Rapid investigating of phase I metabolites of SR9009 in vitro horse liver microsomes via feature-based molecular networking approach: Potential applications in doping control.

SR9009, a peroxisome proliferator-activated receptor δ (PPARδ) agonist, is known for its potential benefits in energy homeostasis. It failed to receive the United States Food and Drug Administration (USFDA) approval and its illegal distribution has raised concerns. As a result, it has been classified as a prohibited substance by the World Anti-Doping Agency and the International Federation of Horseracing Authorities (IFHA). This study emphasizes the application of the in-silico molecular networking technology to analyze phase I drug metabolites in horses, distinguishing it from conventional methodologies in forensic science. Feature-based molecular networking (FBMN) analysis identified 15 metabolites, with novel major N-dealkylated metabolite (-C8H7NO4S), indicative of diverse metabolic modifications in horse liver microsomes incubation assay. Additionally, a proposed metabolic pathway of SR9009 in the in vitro assay was outlined, including the previously known dehydroxylated metabolite. Finally, the metabolic pathways included in this study were as follows: hydroxylation, dehydrogenation, N-dealkylation dihydroxylation, and combinations. Molecular networking provided insights into MS spectra connectivity, facilitating rapid interpretation and accurate detection of previously undiscovered metabolites. In conclusion, this study contributes to the understanding of SR9009 metabolism in horses and underscores the importance of advanced analytical techniques, such as molecular networking, in enhancing the accuracy and efficiency of metabolite analysis for forensic and doping control purposes.

Pharmacokinetic considerations for enhancing drug repurposing opportunities of anthelmintics: Niclosamide as a case study.

Recently, anthelmintics have showcased versatile therapeutic potential in addressing various diseases, positioning them as promising candidates for drug repurposing. However, challenges such as low bioavailability and a lack of a solid pharmacokinetic basis impede successful repurposing. To overcome these flaws, we aimed to investigate the key pharmacokinetic factors of anthelmintics mainly focusing on the absorption, distribution, and metabolism profiles by employing niclosamide (NIC) as a model drug. The intestinal permeability of NIC is significantly influenced by solubility and doesn't function as a substrate for efflux transporters. It showed high plasma protein binding. Also, the metabolism study indicated that NIC would have low metabolic stability by extensively undergoing the intestinal glucuronidation. Additionally, we investigated the CYP-mediated drug-drug interaction potential of NIC in both direct and time-dependent ways. NIC showed strong inhibitory effects on CYP1A2 and CYP2C8 and is not likely to become a time-dependent inhibitor. Our findings could contribute to the identification of essential factors in the pharmacokinetics of anthelmintics, potentially facilitating their repositioning.

Isosorbide, a versatile green chemical: Elucidating its ADME properties for safe use.

Isosorbide, an environmentally friendly and renewable substance, finds extensive application in diverse fields, such as a bisphenol A substitute, polymers, functional materials, organic solvents, fuels, and pharmaceuticals. Despite its increasing interest and widespread usage, there remains a notable absence of available reports regarding its absorption, distribution, metabolism, and excretion (ADME) properties. This study endeavors to investigate the ADME characteristics of isosorbide in rats. Isosorbide levels in biological samples were quantified based on the analytical method using gas chromatography-mass spectrometry (GC-MS). Following administration, isosorbide exhibited rapid absorption and elimination, with a bioavailability of 96.1%. The metabolic stability assay indicated that isosorbide remained stable during metabolism. The majority of absorbed isosorbide was promptly excreted, with urinary excretion as the primary route. This study furnishes valuable insights into the ADME of isosorbide, contributing to its safety assessment and fostering its continued application across various domains.

Evaluating flavonoids as potential aromatase inhibitors for breast cancer treatment: In vitro studies and in silico predictions.

