Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-((14)C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs.

A systematic comparison between two labeling approaches for the investigation of the in vitro metabolic pattern of pharmaceutical drugs was performed by examining the use of (i) radiolabeled drugs analyzed with LC-MS-offline radiodetection and (ii) stable-isotope labeled drugs, used in a defined mixture with the unlabeled drug and analyzed by LC-MS with recognition of the specific isotopic pattern. (14)C was used for the radioisotope-approach and deuterium for the stable-isotope approach. Olanzapine, diclofenac and ketoconazole were chosen as model drugs, as they are commercially available in their non-, radio- and stable-isotope labeled forms. For all three model drugs, liver microsome- and hepatocyte-incubations (both from rat) were performed with various concentrations and incubation times for both, the radio- and the stable-isotope approaches. The metabolic pattern, including structure elucidation of all detected metabolites, was performed independently for all individual compounds and incubations. Subsequently, the metabolic patterns of the radio-, and the stable-isotope approaches were compared. In conclusion, all metabolites found with the radioisotope approach could also be found with the stable-isotope approach. Although the stable-isotope approach does not provide a quantitative result, it can be considered to be a highly suited analytical alternative for early in vitro metabolism investigations, especially when radiolabeled drug analogues are not yet available and quantitative results are not yet necessary.

Investigation of the species-dependent in vitro metabolism of BAL30630 by stable isotope labeling and isotope exchange experiments analyzed by capillary liquid chromatography coupled to mass spectrometry.

The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-D-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide- and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC-QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.

Elucidation of the in vitro metabolic profile of stable isotope labeled BAL19403 by accurate mass capillary liquid chromatography/quadrupole time-of-flight mass spectrometry and isotope exchange.

The in vitro metabolic pattern of BAL19403, a novel macrolide antibiotic, was investigated by capillary liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) in incubations with human microsomes. For the elucidation of the metabolic pathway, BAL19403 labeled with four deuterium atoms (D4) was used, and detection of metabolites performed using mixtures of the unlabeled (H4) BAL19403 and its D4 analogue (1:1) as substrate. All metabolites appeared with similar chromatographic behavior. MS/MS spectra of BAL19403 and its metabolites are dominated by non-informative fragment ions. Therefore, the structure of the metabolites was elucidated mainly by accurate mass measurements with subsequent proposals of elemental compositions. Main biotransformations were N-demethylation, lactone ring hydrolysis, and oxidation. Additionally, N-dealkylation of the aromatic moiety was identified. This dealkylation results not only in formation of an aldehyde, according to the classical pathway, but also in formation of the corresponding alcohol and carboxylic acid. Final elucidation of their structures was possible, since this dealkylation takes place vicinal to the deuterium-labeled part of BAL19403 and interferes with D/H exchange. The degree of D/H exchange, determined by analysis of the metabolite isotopic pattern, was used to elucidate the adjacent functional group.

Investigation of low-abundant in vitro metabolites of stable isotope-labelled BAL4815 by accurate mass capillary-LC-ESI-qTof-MS and MS/MS.

The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.

Single-ascending-dose pharmacokinetics and safety of the novel broad-spectrum antifungal triazole BAL4815 after intravenous infusions (50, 100, and 200 milligrams) and oral administrations (100, 200, and 400 milligrams) of its prodrug, BAL8557, in healthy volunteers.

BAL8557 is the water-soluble prodrug of a novel antifungal triazole, BAL4815. BAL4815 is active against a broad spectrum of major opportunistic and pathogenic fungi, including strains that are resistant to other azoles. Cohorts of healthy male subjects received single-ascending oral (p.o.) doses of BAL8557 that were equivalent to 100, 200, or 400 mg of BAL4815 or single-ascending, 1-h constant-rate intravenous (i.v.) infusions of BAL8557 which were equivalent to 50, 100, or 200 mg of BAL4815. In each cohort, six subjects were randomly assigned to receive active drug and two subjects were assigned to receive the placebo. All doses were well tolerated, and no severe or serious adverse events occurred. Maximum plasma concentrations of BAL4815 were observed 1.5 to 3 h after p.o. drug intake or at the end of the 1-h infusion. After both routes of administration, values for maximum drug concentration observed in plasma and area under the concentration-time curve increased slightly more than proportionally to the administered dose. Mean elimination half-lives were particularly long (56 to 77 h after p.o. administration and 76 to 104 h after i.v. administration). The volume of distribution was large (155 to 292 liters after p.o. administration and 304 to 494 liters after i.v. administration) and systemic clearance was low (1.9 to 2.8 liter/h after p.o. administration and 2.8 to 5.0 liter/h after i.v. administration). Urinary recovery of BAL4815 was less than 0.4% of the infused dose. Based on the exposure data, oral bioavailability of BAL4815 is assumed to be very high. The pharmacokinetics of BAL4815 are well suited to maintaining concentrations of BAL4815 for a long period of time in the body and to enabling an effective treatment of systemic mycoses.

Single-dose pharmacokinetics and safety of a novel broad-spectrum cephalosporin (BAL5788) in healthy volunteers.

