Cephalexin-loaded SLNs with a drug/lipid ratio of 20%, diameter of 180 ± 7 nm, and drug loading 7.9% led to the greatest inhibition zone of Staphylococcus aureus and showed the highest skin permeation capabilities. Cephalexin-loaded SLNs were distributed into poly-iso-butylene adhesive solution and final patches prepared using solvent casting. The physico-chemical characteristics, in vitro drug release, antimicrobial efficacy, and skin cell proliferation properties of patches were evaluated. Results indicated that the optimal transdermal patch formulation containing 90% adhesive solution, 7% cephalexin, and 3% cephalexin-loaded SLNs (with antibiotic content approximately 28% less) inhibited growth of S.aureus better than the formulation containing 90% adhesive solution and 10% cephalexin. In vitro evaluation of the growth of human fibroblast skin cells in media with the optimal patch exhibited greater proliferation (about 25.5%) than those in media without the patch.

Selectivity and kinetic modeling of penicillin G acylase variants for the synthesis of cephalexin under a broad range of substrate concentrations.

The kinetics of cephalexin synthesis and hydrolysis of the activated acyl-donor precursor phenylglycine methyl ester (PGME) were characterized under a broad range of substrate concentrations. A previously developed model by Youshko-Svedas involving the formation of the acyl-enzyme complex followed by binding of the nucleophilic ß-lactam donor does not fully estimate the maximum reaction yields for cephalexin synthesis at different concentrations using initial-rate data. 7-aminodesacetoxycephalosporanic acid (7-ADCA) was discovered to be a potent inhibitor of cephalexin hydrolysis, which may account for the deviation from model predictions. Three kinetic models were compared for cephalexin synthesis, with the model incorporating competitive inhibition due to 7-ADCA yielding the best fit. Additionally, the ßF24A variant and Assemblase® did not exhibit significantly different kinetics for the synthesis of cephalexin compared to the wild-type, for the concentration range evaluated and for both initial-rate experiments and time-course synthesis experiments. Lastly, a continuous stirred-tank reactor for cephalexin synthesis was simulated using the model incorporating competitive inhibition by 7-ADCA, with clear tradeoffs observed between productivity, fractional yield, and PGME conversion.

Effect of Excessive Serotonin on Pharmacokinetics of Cephalexin after Oral Administration: Studies with Serotonin-Excessive Model Rats.

PURPOSE: Serotonin (5-HT) is important for gastrointestinal functions, but its role in drug absorption remains to be clarified. Therefore, the pharmacokinetics and oral absorption of cephalexin (CEX) were examined under 5-HT-excessive condition to understand the role of 5-HT. METHODS: 5-HT-excessive rats were prepared by multiple intraperitoneal dosing of 5-HT and clorgyline, an inhibitor for 5-HT metabolism, and utilized to examine the pharmacokinetics, absorption behavior and the intestinal permeability for CEX. RESULTS: Higher levels of 5-HT in brain, plasma and small intestines were recognized in 5-HT-excessive rats, where the oral bioavailability of CEX was significantly enhanced. The intestinal mucosal transport via passive diffusion of CEX was significantly increased, while its transport via PEPT1 was markedly decreased specifically in the jejunal segment, which was supported by the decrease in PEPT1 expression on brush border membrane (BBM) of intestinal epithelial cells. Since no change in antipyrine permeability and significant increase in FITC dextran-4 permeability were observed in 5-HT-excessive rats, the enhanced permeability for CEX would be attributed to the opening of tight junction, which was supported by the significant decrease in transmucosal electrical resistance. In 5-HT-excessive rats, furthermore, total body clearance of CEX tended to be larger and the decrease in PEPT2 expression on BBM in kidneys was suggested to be one of the reasons for it. CONCLUSIONS: 5-HT-excessive condition enhanced the oral bioavailability of CEX in rats, which would be attributed to the enhanced permeability across the intestinal mucosa via passive diffusion through the paracellular route even though the transport via PEPT1 was decreased.

Diabetes downregulates peptide transporter 1 in the rat jejunum: possible involvement of cholate-induced FXR activation.