Aromatase inhibitors are commonly employed in the treatment of hormone-dependent breast cancers, and flavonoids have emerged as a promising alternative to existing drug classes with unfavorable side effects. In this study, we conducted in vitro investigations into CYP19A1 (aromatase) inhibitory potential of 14 flavonoids, including pinocembrin, sakuranetin, eriodictyol, liquiritigenin, naringenin, hesperetin, flavanone, baicalein, chrysin, nobiletin, luteolin, sinensetin, tricin, and primuletin. Flavonoids displaying inhibitory activity were further assessed using in silico tools, such as molecular docking to predict binding affinities, as well as SwissADME, admetSAR, and QED (Quantitative Estimate of Drug-likeness) for drug-likeness prediction. Flavonoids with IC50 values less than 10 µM, pinocembrin, eriodictyol, naringenin, liquirtigenin, sakuranetin, and chrysin, exhibited favorable physicochemical properties and ADME profiles, suggesting their potential for development as novel flavonoid-based aromatase inhibitors. This study would provide valuable insights for the development of flavonoid-based aromatase inhibitors for the treatment of breast cancer.

The impact of the administration of red ginseng (Panax ginseng) on lipid metabolism and free fatty acid profiles in healthy horses using a molecular networking approach.

This study investigated the potential benefits of the administration of red ginseng (RG) on lipid metabolism and the profiles of individual free fatty acids (FFAs) in healthy horses. Eight healthy horses, raised under similar conditions, were randomly divided into two groups, each comprising four horses. The experimental group received powdered RG (600 mg/kg/day) mixed with a carrier, and the control group received only the carrier. The parameters associated with lipid metabolism and probable adverse effects were evaluated in both groups after 3 weeks. The computational molecular networking (MN) approach was applied to analyze the FFA profiles. The results indicated that RG administration significantly reduced blood triglyceride levels in the experimental group. Analysis of the FFAs using MN revealed significant decreases in specific types of FFAs (C12:0, dodecanoic acid; C14:0, myristric acid; C18:1, oleic acid; C18:2, linoleic acid). RG consumption did not produce significant adverse effects on the renal, hepatic, and immune functions. Thus, RG was found to effectively modulate lipid metabolism and the levels of individual FFAs. The application of the MN for the analysis of FFAs represents a novel approach and can be considered for future research.

Antioxidant Efficacy of Hwangryunhaedok-tang through Nrf2 and AMPK Signaling Pathway against Neurological Disorders In Vivo and In Vitro.

Alzheimer's disease (AD) is a representative cause of dementia and is caused by neuronal loss, leading to the accumulation of aberrant neuritic plaques and the formation of neurofibrillary tangles. Oxidative stress is involved in the impaired clearance of amyloid beta (Aß), and Aß-induced oxidative stress causes AD by inducing the formation of neurofibrillary tangles. Hwangryunhaedok-tang (HHT, Kracie K-09®), a traditional herbal medicine prescription, has shown therapeutic effects on various diseases. However, the studies of HHT as a potential treatment for AD are insufficient. Therefore, our study identified the neurological effects and mechanisms of HHT and its key bioactive compounds against Alzheimer's disease in vivo and in vitro. In a 5xFAD mouse model, our study confirmed that HHT attenuated cognitive impairments in the Morris water maze (MWM) test and passive avoidance (PA) test. In addition, the prevention of neuron impairment, reduction in the protein levels of Aß, and inhibition of cell apoptosis were confirmed with brain tissue staining. In HT-22 cells, HHT attenuates tBHP-induced cytotoxicity, ROS generation, and mitochondrial dysfunction. It was verified that HHT exerts a neuroprotective effect by activating signaling pathways interacting with Nrf2, such as MAPK/ERK, PI3K/Akt, and LKB1/AMPK. Among the components, baicalein, a bioavailable compound of HHT, exhibited neuroprotective properties and activated the Akt, AMPK, and Nrf2/HO-1 pathways. Our findings indicate a mechanism for HHT and its major bioavailable compounds to treat and prevent AD and suggest its potential.

A pharmacokinetic study on red ginseng with furosemide in equine.

