Patients with overactive bladder often exhibit abnormal bladder contractions in response to intravesical cold saline (positive ice-water test). The molecular entity involved in cold sensation within ...the urinary bladder is unknown, but a potential candidate is the ion channel, transient receptor potential (melastatin)-8 (TRPM8). The objective of the present study was to investigate the role of TRPM8 in a bladder-cooling reflex evoked in anaesthetised guinea-pigs that is comparable to the positive ice-water test seen in patients. Guinea-pig TRPM8 was cloned from L6 dorsal root ganglia (DRG) and expressed in HEK293 cells. Functional agonist- and cold-induced Ca2+ influx and electrophysiology assays were performed in these cells, and for comparison in HEK293 cells expressing human TRPM8, using a novel TRPM8 antagonist, the S-enantiomer of 1-phenylethyl 4-(benzyloxy)-3-methoxybenzyl (2-aminoethyl) carbamate hydrochloride (PBMC). Potency data from these assays was used to calculate intravenous infusion protocols for targeted plasma concentrations of PBMC in studies on micturition reflexes evoked by intravesical infusion of menthol or cold saline in anaesthetised guinea-pigs. Tissue expression of TRPM8 in guinea-pig bladder, urethra and in dorsal root ganglia neurones traced from the bladder was also investigated. TRPM8 mRNA and protein were detected in L6 dorsal root ganglia, bladder urothelium and smooth muscle. PBMC antagonised in vitro activation of human and guinea-pig TRPM8 and reversed menthol and cold-induced facilitation of the micturition reflex at plasma concentrations consistent with in vitro potencies. The present data suggest that the bladder-cooling reflex in the guinea-pig involves TRPM8. The potential significance of TRPM8 in bladder disease states deserves future investigation.
Valproic acid glucuronidation kinetics were carried out with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid ...was typified by high
K
m values with microsomes and expressed UGTs (2.3–5.2
mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics
in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively (
K
i
=1.6±0.06
mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.
The preparation of bacterial membranes ("Bactosomes") containing expressed canine (beagle) hepatic cytochromes P450 (P450s) is described. cDNAs from seven canine P450s were subcloned into inducible ...expression plasmids and, for the first time, cotransformed and expressed with a canine P450 oxidoreductase in Escherichia coli to produce active, full-length, native sequence P450s. Enzyme expression levels, although variable, were generally sufficient to enable short incubation times and to limit the total protein present in enzyme incubations. Steady-state kinetics of CYP1A1, 2C21, and 2D15 Bactosomes demonstrated similarities with dog liver microsomes or Baculosomes. However, 3A12 lacked substrate inhibition in the formation of 1'-OH midazolam, and 2B11 displayed non-Michaelis-Menten kinetics, suggesting possible differences in protein interaction effects. In monitoring the metabolites of common P450 substrates, phenacetin deethylation, temazepam demethylation, and bufuralol 1'-hydroxylation were shown to be relatively selective reactions catalyzed by CYP1A1, 2B11, and 2D15, respectively. 1'-OH midazolam was formed in higher quantities by CYP2B11 and 2C21 than by 3A12, raising questions about the use of midazolam as a CYP3A12 probe in vivo. In summary, a panel of recombinant P450s was produced to make up for the lack of commercially available canine P450 isoforms. The Bactosomes are expected to facilitate reaction phenotyping and metabolic drug-drug interaction assessment in canine drug development and to enable the study of interspecies differences in P450-mediated drug metabolism.
