Naotoshi Yamamura

Identification of valproic acid glucuronide hydrolase as a key enzyme for the interaction of valproic acid with carbapenem antibiotics.

Plasma levels of valproic acid (VPA) are decreased by concomitant use with carbapenem antibiotics, such as panipenem (PAPM). One of the plausible mechanisms of this interaction is the inhibition of VPA glucuronide (VPA-G) hydrolysis by carbapenems in the liver. To elucidate this interaction mechanism, we purified VPA-G hydrolase from human liver cytosol, in which the hydrolytic activity was mainly located. After chromatographic purification, the VPA-G hydrolase was identified as acylpeptide hydrolase (APEH). APEH-depleted cytosol, prepared by an immunodepletion method, completely lacked the hydrolytic activity. These results demonstrate that APEH is a single enzyme involved in PAPM-sensitive VPA-G hydrolysis in cytosol. In addition, the hydrolytic activity of recombinant human APEH was inhibited by PAPM and the inhibition profile by typical esterase inhibitors (diisopropyl fluorophosphate, 5,5′-dithiobis(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and d-saccharic acid 1,4-lactone) was similar to that of human liver cytosol. Cytosolic VPA-G hydrolase activity was slightly inhibited by cholinesterase and carboxylesterase inhibitors. β-Glucuronidase activity remained in APEH-depleted cytosol, whereas VPA-G hydrolase activity was completely abolished. Thus, either cholinesterase, carboxylesterase, or β-glucuronidase in cytosol would not be involved in VPA-G hydrolysis. Taken together, APEH plays a major role in the PAPM-sensitive VPA-G hydrolysis in the liver. These findings suggest that APEH could be a key enzyme for the drug interaction of VPA with carbapenems via VPA-G hydrolysis.

Inhibition mechanism of carbapenem antibiotics on acylpeptide hydrolase, a key enzyme in the interaction with valproic acid

We have reported that inhibition of acylpeptide hydrolase (APEH), identified as valproic acid glucuronide hydrolase in human liver cytosol, by carbapenem antibiotics could lead to a decrease of plasma levels of valproic acid. In this study, we examined the inhibition mechanism using human liver cytosol and purified porcine APEH with a similar property to human counterpart. After preincubation of human liver cytosol with panipenem or meropenem for 30 min, the inhibition of APEH activity was 20-fold stronger than that without preincubation. Porcine APEH activity inhibited by meropenem did not recover after dialysis. Meropenem bound to porcine APEH and the binding was blocked by a serine hydrolase inhibitor, diisopropyl fluorophosphate. Open β-lactam ring form of meropenem did not affect APEH activity in human liver cytosol. Likewise, other antibiotics, which have a different heterocycle adjacent to the β-lactam ring with an opposite configuration of the side chain from carbapenems, did not inhibit APEH activity. In conclusion, carbapenems inhibit APEH in both reversible and true irreversible manner and the irreversible inhibition is partially explained by binding to the active serine of APEH. The closed β-lactam ring is essential for inhibition and the heterocycle and/or the configuration of side chain would be important.

In vivo pharmacodynamic activity of tomopenem (formerly CS-023) against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in a murine thigh infection model.

ABSTRACT Tomopenem (formerly CS-023) is a novel carbapenem with broad-spectrum activities against diverse hospital pathogens, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). We examined the in vivo pharmacodynamic characteristics of tomopenem against P. aeruginosa and MRSA by using a neutropenic murine thigh infection model with P. aeruginosa 12467 (MIC, 1 μg/ml) and MRSA 12372 (MIC, 2 μg/ml). The mice had 106 to 107 CFU/thigh of each strain 2 h after inoculation and were treated for 24 h with a fractionated administration of tomopenem given at intervals of 3, 6, 12, and 24 h. The serum protein binding of tomopenem was 17.4%. The efficacy of tomopenem in both infection models was enhanced by frequent dosing, which indicates that the efficacy is driven by the time above MIC (TMIC). In a sigmoid model, the cumulative percentages of the 24-h period that the concentrations of free, unbound fractions of the drug exceeded the MIC under steady-state pharmacokinetic conditions (f%TMICs) were best correlated with efficacy when R2 was 0.79 and 0.86 against P. aeruginosa and MRSA, respectively. Other pharmacokinetic and pharmacodynamic (PK-PD) indexes for the free, unbound fractions, the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC) and the maximum concentration of the drug in serum divided by the MIC (Cmax/MIC), showed poor correlation with efficacy when R2 was ≤0.42. The f%TMIC values required for a static effect, 1-log kill, and 2-log kill against P. aeruginosa were 29, 39, and 51, respectively, which were similar to those for meropenem, for which the values were 24, 33, and 45, respectively. Against MRSA, the values for tomopenem were 27, 35, and 47. In conclusion, the pharmacodynamic characteristics of tomopenem were similar to those of meropenem against P. aeruginosa, and there was no difference between the target values for P. aeruginosa and MRSA required for efficacy in this study.