Peptide transporter 1 (PepT1), highly expressed on the apical membrane of enterocytes, is involved in energy balance and mediates intestinal absorption of peptidomimetic drugs. In this study, we investigated whether and how diabetes affected the function and expression of intestinal PepT1. Diabetes was induced in rats by combination of high-fat diet and low dose streptozocin injection. Pharmacokinetics study demonstrated that diabetes significantly decreased plasma exposures of cephalexin and acyclovir following oral administration of cephalexin and valacyclovir, respectively. Single-pass intestinal perfusion analysis showed that diabetes remarkably decreased cephalexin absorption, which was associated with decreased expression of intestinal PepT1 protein. We assessed the levels of bile acids in intestine of diabetic rats, and found that diabetic rats exhibited significantly higher levels of chenodeoxycholic acid (CDCA), cholic acid (CA) and glycocholic acid (GCA), and lower levels of lithocholic acid (LCA) and hyodeoxycholic acid (HDCA) than control rats; intestinal deoxycholic acid (DCA) levels were unaltered. In Caco-2 cells, the 6 bile acids remarkably decreased expression of PepT1 protein with CDCA causing the strongest inhibition, whereas TNF-α, LPS and insulin little affected expression of PepT1 protein; short-chain fatty acids induced rather than decreased expression of PepT1 protein. Farnesoid X receptor (FXR) inhibitor glycine-ß-muricholic acid or FXR knockdown reversed the downregulation of PepT1 expression by CDCA and GW4064 (another FXR agonist). In diabetic rats, the expression of intestinal FXR protein was markedly increased. Oral administration of CDCA (90, 180 mg·kg-1·d-1, for 3 weeks) dose-dependently decreased the expression and function of intestinal PepT1 in rats. In conclusion, diabetes impairs the expression and function of intestinal PepT1 partly via CDCA-mediated FXR activation.

Adsorption of cephalexin in aqueous media by graphene oxide: kinetics, isotherm, and thermodynamics.

The present study proposes the synthesis and characterization of graphene oxide (GO) and its application in the adsorption of the antibiotic cephalexin (CFX) in aqueous solution. The characterization of graphene oxide was obtained by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and zeta potential. The influence of pH on the batch adsorption process was investigated by analysing adsorption equilibrium isotherms and adsorption kinetics. The images obtained by SEM and TEM presented the typical morphology attributed to GO sheets. The kinetic adsorption tests showed that equilibrium was reached in 420 min, and an adsorption capacity of 164 mg g-1 was obtained. The models that best fit the experimental data were pseudo-second as well as the Langmuir isotherm. Therefore, GO was effective for removing the CFX antibiotic from aqueous solution by using a batch adsorption process.

Cometabolic biodegradation of cephalexin by enriched nitrifying sludge: Process characteristics, gene expression and product biotoxicity.

The nitrifying systems have been reported to be able to biodegrade micropollutants, yet it is still unclear about the cometabolism of ammonia-oxidizing bacteria (AOB) towards micropollutants, in particular their enzyme and transcriptional responses under exposure of micropollutants. This study investigated cometabolic biodegradation of a selected antibiotic, cephalexin (CFX), by an enriched nitrifying culture through a series of batch experiments, together with the assessments of enzymatic activity, key gene expression, and biotoxicity of the degradation products. More than 99% CFX with an initial concentration of 50 µg/L could be removed with the presence of ammonium, while <44% of CFX removal was observed in the absence of ammonium, suggesting the cometabolic degradation of CFX by ammonia-oxidizing bacteria (AOB). After the addition of 50 µg/L CFX, the ammonia oxidizing rate (AOR) decreased from 36.6 to 11.0 mg N/(L·h·g VSS), followed by a slight recovery when CFX concentration decreased to below 8 µg/L. Ammonia monooxygenase (AMO) activity showed a similar trend with that of AOR. The quantitative reverse transcription PCR assay indicated that the expression level of amoA gene was significantly upregulated (up to 3-fold, p < 0.05) due to the addition of CFX, while decreased to the normal level once CFX was degraded, suggesting a mechanism of AOB to neutralize the toxicity of CFX by metabolizing ammonia more effectively. Meanwhile, the biotoxicity test showed the degradation products of CFX did not exhibit any antibacterial impacts in terms of cell viability, compared to the parent compounds. Our finding shed a light on AMO-mediated cometabolic biodegradation of antibiotics in nitrifying cultures.

