The objective is to determine the effect of anti-ishemia agents on metabolism of adenosine-5'-triphosphate (ATP) in red blood cell (RBC) in a normotensive rat model. Male Sprague Dawley (SD) rats weighing between 300 - 400 g were used. Each rat received either saline (control), or 5 mg/kg of diltiazem (DTZ), losartan, amlodipine or dipyrida- mole by subcutaneous injection (sc) twice daily for 5 doses. Blood samples were collected using a "Stopping Solution" from each rat at time 0 (before the last dose), and sequentially after over 6 hours following the last dose via an indwelling carotid artery catheter. In addition, hemodynamic recordings were collected throughout the experiment. Concentrations of ATP and other purine nucleotides in the RBC were determined by a validated HPLC. Data between groups were analyzed by ANOVA and paired t-test, and differences between groups considered significant when p < 0.05. The results showed that the concentrations of ATP and the other purine nucleotides in RBC of the rats treated with the anti-ischemia drugs were not different from the control rats (Table 1). The concentrations of ATP and guanosine-5'-triphosphate (GTP) were higher towards the end of the experiment, but the increase was significant only after amlodipine and losartan (p < 0.05). The increase of ATP and AMP concentrations correlated with decrease of diastolic blood pressure (DBP) in the rats. The study concluded that the anti-ischemia drugs tested in the current study have no effect on RBC concentrations of ATP in the restraining rat model.
Laromustine (VNP40101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino) carbonylhydrazine) is a novel sulfonylhydrazine alkylating agent. For the first time In vitro profiling and mass balance of ( 14 C)-VNP40101M in rat, dog, monkey and human liver microsomes was investigated. Also, the role of human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes in the conversion of ( 14 C)-VNP40101M by NADPH-fortified human liver microsomes was determined. In this study, ( 14 C)-VNP40101M was converted to five radioactive components (C-1, C-2, C-3, C-4 and C-7) after 60 min of incubation with dog, monkey and human liver microsomes. With the exception of C-3, the same components were detected with rat liver microsomes. In the presence of NADPH, after 60 min of incuba- tion, the loss of substrate for rat, dog, monkey and human was 63, 82, 76 and 64%, respectively and mass balance ranged from 91.0 - 99.3%. In the absence of NADPH, after 60 min of incubation with ( 14 C)-VNP40101M (100 �M), the loss of substrate for rat, dog, monkey and human liver microsomes was 59, 53, 61 and 59%, respectively and mass balance ranged from 100.6 - 116.4%. The profiles of metabolites were similar. The relative abundance of individual metabolites was not species dependent. The formation of C-7 was not observed in zero-cofactor (no NADPH) or zero-protein samples, suggesting that its formation was enzymatic. The formation of C-1, C-2, C-3, and C-4 increased with respect to incubation time. Using a panel of CYP enzymes including rCYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4, it was shown that C- 7 formation was catalyzed by CYP2B6 and CYP3A4/5. The results of this study suggest that (1) P450 plays a role in C-7 formation but plays little or no role in the conversion of ( 14 C)-VNP40101M to C-1 through C-4, and (2) the relative abun- dance of individual degradation/metabolite products were not species dependent. These findings provide a comprehensive understanding of the metabolism of this new agent.
The pharmacodynamics and elimination kinetics of escalating doses (1.5-12 mg/kg) of hyaluronan (HA) infusions were studied in healthy human volunteers.Metabolic breakdown of serum HA and associated adverse events were monitored throughout the study.The HA-binding capacities of circulating CD4+ and CD8+ T lymphocytes, CD19+ Blymphocytes and CD14+ peripheral blood monocytes (PBMC) were also quantified.Breakdown of infused HA into small fragments (<37 kDa) were not detected and adverse events related to HA infusions were infrequent and non-serious in nature.Binding of FITC-HA was greatest to CD14+ monocytes and the binding capacity of these cells for FITC-HA was significantly increased by the final HA infusion.At that time, binding to CD14+ monocytes was related to serum HA levels suggesting a close relationship between PK and PD of serum HA.Drug level analysis demonstrated a disproportional increase in the area under the serum concentration vs. time curve with increasing HA dose.The observed non-linear HA kinetics appears to result from a saturable elimination process as revealed by pharmacokinetic modeling.These results have implications for the use of injected HA for drug delivery or in imaging applications.Participation a drug study 4 weeks prior to study entry C. Prohibited (for duration of study) d Medication (including over-the-counter products) Use of alcohol-containing or xanthine-containing beverages through sample collection See Methods and Results sections for descriptions of procedures.a Subjects could not have used nicotine within 3 months of study entry.b Patients were within 15% of their ideal weight ("Table of Desirable Weights of Adults", Metropolitan Life Insurance Company, 1983).c Includes disorders that would preclude informed consent or compliance with protocol requirements.d If medication other than that specified in the protocol was required, decision to continue subject was made by L.A.B. Medical Personnel.