Identification of novel metabolic pathways of pioglitazone in hepatocytes: N-glucuronidation of thiazolidinedione ring and sequential ring opening pathway

The metabolism of [14C]pioglitazone was studied in vitro in incubations with freshly isolated human, rat, and monkey hepatocytes. Radioactivity detection high-performance liquid chromatography analysis of incubation extracts showed the detection of 13 metabolites (M1–M13) formed in incubations with human hepatocytes. An identical set of metabolites (M1–M13) was also detected in monkey hepatocytes. However, in rat hepatocytes, M1 through M3, M5 through M7, M9 through M11, and M13 were also detected, but M4, M8, and M12 were not detected. The structures of the metabolites were elucidated by liquid chromatography/tandem mass spectrometry using electrospray ionization. Novel metabolites of pioglitazone detected using these methods included thiazolidinedione ring-opened methyl sulfoxide amide (M1), thiazolidinedione ring-opened N-glucuronide (M2), thiazolidinedione ring-opened methyl sulfone amide (M3), thiazolidinedione ring N-glucuronide (M7), thiazolidinedione ring-opened methylmercapto amide (M8), and thiazolidinedione ring-opened methylmercapto carboxylic acid (M11). In summary, based on the results from these studies, two novel metabolic pathways for pioglitazone in hepatocytes are proposed to be as follows: 1) N-glucuronidation of the thiazolidinedione ring of pioglitazone to form M7 followed by hydrolysis to M2, and methylation of the mercapto group of the thiazolidinedione ring-opened mercapto carboxylic acid to form M11; and 2) methylation of the mercapto group of the thiazolidinedione ring-opened mercapto amide to form M8, oxidation of M8 to form M1, and oxidation of M1 to form M3.

In Vitro Metabolism of Rivoglitazone, a Novel Peroxisome Proliferator-Activated Receptor γ Agonist, in Rat, Monkey, and Human Liver Microsomes and Freshly Isolated Hepatocytes

The in vitro metabolism of rivoglitazone, (RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride, a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, was studied in liver microsomes and freshly isolated hepatocytes of rat, monkey, and human as well as cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes. Fourteen metabolites were detected, and these structures were elucidated by liquid chromatography-tandem mass spectrometry. Five initial metabolic pathways of rivoglitazone consisting of four oxidation pathways and one N-glucuronidation pathway were predicted in correspondence with those proposed for in vivo studies using rats and monkeys. In metabolization using liver microsomes, the TZD ring-opened mercapto amide (M22) and TZD ring-opened mercapto carboxylic acid (M23) were identified as the primary metabolite of the TZD ring-opening pathway and its sequential metabolite, which have not been detected previously from in vivo studies. Combination with S-adenosyl-l-methionine was useful to obtain the sequential S-methylated metabolites from the oxidative metabolites. N-Glucuronide and sequential TZD ring-opened metabolites were also found in liver microsomes in the presence of UDP-glucuronic acid. The O-demethyl-O-sulfate (M11), which is the major in vivo metabolite in rats and monkeys, was detected in all species of hepatocytes. In addition, a TZD ring-opened S-cysteine conjugate (M15) was detected in human hepatocytes. From these results, the in vivo metabolic pathways in humans were predicted to be the four oxidation and one N-glucuronidation pathways. The four oxidative metabolites were formed by multiple human P450 enzymes, and N-glucuronide was formed by UGT1A3 and UGT2B7.

Isolation and Identification of Diglucuronides of Some Endogenous Steroids in Dogs

Diglucuronidation is a novel glucuronidation reaction where the second glucuronosyl moiety is attached at the C2′ position of the first glucuronosyl moiety. To examine whether diglucuronidation takes place in endogenous substrates in vivo, control urine and bile samples were collected from male Crl:CD(SD) IGS rats, beagle dogs, and cynomolgus monkeys and analyzed by liquid chromatography-mass spectrometry (LC-MS) after solid phase extraction. Several diglucuronides of C19 steroids, including M1 (C31H46O14) and M2 (C31H44O14), were detected in the urine and bile of the dogs but not in the excreta of the rats and monkeys. A milligram quantity of M1 was successfully isolated from the pooled dog urine and analyzed by nuclear magnetic resonance (NMR) spectroscopy. M1 was unambiguously identified as epiandrosterone 3-O-diglucuronide by comparing the LC-MS and two-dimensional NMR data of M1 with those of the biosynthesized epiandrosterone 3-O-diglucuronide. M2 was identified as dehydroepiandrosterone 3-O-diglucuronide. According to these findings, the diglucuronidation reaction was proven to be occurring on steroid hormones in vivo in dogs.

