Novel carbapenem antibiotics for parenteral and oral applications: in vitro and in vivo activities of 2-aryl carbapenems and their pharmacokinetics in laboratory animals.

SM-295291 and SM-369926 are new parenteral 2-aryl carbapenems with strong activity against major causative pathogens of community-acquired infections such as methicillin-susceptible Staphylococcus aureus, Streptococcus pneumoniae (including penicillin-resistant strains), Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Moraxella catarrhalis, Haemophilus influenzae (including ß-lactamase-negative ampicillin-resistant strains), and Neisseria gonorrhoeae (including ciprofloxacin-resistant strains), with MIC(90)s of ≤ 1 µg/ml. Unlike tebipenem (MIC(50), 8 µg/ml), SM-295291 and SM-369926 had no activity against hospital pathogens such as Pseudomonas aeruginosa (MIC(50), ≥ 128 µg/ml). The bactericidal activities of SM-295291 and SM-369926 against penicillin-resistant S. pneumoniae and ß-lactamase-negative ampicillin-resistant H. influenzae were equal or superior to that of tebipenem and greater than that of cefditoren. The therapeutic efficacies of intravenous administrations of SM-295291 and SM-369926 against experimentally induced infections in mice caused by penicillin-resistant S. pneumoniae and ß-lactamase-negative ampicillin-resistant H. influenzae were equal or superior to that of tebipenem and greater than that of cefditoren, respectively, reflecting their in vitro activities. SM-295291 and SM-369926 showed intravenous pharmacokinetics similar to those of meropenem in terms of half-life in monkeys (0.4 h) and were stable against human dehydropeptidase I. SM-368589 and SM-375769, which are medoxomil esters of SM-295291 and SM-369926, respectively, showed good oral bioavailability in rats, dogs, and monkeys (4.2 to 62.3%). Thus, 2-aryl carbapenems are promising candidates that show an ideal broad spectrum for the treatment of community-acquired infections, including infections caused by penicillin-resistant S. pneumoniae and ß-lactamase-negative ampicillin-resistant H. influenzae, have low selective pressure on antipseudomonal carbapenem-resistant nosocomial pathogens, and allow parenteral, oral, and switch therapies.

Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19.

Toxic and pharmacokinetic profiles of drug candidates are evaluated in vivo often using monkeys as experimental animals, and the data obtained are extrapolated to humans. Well understanding physiological properties, including drug-metabolizing enzymes, of monkeys should increase the accuracy of the extrapolation. The present study was performed to compare regio- and stereoselectivity in the oxidation of propranolol (PL), a chiral substrate, by cytochrome P450 2D (CYP2D) enzymes among humans, cynomolgus monkeys and marmosets. Complimentary DNAs encoding human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19 were cloned, and their proteins expressed in a yeast cell expression system. The regio- and stereoselective oxidation of PL enantiomers by yeast cell microsomal fractions were compared. In terms of efficiency of expression in the system, the holo-proteins ranked CYP2D6=CYP2D17>>CYP2D19. This may be caused by the bulky side chain of the amino acid residue at position 119 (leucine for CYP2D19 vs. valine for CYP2D6 and CYP2D17), which can disturb the incorporation of the heme moiety into the active-site cavity. PL enantiomers were oxidized by all of the enzymes mainly into 4-hydroxyproranolol (4-OH-PL), followed by 5-OH-PL and N-desisopropylpropranolol (NDP). In the kinetic analysis, apparent K(m) values were commonly in the µM range and substrate enantioselectivity of R-PL

Experimental verification of the efficacy of optimized two-step infusion therapy with meropenem using an in vitro pharmacodynamic model and Monte Carlo simulation.

In this study we compared the efficacy of a theoretically optimized two-step infusion therapy (OTIT; rapid first-step infusion and slow second-step infusion) to the efficacies of prolonged infusion therapy (PIT) and traditional 0.5 h infusion therapy (TIT) with meropenem against Pseudomonas aeruginosa using an in vitro pharmacodynamic model and a Monte Carlo simulation. In the in vitro pharmacodynamic model, the bactericidal effect against P. aeruginosa was evaluated for 8 h, which is the usual dosing interval of meropenem. It was confirmed that the durability of the bactericidal effect of OTIT (0.25-1 g/0.5 h + 0.25-1 g/4 h t.i.d.) was almost equal to that of PIT and superior to that of TIT (0.5-2 g/0.5-4 h t.i.d.). In addition, the initial bactericidal effects of OTIT were superior to those of the prolonged 4 h infusion. In the Monte Carlo simulation study, the probability of target attainments (PTAs) of all dosing regimens of OTIT at MICs of 2-8 µg/ml were apparently superior to those of TIT and 4- and 6 h-PIT, when the target therapeutic condition for serious life-threatening infections was the achievement of both the percentage of the dosing interval at which the drug concentration exceeds the MIC (%T(>MIC)) ≥ 50% and the peak level divided by the MIC (Cmax/MIC) ≥ 4. Especially, the PTAs of the dosing regimens of 0.25-1 g/0.5 h + 0.25-1 g/4-6 h were excellent at MICs of 2-8 µg/ml. Against recent clinical isolates of P. aeruginosa in Japan, the dosing regimens of OTIT provided higher PTAs compared with those of TIT and 4- and 6 h-PIT. These results suggested that OTIT with sufficient pharmacokinetic conditions could be useful for enhancing the therapeutic efficacy of meropenem against serious life-threatening infections.