In vitro determination of metabolic stability is routinely used to assess the overall metabolic liability of com- pounds and for prioritization for in vivo studies. If in vitro metabolic stability data could be used to reliably predict in vivo clearance (CL), it would add significant value in the selection of compounds for in vivo pharmacokinetic and pharmacol- ogy studies. We have evaluated the utility of our in vitro metabolic stability screening assay to estimate in vivo CL in the mouse. The in vitro mouse clearances (CLin vitro) of 146 structurally diverse compounds with metabolic stabilities > 30 %, were compared to mouse in vivo CL data. Approximately 45 % of the compounds showed agreement between in vivo CL and predicted CLin vitro within a 2-fold error criteria. The correlation appeared worse when correction for the extent of in- corporation of plasma protein binding or both plasma and S9 bindings (i.e. ~14 % and~ 28 % agreement, respectively). Classification of the compounds into three groups based on in vivo CL ( 70 mL/min/kg) did not show any improvement between in vivo CL and predicted CLin vitro. The percentage of compounds fal- ling within the 2-fold error criteria for low CL, moderate CL and high CL groups were 54, 31 and 24 %, respectively. In conclusion, our analysis suggests that in vitro metabolic stability data, as routinely obtained in early ADME screening protocols, does not demonstrate a strong correlation with or predictivity for, absolute in vivo CL in the mouse.
The ability of four mammalian cytochromes P450 (CYP) of the CYP1A subfamily, human and mouse CYP1A1s and human and rabbit CYP1A2s, to metabolize a series of steroids and related compounds was investigated using high throughput approaches. Oxidation rates and metabolite patterns for 16 steroid substrates and for 20 polycyclic aromatic hydrocarbon (PAH) substrates were determined in standardized automated conditions. Multivariate statistics of normalized activity data sets was used to sort out significant information and to compare functional signatures of assayed enzymes. Interestingly, for steroid substrates, rabbit CYP1A2 unambiguously aggregates with human and mouse CYP1A1s and appears functionally divergent from human CYP1A2. In contrast, the functional classification was found consistent with the sequence classification when exogenous PAH substrates were tested. The observed features rely on a large set of substrates, all presenting a similar chemical scaffold but decorated with different substituents similar to chemical series used in drug development. Differential functional clusters are thus evidenced for endogenous and exogenous substrates with CYP1A enzymes. A few residues on rabbit CYP1A2 that may account for its unusual 1A1-like specificity toward steroids have been identified both within the active site and at the protein surface. These specific residues thus seem to play a controlling role for global substrate class discrimination, potentially by involving substrate bulkiness and shape sensing.
Polypharmacy as a result of combating co-infections, or combination therapy for better efficacy and reducing the emergency of drug resistance, is on the increase in the African clinical setting in the advent of HIV/AIDS, and tuberculosis (TB) co-infections, and increasing incidences of malaria and other tropical infections. The clinicians and pharmacists are therefore faced with the challenge of prescribing drugs in combinations that are likely to result in severe adverse effects or compromising treatment success. The aim of this study was, therefore, to develop a simple stand alone or network based experimental computational tool to assist doctors and pharmacists in detecting drug combinations likely to result in undesirable metabolism based drug-drug interactions (DDIs) and offer alternate safe prescription options. The mechanism of most drug-drug interactions is through inhibition and induction of drug metabolising enzymes. Models for the prediction of reversible and irreversible inhibitors of the major drug metabolising enzyme system, cytochrome P450, were used in developing the pharmacoinformatic tool. These models enable the prediction of likely in vivo drug-drug interactions from in vitro data. In vivo drug-drug interaction data from the literature was also loaded into the software to validate the system and to give clinical guidance on specific drug-drug interactions. In this first phase of the project, focus was on medications used in the treatment of HIV/AIDS, TB, malaria and other diseases common in Africa. The prototypic tool was based on a Standard Query Language (SQL) database with DELPHI 6.0 as the user interface. Its user friendly pages lead the doctor or pharmacist through drug combination entry functions and gives warning if an interaction is likely. Subsequent actions enable the operator to retrieve more information on the mechanism of interactions, the quantitative measure of the interaction, access to published abstracts on studies, and possible prescription options to minimise DDIs. The software currently has data for 50 drugs used in the design and focuses on the treatment of tropical diseases in addition to classical cases of drug-drug interactions involving other general classes of drugs. The tool can be distributed on Compaq Disk (CD) and be run on any Personal Computer (PC) on windows. We have successfully developed a pharmacokinetic- based tool with a potential to assist clinicians and pharmacists in detecting and rationalizing DDIs. The tool has proved very useful as a teaching tool on DDIs by using the more advanced functions that explore the performance of current drug-drug interactions prediction models. From the available literature, it is clear that more studies need to be done to establish the prevalence and mechanisms of DDIs in the treatment of infectious diseases. We are now adding more data, validating the tool and finally testing the acceptability of this tool among clinicians and pharmacists for routine use.