The aim of this study was to elucidate the metabolic pathways for dihydroartemisinin (DHA), the active metabolite of the artemisinin derivative artesunate (ARTS). Urine was collected from 17 ...Vietnamese adults with falciparum malaria who had received 120 mg of ARTS i.v., and metabolites were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Human liver microsomes were incubated with 12-(3)HDHA and cofactors for either glucuronidation or cytochrome P450-catalyzed oxidation. Human liver cytosol was incubated with cofactor for sulfation. Metabolites were detected by HPLC-MS and/or HPLC with radiochemical detection. Metabolism of DHA by recombinant human UDP-glucuronosyltransferases (UGTs) was studied. HPLC-MS analysis of urine identified alpha-DHA-beta-glucuronide (alpha-DHA-G) and a product characterized as the tetrahydrofuran isomer of alpha-DHA-G. DHA was present only in very small amounts. The ratio of the tetrahydrofuran isomer, alpha-DHA-G, was highly variable (median 0.75; range 0.09-64). Nevertheless, alpha-DHA-G was generally the major urinary product of DHA glucuronidation in patients. The tetrahydrofuran isomer appeared to be at least partly a product of nonenzymic reactions occurring in urine and was readily formed from alpha-DHA-G by iron-mediated isomerization. In human liver microsomal incubations, DHA-G (diastereomer unspecified) was the only metabolite found (V(max) 177 +/- 47 pmol min(-1) mg(-1), K(m) 90 +/- 16 microM). Alpha-DHA-G was formed in incubations of DHA with expressed UGT1A9 (K(m) 32 microM, V(max) 8.9 pmol min(-1) mg(-1)) or UGT2B7 (K(m) 438 microM, V(max) 10.9 pmol mg(-1) min(-1)) but not with UGT1A1 or UGT1A6. There was no significant metabolism of DHA by cytochrome-P450 oxidation or by cytosolic sulfotransferases. We conclude that alpha-DHA-G is an important metabolite of DHA in humans and that its formation is catalyzed by UGT1A9 and UGT2B7.
Long acting inhaled muscarinic receptor antagonists, such as tiotropium, are widely used as bronchodilator therapy for chronic obstructive pulmonary disease (COPD). Although this class of compounds ...is generally considered to be safe and well tolerated in COPD patients the cardiovascular safety of tiotropium has recently been questioned. We describe a rat in vivo model that allows the concurrent assessment of muscarinic antagonist potency, bronchodilator efficacy and a potential for side effects, and we use this model to compare tiotropium with NVA237 (glycopyrronium bromide), a recently approved inhaled muscarinic antagonist for COPD. Anaesthetized Brown Norway rats were dosed intratracheally at 1 or 6h prior to receiving increasing doses of intravenous methacholine. Changes in airway resistance and cardiovascular function were recorded and therapeutic indices were calculated against the ED50 values for the inhibition of methacholine-induced bronchoconstriction. At both time points studied, greater therapeutic indices for hypotension and bradycardia were observed with glycopyrronium (19.5 and 28.5 fold at 1h; >200 fold at 6h) than with tiotropium (1.5 and 4.2 fold at 1h; 4.6 and 5.5 fold at 6h). Pharmacokinetic, protein plasma binding and rat muscarinic receptor binding properties for both compounds were determined and used to generate an integrated model of systemic M2 muscarinic receptor occupancy, which predicted significantly higher M2 receptor blockade at ED50 doses with tiotropium than with glycopyrronium. In our preclinical model there was an improved safety profile for glycopyrronium when compared with tiotropium.
•We use an in vivo rat model to study CV safety of inhaled muscarinic antagonists.•We integrate protein and receptor binding and PK of tiotropium and glycopyrrolate.•At ED50 doses for bronchoprotection we model systemic M2 receptor occupancy.•Glycopyrrolate demonstrates lower M2 occupancy at bronchoprotective doses.•Glycopyrrolate demonstrates an improved CV safety profile, versus tiotropium.
The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were ...characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes.
Colon cancer exhibits inherent insensitivity to chemotherapy by mechanisms that are poorly characterized. We have shown that human colon cancer cells are efficient in drug conjugation catalyzed by ...UDP-glucuronosyltransferases (UGTs) and now report on the role of glucuronidation in de novo resistance to two topoisomerase I inhibitors. Identification of the UGT responsible for glucuronidation of SN-38 and the anthraquinone NU/ICRF 505 was achieved by first using a panel of human cDNA-expressed isozymes to measure conjugating activity. HT29 colon cancer cells were then probed by reverse transcriptase-PCR, Western Blot analysis, and liquid chromatography with mass spectrometry for their profile and activity of UGT isozymes and screened for effective inhibitors of glucuronidation. Expression analysis was also conducted in colon cancer biopsies and paired adjacent normal colon specimens. UGT1A9 was identified as the isozyme catalyzing biotransformation of the two compounds in HT29 cells and propofol as an effective competitive inhibitor of this metabolism. Inhibition of glucuronidation resulted in up to a 5-fold enhancement in drug activity. The majority of colon cancer biopsies studies expressed UGT protein at levels greater than in HT29 cells but with marked interpatient variations and proficiently glucuronidated the two anticancer drugs. A range of UGT aglycones were capable of modulating glucuronidation in the biopies with octylgallate being 10-fold more potent (ID(50) 24 microM) than propofol. In a subset of tumors (33%), UGT protein levels and activity exceeded that of paired normal colon. Glucuronidation may represent a mechanism of intrinsic drug resistance in colon cancer open to modulation by a range of food additives and proprietary medicines.