Comparative study of the oxidation of propranolol enantiomers in hepatic and small intestinal microsomes from cynomolgus and marmoset monkeys.

Oxidative metabolism of propranolol (PL) enantiomers (R-PL and S-PL) to 4-hydroxypropranolol (4-OH-PL), 5-OH-PL and N-deisopropylpropranolol (NDP) was examined in hepatic microsomes from cynomolgus and marmoset monkeys and in small intestinal microsomes from monkeys and humans. In hepatic microsomes, levels of oxidation activities were similar between the two monkey species, and substrate enantioselectivity (R-PLS-PL) was seen in the formation of NDP in cynomolgus monkeys and humans and in the formation of 5-OH-PL in marmosets. The formation of the three metabolites in cynomolgus monkeys and the formation of NDP in marmosets were biphasic, while the formation of 4-OH-PL in humans was monophasic. From the inhibition experiments using CYP antibodies, CYP2C9 and 2C19 were thought to be involved as N-deisopropylases and CYP2D6 and 3A4 as 4-hydroxylases in human small intestine. Furthermore, CYP1A, 2C and 3A enzymes could be involved in cynomolgus monkeys and CYP2C and 3A enzymes in marmosets. These results indicate that the oxidative profile of PL in hepatic and small intestinal microsomes differ considerably among cynomolgus monkeys, marmosets and humans.

A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes.

Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.

Stereoselective glucuronidation of propranolol in human and cynomolgus monkey liver microsomes: role of human hepatic UDP-glucuronosyltransferase isoforms, UGT1A9, UGT2B4 and UGT2B7.

The stereoselective glucuronidation of propranolol (PL) in human and cynomolgus monkey liver microsomes, and the roles of human hepatic UDP-glucuronosyltransferase (UGT) isoforms involved in the enantiomeric glucuronidation of PL using recombinant UGT enzymes were investigated. In Michaelis-Menten plots, R- and S-PL glucuronidation by human liver microsomes showed sigmoidal kinetics whereas the kinetics of enantiomeric PL glucuronidation by cynomolgus monkey liver microsomes was monophasic. The Km, Vmax and CLint values of cynomolgus monkey liver microsomes were generally higher than the S50, Vmax and CLmax values of human liver microsomes in R- and S-PL glucuronidation. The glucuronidation of R- and S-PL was catalyzed by at least 3 UGT isoforms: UGT1A9, UGT2B4 and UGT2B7. Michaelis-Menten plots for R- and S-PL glucuronidation by UGT1A9 were monophasic, whereas the kinetics of UGT2B7 showed sigmoidal curves. Enantiomeric R-PL glucuronidation by UGT2B4 showed sigmoidal kinetics, whereas S-PL glucuronidation displayed monophasic kinetics. UGT1A9 showed remarkable stereoselectivity in Vmax and CLint values of R-PL < S-PL. These findings demonstrate that the profiles of enantiomeric PL glucuronidation in human and cynomolgus monkey liver microsomes are largely different and suggest that the human hepatic UGT isoforms UGT1A9, UGT2B4 and UGT2B7 play distinctive roles in enantiomeric PL glucuronidation.

Pharmacokinetics of a novel N-methyl-D-aspartate receptor antagonist (SM-18400): identification of an N-acetylated metabolite and pre-clinical assessment of N-acetylation polymorphism.

(S)-9-chloro-5-[p-aminomethyl-o-(carboxymethoxy)phenylcarbamoylmethyl]-6,7-dihydro-1 H,5 H-pyrido[1,2,3-de]quinoxaline-2,3-dione hydrochloride trihydrate (SM-18400) was given intravenously to rats and dogs and its pharmacokinetics was investigated. By LC/MS/MS analysis, the major metabolite in the rat serum was identified as N-acetylated SM-18400 (SM-NAc). In rats, AUC ratio of SM-NAc to SM-18400 was approximately 50%. However, 71% of the dose was excreted as unchanged SM-18400 and only 9.8% as SM-NAc in the urine and bile, indicating that the contribution of N-acetylation clearance (CL(NAc)) to the total clearance (CL(tot)) is limited to 10-30% in rats. No SM-NAc or other metabolites were detected in the dog serum, urine or bile. The in vitro intrinsic clearance (CL(int), ml/min/mg cytosolic protein) of N-acetyltransferase (NAT) activities of dog liver cytosol towards SM-18400 and hepatic N-acetylation clearance (CL(NAc), ml/min/kg body weight) estimated by well-stirred model were both only 5% of the respective rat value, well reflecting the relative in vivo CL(NAc)/CL(tot) ratios. CL(int) values for human liver cytosol samples (n = 4) and estimated CL(NAc) were all less than 18% and 7% of the rat, respectively. Based on these results, we concluded that the CL(NAc)/CL(tot) of human would be small enough to avoid major inter-individual variance in SM-18400 pharmacokinetics due to N-acetylation polymorphism. In addition, even a human liver cytosol sample lacking polymorphic NAT2 activity as determined by sulfamethazine (SMZ) N-acetylation analysis, proved capable of acetylating SM-18400, suggesting that NAT2 is not the major enzyme responsible for N-acetylation of SM-18400 in human. This fact would also reduce the risk of N-acetylation polymorphism playing a role in clinical use of this drug.