Red ginseng (RG) is a popular ingredient in traditional Korean medicine that has various health benefits. It is commonly taken orally as a dietary supplement; however, its potential interactions with concomitantly administered drugs are unclear. In this study, we examined the pharmacokinetic interaction between furosemide and RG in equine plasma. Liquid chromatography with tandem mass spectrometry analysis was performed to evaluate ginsenosides in the plasma of horses after feeding them RG and furosemide and validate the results. A single bolus of furosemide (0.5 mg/kg) was administered intravenously to female horses that had consumed RG (600 mg/kg/day) every morning for 3 weeks (experimental group), and blood samples were collected from 0 to 24 h, analyzed, and compared with those from female horses that did not consume RG (control group). Four (20s)-protopanaxadiol ginsenosides (Rb1, Rb2, Rc, and Rd) were detected in the plasma. Rb1 and Rc individually showed a high concentration distribution in the plasma. The Cmax, AUC0-t, and AUC0-∞ of furosemide was significantly increased in the experimental group (p < 0.05), while the CL, Vz, and Vss was decreased (p < 0.05, p < 0.01). These changes indicate the potential for pharmacokinetic interactions between furosemide and RG.

Exploring Metabolic Pathways of Anamorelin, a Selective Agonist of the Growth Hormone Secretagogue Receptor, via Molecular Networking.

In this study, we delineated the poorly characterized metabolism of anamorelin, a growth hormone secretagogue receptor agonist, in vitro using human liver microsomes (HLM), based on classical molecular networking (MN) and feature-based molecular networking (FBMN) from the Global Natural Products Social Molecular Networking platform. Following the in vitro HLM reaction, the MN analysis showed 11 neighboring nodes whose information propagated from the node corresponding to anamorelin. The FBMN analysis described the separation of six nodes that the MN analysis could not achieve. In addition, the similarity among neighboring nodes could be discerned via their respective metabolic pathways. Collectively, 18 metabolites (M1-M12) were successfully identified, suggesting that the metabolic pathways involved were demethylation, hydroxylation, dealkylation, desaturation, and N-oxidation, whereas 6 metabolites (M13a*-b*, M14a*-b*, and M15a*-b*) remained unidentified. Furthermore, the major metabolites detected in HLM, M1 and M7, were dissimilar from those observed in the CYP3A4 isozyme assay, which is recognized to be markedly inhibited by anamorelin. Specifically, M7, M8, and M9 were identified as the major metabolites in the CYP3A4 isozyme assay. Therefore, a thorough investigation of metabolism is imperative for future in vivo studies. These findings may offer prospective therapeutic opportunities for anamorelin.

Investigation of Drug-Interaction Potential for Arthritis Dietary Supplements: Chondroitin Sulfate, Glucosamine, and Methylsulfonylmethane.

Osteoarthritis is one of the leading conditions that promote the consumption of these dietary supplements. Chondroitin sulfate, glucosamine, and methylsulfonylmethane are among the prominent alternative treatments for osteoarthritis. In this study, these dietary supplements were incubated with cytochrome P450 isozyme-specific substrates in human liver microsomes, and the formation of marker metabolites was measured to investigate their inhibitory potential on cytochrome P450 enzyme activities. The results revealed no significant inhibitory effects on seven CYPs, consistent with established related research data. Therefore, these substances are anticipated to have a low potential for cytochrome P450-mediated drug interactions with osteoarthritis medications that are likely to be co-administered. However, given the previous reports of interaction cases involving glucosamine, caution is advised regarding dietary supplement-drug interactions.

The Next Generation COVID-19 Antiviral; Niclosamide-Based Inorganic Nanohybrid System Kills SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic is a serious global threat with surging new variants of concern. Although global vaccinations have slowed the pandemic, their longevity is still unknown. Therefore, new orally administrable antiviral agents are highly demanded. Among various repurposed drugs, niclosamide (NIC) is the most potential one for various viral diseases such as COVID-19, SARS (severe acute respiratory syndrome), MERS (middle east respiratory syndrome), influenza, RSV (respiratory syncytial virus), etc. Since NIC cannot be effectively absorbed, a required plasma concentration for antiviral potency is hard to maintain, thereby restricting its entry into the infected cells. Such a 60-year-old bioavailability challenging issue has been overcome by engineering with MgO and hydroxypropyl methylcellulose (HPMC), forming hydrophilic NIC-MgO-HPMC, with improved intestinal permeability without altering NIC metabolism as confirmed by parallel artificial membrane permeability assay. The inhibitory effect on SARS-CoV-2  replication is confirmed in the Syrian hamster model to reduce lung injury. Clinical studies reveal that the bioavailability of NIC hybrid drug can go 4 times higher than the intact NIC. The phase II clinical trial shows a dose-dependent bioavailability of NIC from hybrid drug  suggesting its potential applicability as a game changer in achieving the much-anticipated endemic phase.