Optimizing of pharmaceutical active compounds biodegradability in secondary effluents by ß-lactamase from Bacillus subtilis using central composite design.

Biodegradation of pharmaceuticals active compounds (PACs) in secondary effluents by using B. subtilis 2012WTNC as a function of ß-lactamase was optimized using response surface methodology (RSM) designed by central composite design (CCD). Four factors including initial concentration of bacteria (1-6 log10 CFU mL-1), incubation period (1-14 days), incubation temperature (20-40 °C) and initial concentration of PACs (1-5 mg L-1) were investigated. The optimal operating factors for biodegradation process determined using response surface methodology (RSM) was recorded with 5.57 log10 CFU mL-1 of B. subtilis, for 10.38 days, at 36.62 °C and with 4.14 mg L-1 of (cephalexin/amoxicillin) with R2 coefficient of 0.99. The biodegradation was 83.81 and 93.94% respectively. The relationship among the independent variables was significant (p < 0.05) with 95% of confidence level at the best operating parameters. The bioassay for PACs after the degradation process revealed that no residual antibiotic activity was detected of amoxicillin and cephalexin against E. coli and S. aureus after degradation using B. subtilis which reflects the higher potential of bacteria to biodegrade PACs in secondary effluents. B. subtilis has the potential for biodegradation of PACs in the secondary effluents.

Draft genome sequence of Xenophilus sp. E41 isolated from an activated sludge reactor treating wastewater with high cephalexin concentration.

OBJECTIVES: This study reports the draft genome sequence of Xenophilus sp. E41, a strain resistant to multiple antimicrobials isolated from an activated sludge reactor treating wastewater with a high cephalexin concentration. METHODS: Genomic DNA of Xenophilus sp. E41 was extracted and sequenced using an Illumina NovaSeq 6000 system. The generated sequence reads were assembled using MEGAHIT in combination with SOAPdenovo. Mauve and CompareM were used to align the Xenophilus sp. E41 genome to other draft genomes of the genus Xenophilus in order to determine their evolutionary relationships. The draft genome was annotated using the Rapid Annotation using Subsystem Technology (RAST) server and the nr database, whilst antimicrobial resistance genes (ARGs) were identified using the SARG 2.0 database, RAST server and nr database. RESULTS: Xenophilus sp. E41, with a genome length of 5919552bp, harbours seven types of ARGs involving resistance to ß-lactams, tetracycline, aminoglycosides, sulfonamides, chloramphenicol, teicoplanin and bleomycin. No virulence factors or plasmids were identified. CONCLUSION: The genome sequence reported here will provide useful information for a better understanding of antimicrobial resistance profiles in this strain and the genus Xenophilus.

Hydrolysis of cephalexin and meropenem by New Delhi metallo-ß-lactamase: the substrate protonation mechanism is drug dependent.

Emergence of antibiotic resistance due to New Delhi metallo-ß-lactamase (NDM-1) bacterial enzymes is of great concern due to their ability to hydrolyze a wide range of antibiotics. There are ongoing efforts to obtain the atomistic details of the hydrolysis mechanism in order to develop inhibitors for NDM-1. In particular, it remains elusive how drug molecules of different families of antibiotics are hydrolyzed by NDM-1 in an efficient manner. Here we report the detailed molecular mechanism of NDM-1 catalyzed hydrolysis of cephalexin, a cephalosporin family drug, and meropenem, a carbapenem family drug. This study employs molecular dynamics (MD) simulations using hybrid quantum mechanical/molecular mechanical (QM/MM) methods at the density functional theory (DFT) level, based on which reaction pathways and the associated free energies are obtained. We find that the mechanism and the free energy barrier for the ring-opening step are the same for both the drug molecules, while the subsequent protonation step differs. In particular, we observe that the mechanism of the protonation step depends on the R2 group of the drug molecule. Our simulations show that allylic carbon protonation occurs in the case of the cephalexin drug molecule where Lys211 is the proton donor, and the proton transfer occurs via a water chain formed (only) at the ring-opened intermediate structure. Based on the free energy profiles, the overall kinetics of drug hydrolysis is discussed. Finally, we show that the proposed mechanisms and free energy profiles could explain various experimental observations.