BAL5788 is the water-soluble prodrug of BAL9141, a novel broad-spectrum cephalosporin with potent bactericidal activities against methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae. We investigated the safety and pharmacokinetics of BAL5788 in a double-blind, single-ascending-dose study with 40 healthy male subjects. The subjects were randomized to receive placebo (n = 2 subjects per dose) or BAL5788 (n = 6 subjects per dose) as a 200-ml intravenous infusion over 30 min. The BAL5788 doses used were 125, 250, 500, 750, and 1,000 mg (BAL9141 equivalents). All doses were well tolerated, with no severe or serious adverse events (AEs). The most frequent AE was taste disturbance. No electrocardiographic abnormalities and no trends or clinically significant changes in laboratory parameters or vital signs were observed. The maximum concentration of drug in serum and the area under the concentration-time curve for BAL9141 were dose proportional over the dosing range. The elimination half-life of BAL9141 was about 3 h. The volume of distribution at steady state was equal to the volume of the adult extracellular water compartment, and the rate of renal clearance of free drug corresponded to the normal glomerular filtration rate for adults. More than 70% of the administered dose was excreted as BAL9141 in the urine, and almost no prodrug was detected. After the infusion of 750 mg, the mean plasma BAL9141 concentrations exceeded the MIC at which 100% of MRSA isolates are inhibited (4 microg/ml) for approximately 7 h, or 58% of a 12-h dosing interval. These results indicate that infusions of 750 mg twice a day should be adequate for the treatment of infections caused by MRSA.

Multiple-dose pharmacokinetics and safety of a novel broad-spectrum cephalosporin (BAL5788) in healthy volunteers.

BAL5788 is the water-soluble prodrug of BAL9141, a novel broad-spectrum cephalosporin with potent bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae. Safety and pharmacokinetic data from a multiple-dose study with 16 healthy male volunteers are reported. Subjects were randomized to receive BAL5788 at 500 or 750 mg (as BAL9141 equivalents; n = 6 subjects per dose) or placebo (n = 2 subjects per dose). The doses were given as 200-ml infusions over 30 min once daily on days 1 and 8 and twice daily on days 2 to 7. BAL5788 was well tolerated, with no severe or serious adverse events (AEs) or dosing-related changes in laboratory parameters, electrocardiographic findings, or vital signs. Drug accumulation in plasma was negligible during the dosing period. The results of pharmacokinetic analyses agreed well with data reported from a previous single-ascending-dose study. The elimination half-life of BAL9141 was about 3 h. The volume of distribution at steady state was equal to the volume of the adult extracellular water compartment. BAL9141 was predominantly eliminated in urine, and renal clearance of the free drug corresponded to the normal glomerular filtration rate in adults. After multiple infusions of 750 mg, the mean concentrations of BAL9141 in plasma exceeded the MIC at which 100% of MRSA isolates are inhibited (4 microg/ml) for approximately 7 to 9 h, corresponding to 58 to 75% of a 12-h dosing interval.

Performance and reliability of splitless microliter gradient pumps in a metabolic stability study using cytochrome P450/3A4 and capillary liquid chromatography-mass spectrometry.

The performance of two commercially available capillary LC pumps (MicroPro (Eldex, USA), Evolution 200 (ProLab, Switzerland)) generating really splitless gradients in the microliter per minute range was tested in detail concerning their applicability for routine drug discovery. A standard method to study metabolic stability against CYP450 isoform 3A4 was selected. This method was transformed into a fast splitless capillary LC-MS method. Both pumps generated reproducible gradients at flows of 5-10 microl/min within 10-15 min. Although gradient formation of the MicroPro system was very reproducible, its equilibration time was too long for fast gradients around 5 microl/min. The Evolution 200 pump offered a good performance with 180 microm i.d. columns at a flow rate of 6 microl/min. The precision of the retention time of the internal standard (ISTD) varied between 1.4 and 3.4% (n = 131-152, three different columns tested). Up to 800 injections of sufficient performance on one column and a stable enough response of the ISTD for 16 h sequence duration were obtained. Accuracy between 95 and 105% and precision < or = 8.4% for 1'-hydroxylated midozolam were reached. The IC50 values of the miniaturized assay (drug candidate BAL4815 1.7 +/- 0.5, itraconazole 0.46 +/- 0.06, and ketoconazole 0.12 +/- 0.01 microM) agreed well with those of the conventional approach. Details concerning method optimization and limitations in operation are discussed in detail. Still, the overall performance of the capillary LC pumps cannot cope completely with that of conventional HPLC pumps in terms of user-friendliness.

Preformulation approaches to improve the oral bioavailability of two novel piperidine renin inhibitors in dog.

Different experimental formulations based on aqueous and oily systems, water miscible solvents, and solid dispersions were investigated for their potential to increase the oral bioavailability (F) of two novel piperidine renin inhibitors (Ro-X1: (R)-1-methoxy-3-[(3S,4R,5R)-4-[4-[3-(2-methoxy-benzyloxy)-propoxy]-phenyl]- 5-(4-methoxy-naphthalen-2-ylmethoxy)-piperidin-3-yloxy]-propan-2-ol; Ro-X2: (R)-3-[(3S,4R,5R)-4-[4-[3-(2-methoxy-benzyloxy)-propoxy]-phenyl]-5-(4- methoxy-naphthalen-2-ylmethoxy)-piperidin-3-ylmethoxy]-propane-1,2-diol) in dogs compared to their administration as acidic aqueous solution. The compounds were characterized by a low solubility at pH 7 (Ro-X1: 3 micrograms/ml, Ro-X2: 24 micrograms/ml) and a high lipophilicity (Ro-X1: LogP = 5.7, Ro-X2: LogP = 3.7). For Ro-X1 oil-based vehicles resulted in an improvement in the oral bioavailability compared to the aqueous solution (F = 6 +/- 1.2%) with the best result being achieved with a solution in Capmul (F = 14.6 +/- 3.5%). By contrast, for Ro-X2 the highest bioavailability (F = 27.1 +/- 8.4%) was achieved using an aqueous solution. Computer simulations based on the physicochemical parameters of the compounds only predicted that the fraction of compound absorbed in man should be almost quantitative for Ro-X2 and only about 28% for Ro-X1. These results suggest that other factors such as extensive gut and/or hepatic metabolism as well as exclusion by intestinal transporters such as p-glycoprotein, rather than incomplete solubilization in the gut, are the major reasons for the limited oral bioavailability of both compounds.