Effects of Plant-Based Extract Mixture on Alcohol Metabolism and Hangover Improvement in Humans: A Randomized, Double-Blind, Paralleled, Placebo-Controlled Clinical Trial.

Hangovers are uncomfortable physiological symptoms after alcohol consumption caused by acetaldehyde, a toxic substance in which alcohol is metabolized by alcohol dehydrogenase (ADH). Rapid alcohol and acetaldehyde decomposition are essential to alleviate alcohol handling symptoms. This study investigated the effects of HY_IPA combined with Mesembryanthemum crystallinum, Pueraria lobata flower, and Artemisia indica on alleviating hangovers. A randomized, double-blind, parallel-group, placebo-controlled clinical study was conducted on 80 individuals with hangover symptoms. Alcohol intake was 0.9 g/bw with 40% whiskey, adjusted proportionately to body weight. The Acute Hangover Scale total score was 5.24 ± 5.78 and 18.54 ± 18.50 in the HY_ IPA and placebo groups, respectively (p < 0.0001). All nine indicators of the hangover symptom questionnaire were significantly improved in the HY_IPA group (p < 0.01). Blood alcohol and acetaldehyde concentrations rapidly decreased from 30 min in the HY_IPA group (p < 0.05). ADH and acetaldehyde dehydrogenase (ALDH) activities in the blood of the HY_IPA group were significantly higher than those in the placebo group at 0, 1, and 2 h after alcohol consumption (p < 0.01). The rapid hangover relief was due to increased ADH and ALDH. Therefore, HY_IPA effectively relieves hangover symptoms by decomposing alcohol and acetaldehyde when consumed before alcohol consumption.

Preclinical Bioavailability Assessment of a Poorly Water-Soluble Drug, HGR4113, Using a Stable Isotope Tracer.

Drug solubility limits intravenous dosing for poorly water-soluble medicines, which misrepresents their bioavailability estimation. The current study explored a method using a stable isotope tracer to assess the bioavailability of drugs that are poorly water-soluble. HGR4113 and its deuterated analog, HGR4113-d7, were tested as model drugs. To determine the level of HGR4113 and HGR4113-d7 in rat plasma, a bioanalytical method using LC-MS/MS was developed. The HGR4113-d7 was intravenously administered to rats that were orally pre-administered HGR4113 at different doses; subsequently, the plasma samples were collected. HGR4113 and HGR4113-d7 were simultaneously determined in the plasma samples, and bioavailability was calculated using plasma drug concentration values. The bioavailability of HGR4113 was 53.3% ± 19.5%, 56.9% ± 14.0%, and 67.8% ± 16.7% after oral dosages of 40, 80, and 160 mg/kg, respectively. By eliminating the differences in clearance between intravenous and oral dosages at different levels, acquired data showed that the current method reduced measurement errors in bioavailability when compared to the conventional approach. The present study suggests a prominent method for evaluating the bioavailability of drugs with poor aqueous solubility in preclinical studies.

Pharmacokinetic Profiling of Ginsenosides, Rb1, Rd, and Rg3, in Mice with Antibiotic-Induced Gut Microbiota Alterations: Implications for Variability in the Therapeutic Efficacy of Red Ginseng Extracts.

Ginsenoside Rg3 is reported to contribute to the traditionally known diverse effects of red ginseng extracts. Significant individual variations in the therapeutic efficacy of red ginseng extracts have been reported. This study aimed to investigate the effect of amoxicillin on the pharmacokinetics of ginsenosides Rb1, Rd, and Rg3 in mice following the oral administration of red ginseng extracts. We examined the α-diversity and ß-diversity of gut microbiota and conducted pharmacokinetic studies to measure systemic exposure to ginsenoside Rg3. We also analyzed the microbiome abundance and microbial metabolic activity involved in the biotransformation of ginsenoside Rb1. Amoxicillin treatment reduced both the α-diversity and ß-diversity of the gut microbiota and decreased systemic exposure to ginsenoside Rg3 in mice. The area under the curve (AUC) values for Rg3 in control and amoxicillin-treated groups were 247.7 ± 96.6 ng·h/mL and 139.2 ± 32.9 ng·h/mL, respectively. The microbiome abundance and microbial metabolic activity involved in the biotransformation of ginsenoside Rb1 were also altered by amoxicillin treatment. The metabolizing activity was reduced from 0.13 to 0.05 pmol/min/mg on average. Our findings indicate that amoxicillin treatment potentially reduces the gut-microbiota-mediated metabolism of ginsenoside Rg3 in mice given red ginseng extracts, altering its pharmacokinetics. Gut microbiome variations may thus influence individual ginsenoside pharmacokinetics, impacting red ginseng extract's efficacy. Our results suggest that modulating the microbiome could enhance the efficacy of red ginseng.