Binding of TEM-1 beta-lactamase to beta-lactam antibiotics by frontal affinity chromatography.

TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278-288K under simulated physiological conditions. The results showed that all the binding of TEM-1 beta-lactamase to the three antibiotics were spontaneously exothermic processes with the binding constants of 8.718×103, 6.624×103 and 2.244×103 (mol/L), respectively at 288K. All the TEM-1 beta-lactamases were immobilized on the surface of the stationary phase in the mode of monolayer and there existed only one type of binding sites on them. Each TEM-1 beta-lactamase bound with only one beta-lactam antibiotic and hydrogen bond interaction and Van der Waals force were the main forces between them. This work provided an insight into the binding interactions between TEM-1 beta-lactamases and beta-lactam antibiotics, which may be beneficial for the designing and developing of new substrates resistant to TEM-1 beta-lactamases.

Recognition and binding of ß-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical ß-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three ß-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with ß-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties.

Exploring the potential impact of an expanded genetic code on protein function.

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 ß-lactamase. A screen for growth on the ß-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acyl-enzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

Characterization of cefalexin degradation capabilities of two Pseudomonas strains isolated from activated sludge.

Pharmaceuticals have recently been regarded as contaminants of emerging concern. To date, there is limited knowledge about antibiotic-degrading microorganisms in conventional activated sludge treatment systems and their characteristics toward antibiotic degradation especially in the presence of a pharmaceutical mixture. As such, antibiotic-degrading microorganisms were investigated and isolated from the activated sludge, and their degradation capabilities were evaluated. Two strains of cefalexin-degrading bacteria CE21 and CE22 were isolated and identified as Pseudomonas sp. in the collected activated sludge. Strain CE22 was able to degrade over 90% of cefalexin, while CE21 was able to remove 46.7% of cefalexin after incubation for 24h. The removal efficiency of cefalexin by CE22, different from that of CE21, was not significantly affected by an increase in cefalexin concentration, even up to 10ppm, however the presence of 1ppm of other pharmaceuticals had a significant effect on the degradation of cefalexin by CE22, but no significant effect on CE21. The degradation product of cefalexin by the two strains was identified to be 2-hydroxy-3-phenyl pyrazine. Our results also indicated that CE21 and CE22 were able to degrade caffeine, salicylic acid and chloramphenicol. Moreover, CE21 was found to be capable of eliminating sulfamethoxazole and naproxen.

Polymer nanoreactors shown to produce and release antibiotics locally.

We designed and prepared nanoreactors based on a poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymer encapsulating the enzyme penicillin acylase for local and controlled production of antibiotics.

Crystal structures of penicillin-binding protein 3 (PBP3) from methicillin-resistant Staphylococcus aureus in the apo and cefotaxime-bound forms.

Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the ß-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin-binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of "dimerization domain", the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.

Biodegradability enhancement of wastewater containing cefalexin by means of the electro-Fenton oxidation process.

The comparative degradation behavior of cefalexin (CLX) using advanced oxidation processes (AOPs) with the aim of improving CLX biodegradability was studied. Among the AOPs used, RuO(2)/Ti anodic oxidation (AO), AO in the presence of electro-generated H(2)O(2) (AO-H(2)O(2)), and the electro-Fenton (EF) process, the EF process was the most effective. In the EF process an activated carbon fiber (ACF) was used as a cathode. Different input variables, including catalyst concentration, pH, and current density were evaluated to find the optimum conditions for the EF. The most suitable operational conditions were as follows: a current density of 6.66 mA/cm(2), a pH of 3, and a concentration of 1 mM of Fe(2+) as the catalyst. Different CLX concentrations were analyzed as well of different reaction times to assess the degree of mineralization. The change in biodegradability was evaluated by the BOD(5)/COD and the BOD(14)/COD ratios. The EF did not effectively remove the COD, but removed enough to achieve suitable biodegradability for a further biological process.

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2.

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.

Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.

Cimetidine, an H2 receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 µM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 µM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (µM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 µM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 µM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.