UGT1A6 and UGT1A9 have both been demonstrated to rapidly glucuronidate simple phenolic compounds. A series of simple phenols were selected and screened with both isoforms and then used as model ...substrates for the generation of V(max) and K(m) values. UGT1A6 showed a more restricted acceptance of phenolic substrates compared with UGT1A9. However, the affinity of UGT1A6 for these compounds exhibited higher K(m) values than UGT1A9, although rates of turnover were similar. Molecular surface-weighted holistic invariant molecular descriptors were generated for each substrate and used to produce the first quantitative structure activity relationship models generated for expressed human UGTs. Models relating log of the K(m) value to the generated descriptors correlated well with the experimental data r(2) value of 0.996 for UGT1A6 and r(2) value of 0.83 for UGT1A9. Cross validation by a leave-one-out method also showed good predictive capability within the subset with a q(2) value of 0.98 for UGT1A6 and q(2) value of 0.73 for UGT1A9. Empirically, UGT1A6 V(max) decreased as the 4-substituent increased in size, and a trend was observed when UGT1A6 V(max) was plotted against molecular volume. The larger UGT1A6 substrates were typified by low activity and lower K(m) values than their smaller counterparts. Extrapolating from this, it was demonstrated that phenols with large 4-substituents, which were not UGT1A6 substrates, could inhibit 4-ethylphenol glucuronidation. The K(m) values for UGT1A9 showed a similar relationship to UGT1A6 but with much lower K(m) values and greater variability in range of this value.
Background: Glucuronidation represents a novel mechanism of intrinsic drug resistance in colon cancer cells. To safely reverse
this mechanism in vivo, it is essential to identify which isoforms of ...UDP-glucuronosyltransferases are responsible for catalysing
this drug metabolism in tumour tissue. Materials and Methods: LC-MS was applied to measure rates of glucuronidation of two
anticancer compounds (SN-38 and NU/ICRF 505) by patient colon cancer biopsies and paired normal colon. Results: Three independent
lines of enquiry indicated that, in the tumour specimens, SN-38 was glucuronidated primarily by UGT1A1, the isozyme generally
recognised as being responsible for hepatic detoxification of this compound, while with NU/ICRF 505 two candidate isoforms
emerged - UGT1A8 and/or UGT1A10 - both of which are not normally expressed in the liver. Conclusion: These data suggest that
tumour selective modulation of this drug resistance mechanism in patients may be feasible with NU/ICRF 505 but more difficult
to realise with SN-38.
Glucuronidation is an important pathway for human drug metabolism. Four cloned and expressed human UDP-glucuronosyltransferases (UGT1A1, UGT1A6, UGT1A9, and UGT2B15) were used to screen a series of ...three potential drug substrates differing only in position of the phenol moiety. The meta and para phenols, UK-156,037 and UK-157,147, were found to be substrates for UGT1A1 with K(m) values of 256 and 105 microM, respectively. The ortho phenol UK-157,261 was glucuronidated predominantly by UGT1A9 with a K(m) of 45 microM. The latter K(m) compares favorably with the known UGT1A9 substrate propofol (K(m) = 200 microM). In a series of competition experiments, UK-157,261 was shown to inhibit the glucuronidation of propofol by UGT1A9 with a K(i) value of 65 microM. This result indicates that even the most potent of these compounds is extremely unlikely to interact in the clinic with the glucuronidation of propofol. This study shows the utility of the expressed human UDP-glucuronosyltransferases in determining substrate structure-activity relationships and potential drug-drug interactions.