PEGylation of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) results in decreased uptake into rats and human liver.

Objectives  Our aim was to investigate the effect of PEGylation on the uptake of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) into rat liver, kidney and spleen, and human liver.

Generation and Characterization of a Novel Small Biologic Alternative to PCSK9 Antibodies, DS-9001a, Albumin Binding Domain-Fused Anticalin Protein

Since it was recently reported that an antibody for proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces the risk of cardiovascular events in a clinical context, PCSK9 inhibition is thought to be an attractive therapy for dyslipidemia. In the present study, we created a novel small biologic alternative to PCSK9 antibodies called DS-9001a, comprising an albumin binding domain fused to an artificial lipocalin mutein (ABD-fused Anticalin protein), which can be produced by a microbial production system. DS-9001a strongly interfered with PCSK9 binding to low-density-lipoprotein receptor (LDL-R) and PCSK9-mediated degradation of LDL-R. In cynomolgus monkeys, single DS-9001a administration significantly reduced the serum LDL-C level up to 21 days (62.4% reduction at the maximum). Moreover, DS-9001a reduced plasma non–high-density-lipoprotein cholesterol and oxidized LDL levels, and their further reductions were observed when atorvastatin and DS-9001a were administered in combination in human cholesteryl ester transfer protein/ApoB double transgenic mice. Additionally, their reductions on the combination of atorvastatin and DS-9001a were more pronounced than those on the combination of atorvastatin and anacetrapib. Besides its favorable pharmacologic profile, DS-9001a has a lower molecular weight (about 22 kDa), yielding a high stoichiometric drug concentration that might result in a smaller administration volume than that in existing antibody therapy. Since bacterial production systems are viewed as more suited to mass production at low cost, DS-9001a may provide a new therapeutic option to treat patients with dyslipidemia. In addition, considering the growing demand for antibody-like drugs, ABD-fused Anticalin proteins could represent a promising new class of small biologic molecules.

Renal handling of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, in rabbits in comparison with meropenem

The plasma half-life of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, is longer than that of meropenem in animals and humans. To address this issue, renal clearance studies were conducted in rabbits. A constant rate infusion of CS-023 and meropenem was conducted in male Japanese White rabbits. Concentrations in the plasma, urine and renal cortex were measured to evaluate renal clearance and renal tissue uptake. CS-023 showed a clearance ratio (renal clearance/glomerular filtration rate) of around 1, which was not affected by co-administration of probenecid or p-aminohippurate. On the other hand, meropenem exhibited a clearance ratio of around 3, which was significantly decreased to 1 by co-administration of probenecid. p-Aminohippurate, in contrast, had no effect. The renal cortex/plasma concentration ratio of CS-023 was around 0.6 with or without probenecid co-administration. This ratio of meropenem was around 3, which was decreased significantly by co-administration of probenecid to around 0.6. These data suggest that meropenem is secreted in the renal tubules via organic anion transporters, but CS-023 is not. The present findings in rabbits would indicate that a lack of renal tubular secretion of CS-023 is a reason for the long plasma half-life compared with meropenem.

In vitro metabolism of human and salmon calcitonins in rat liver and kidney evaluated by liquid chromatography-tandem mass spectrometry.

1. Using LC-MS and LC-MS/MS, an in vitro study was conducted on the metabolism of human calcitonin (hCT) and salmon calcitonin (sCT) in rat liver and kidney to determine the rates of metabolism and the positions of hydrolytic cleavage in both peptides. 2. In lysosomal fractions of rat liver and kidney, hCT was degraded 9-12 times faster than sCT. Many metabolites of hCT were produced in the lysosomal fractions, whereas the metabolites of sCT were scarcely found. 3. In the case of the cytosolic fractions, three positions of initial endoproteolytic cleavage were found in hCT, leading to the production of many peptide fragments via subsequent exoproteolytic metabolism. The initial cleavage position of sCT could not be identified precisely, but it was postulated that the rate-determining step in the metabolism of sCT is the endoproteolytic hydrolysis. 4. The studies using pure proteases and protease inhibitors indicated that the metabolism of calcitonins proceeds by initial endoproteolytic cleavage and subsequent exoproteolytic digestion, catalysed by an aspartate-protease in lysosomes and by a metalloprotease and cysteine-protease in combination in the cytosol. 5. The result suggested that the higher in vivo pharmacological activity of sCT compared with that of hCT may be due to a slower metabolism of the former.