Objective: To investigate the effects of rifampin on the steady-state pharmacokinetics of gepirone and metabolites after multiple dosing of both drugs.Methods: 24 subjects completed a randomized crossover study with 2 study phases separated by a washout period of at least 4 weeks.The subjects received multiple dosing of gepirone extended-release (gepirone ER) (20 mg daily for 2 days titrated to 40 mg daily for 5 days) with and without concurrent use of rifampin 600 mg daily.Plasma concentrations of gepirone and two principal metabolites were determined for up to 48 hours after dosing on day 7. Urinary 6hydroxycortisol:cortisol ratio was also used to assess the extent of enzyme induction during both study periods.Results: Rifampin significantly decreased the area under the plasma concentration-time curves (AUC) of gepirone and 3´-OH-gepirone by 95% and 65%, respectively.The peak concentration (C max ) values also were reduced by 92% and 58%, respectively.On the other hand, there were minimal changes in AUC and C max of 1-PP during concurrent use of rifampin.Gepirone dosing did not change the urinary 6 -hydroxycortisol:cortisol ratio, in contrast to a 4.1-fold increase in the ratio with concurrent use of rifampin.Conclusions: Rifampin significantly decreased the systemic exposure of gepirone and 3´-OH-gepirone.The likely mechanism is induction of CYP3A4-mediated first-pass metabolism in the intestine and the liver.Concurrent use of potent CYP3A4 enzyme inducers might lead to significant reduction in pharmacologic effect of gepirone.On the other hand, gepirone does not appear to have CYP3A4 induction or inhibition effects.
The objective of the study was to compare the pharmacokinetics and hemodynamic effects of diltiazem (DTZ) after single dose and multiple doses using an in vivo rat model. Male SD rats (n = 6 - 8 per group) weighing between 350 - 450 g were used. Each rat received either a single 20mg/kg dose of DTZ or 5mg/kg sc twice daily for 5 doses by subcuta- neous (sc) injection. Plasma concentrations of DTZ and its major metabolites were determined by HPLC for up to 8 h. In addition, Systolic Blood Pressure, Diastolic Blood Pressure and Heart Rate were continuously recorded, and analysed using WinNonLin and considered significant when p < 0.05. The results indicate that after the single 20 mg/kg subcutaneous in- jection, SBP fell from 138 ± 4 to 125 ± 3 mmHg (-9.4%), DBP from 105 ± 3 to 78 ± 4 mmHg (-26%), and HR from 442 ± 12 to 396 ± 7 bpm (-10%). After 5 mg/kg twice daily for 5 doses, the observed SBP was reduced from 127 ± 5 to 111 ± 7 mmHg (-13%), DBP from 108 ± 6 to 88 ± 7 mmgHg (-19%), and HR from 458 ± 11 to 407 ± 22 bpm (-11%). The phar- macokinetics and hemodynamic data were characterized by an Inhibitory Emax model, which showed a similar profile fol- lowing the single and multiple doses. Multiple regression analyses of the data predicted that the metabolites in particular deacetyl diltiazem (M1) contributed significantly to the blood pressure lowering effects following multiple doses, but the effects of metabolite were minimal after single dose.
Epothilones are natural macrolides displaying potent antiproliferative properties against various cell lines and capable to bind tubulin and acting as microtubule-stabilizing agents like taxoids. We intended to isolate and characterize epothilone metabolites and identify enzymes implicated in the biotransformation process. In the presence of NADPH, liver microsomes from phenobarbital-treated rats produce two metabolites resulting from the oxidation of either epothilone A or B by CYP isoforms. Similarly, the oxidative biotransformation of epothilones A and B by human liver microsomes generates three metabolites with Km values ranged from 61 to 86� M. The two major metabolites (m1 and m2) are hydroxylated on the macrolide ring essentially by CYP3A4, whereas 3A5, 3A7 and 2B6 are minor contributors to the reaction. M3 is formed by CYP2C19 and 2C9 and results from the hydroxylation of the methyl on carbon 17 of the lateral chain. Inhibition of CYP3A almost completely abolished the formation of m1 and m2, whereas inhibition of CYP2C19 substantially reduced the production of m3. Collectively these data suggest that the oxidative metabolism of epothilones is principally mediated by CYP3A4 and CYP2C19. Epothilone B was found to be an in vitro inhibitor of CYP2C9 (IC50� 25� M), CYP2C19 (Ki� 1.7� M) and CYP3A4/5 (Ki� 1.85� M) whereas conversely taxanes or Vinca alka- loids significantly reduced oxidation of epothilone B. However, clinically relevant inhibition in patients undergoing che- motherapy is unlikely due to low therapeutic epothilone B blood concentrations.
Glutamate dehydrogenase (GHD) synthesizes some RNAs that regulate mRNA abundance in response to the environment. The connection of gene expression and drug metabolism by the GDH-synthesized RNA has not been demonstrated experimentally. The regulation of the mRNAs encoding the drug-metabolizing enzymes was studied by northern hybridization using the GDH-synthesized RNAs as probes. The mRNAs encoding cytochrome P-450 reductase, UDPglucosyltransferase, alternative oxidase, and ABC-transporters were upregulated by the administered ATP+UTP+GTP. Also superoxide dismutase and GSH S-transferase were upregulated by administered ATP. The untreated control, GTP, and UTP did not upregulate any of the mRNAs. The mRNAs encoding the enzymes were coordinately regulated at the molecular level. All the enzymes are also active in drug detoxication in mammals. Photometric assays of enzyme activities confirmed that the enzymes were present at levels proportional to their respective encoding mRNAs as detected by the GDH-synthesized RNA probes. Genetic code-based nucleic acid probes were partially accurate in detecting the mRNAs encoding the enzymes. Therefore, GDH-synthesized RNAs are important genetic metabolic probes for the screening of mRNAs encoding the drug metabolizing enzymes. Nucleoside triphosphates and analogs are antihypertensives, antineoplastics, antiarrhythmics, antimetabolites, antiviral agents etc and they induce GDH isomerization.
Soluble epoxide hydrolase (sEH) is becoming an attractive therapeutic target in cardiovascular disease. Recently, known human sEH polymorphisms were associated with elevated plasma cholesterol and atherosclerosis. In this study we evaluated the potential role of sEH in regulating cholesterol metabolism through modulating the levels of fatty acid epoxide substrates and/or their corresponding diol products known to activate peroxisome proliferator activated receptors (PPARs). We measured changes in cholesterol levels induced by expressing sEH proteins in mammalian cell lines and in response to treatment with various sEH-related compounds. Our results indicate that sEH has a cholesterol lowering effect that is mediated at least in part through its C-terminal hydrolase activity. In addition, several fatty acid epoxides and their corresponding diols showed cholesterol lowering effects in the current study. In conclusion, this study provides evidence that fatty acid epoxides and diols are endogenous cholesterol lowering molecules and that sEH may be involved in cholesterol regulation by modulating their levels.
Highly variable drugs have been defined as drugs with a residual variability of more than 30% in terms of the ANOVA coefficient of variation. Different approaches have been proposed during the last years to deal with this problem but the topic remains controversial. Itraconazole, a highly variable drug, has low bioavailability with a high CYP3A4 presystemic biotransformation. Also, it has a very poor aqueous solubility which is very dependent on the pH of the dissolution medium. The pregnane X receptor (PXR) has been shown to mediate the genomic effects of progesterone and estradiol in the expression of the cytochrome P-450 gene family, which plays an important role in the metabolism of hormones and xenobiotics. During the menstrual cycle both hormone concentrations vary, providing a rationale for the more variable CYP3A4 activity in women. The analysis of the data of an itraconazole bioequivalence study involving 24 healthy volunteers (12 men and 12 women) carried out by other investigators enables us to conclude that women have less oral bioavailability and more variable AUC than men. Low bioavailability seems to be related with the higher stomach pH observed in women and variability with the aforementioned menstrual cycle incidence on both pH and CYP3A4 expression. The lower variability observed in men made it possible to discriminate differences in AUC’s variability displayed by each brand.