The Intake of Coffee Increases the Absorption of Aspirin in Mice by Modifying Gut Microbiome.

The absorption of orally administered aspirin into the blood was affected by gastrointestinal environmental factors such as gut pH, digestive enzymes, and microbiota. The intake of coffee affects the pharmacological effects of aspirin. Therefore, we examined the gut microbiota-mediated effect of coffee bean extract (CBE) intake on the pharmacokinetics of aspirin in mice. The intake of CBE modified the gut microbiota composition and their α- and ß-diversities: It decreased the Proteobacteria, Helicobacteriaceae, and Bacteroidaceae populations in the fecal microbiota composition, while the S24-7_f (Muribaculaceae) and Lactobacillaceae populations increased. The fecal aspirin-hydrolyzing activities of humans and mice to salicylic acid were 0.045 ± 0.036 µmole/h/g and 0.032 ± 0.003 µmole/h/g, respectively. However, CBE treatment significantly suppressed the aspirin-hydrolyzing activity in mice. Furthermore, the area under the serum concentration-time curves (AUCs) of aspirin and salicylic acid were 0.265 ± 0.050 µg·h/mL and 16.224 ± 5.578 µg·h/mL in CBE-treated mice, respectively, and 0.248 ± 0.042 µg·h/mL and 10.756 ± 2.071 µg·h/mL in control mice, respectively. Moreover, CBE treatment suppressed the multidrug resistance protein 4 (Mrp4) expression in the intestines of mice, while the P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) expression was not affected. Furthermore, the CBE-treated mouse fecal lysate suppressed Mrp4 expression in Caco-2 cells compared to that of vehicle-treated mice, while CBE treatment did not affect Mrp4 expression. Oral gavage of caffeine also suppressed the Mrp4 expression in the intestines of mice. These findings suggest that intake of coffee can increase the absorption of aspirin by modifying the gut microbiome.

Molecular networking-guided strategy for the pharmacokinetic study of herbal medicines: Cudrania tricuspidata leaf extracts.

In this study, the pharmacokinetic profiles of the bioactive components in the leaf extract of the medicinal herb, Cudrania tricuspidate, were investigated using an MS/MS-based molecular networking system. To identify the major active components of the C. tricuspidate leaf extract (CLE), HPLC-DAD analysis was conducted with a standard mixture of six flavonoids (rutin, isoquercitrin, nicotiflorin, kaempferol 3-O-glucoside, quercetin, and kaempferol). The unknown peaks were determined via molecular networking analysis using the mass dataset obtained by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). For the subsequent pharmacokinetic study, CLE (1 g/kg) was orally administered to rats, and plasma samples were collected. The product ion mass data of plasma samples using LC-QTOF/MS were obtained and subjected to molecular networking analysis. The resulting molecular networking map indicated that the glucuronide metabolites of quercetin and kaempferol were the major circulating species. Accordingly, quercetin and kaempferol were determined following ß-glucuronidase treatment, and their pharmacokinetic parameters were calculated. These findings indicate that the proposed molecular network-based approaches are potential and efficient methods for the pharmacokinetic study of herbal medicines.

A quantitative method for the simultaneous detection of SR9009 and SR9011 in equine plasma using liquid chromatography-electrospray ionization-tandem mass spectrometry.

SR9009 and SR9011 are metabolic modulators pharmacologically targeting REV-ERB receptors as synthetic agonists. A liquid chromatography-tandem mass spectrometry method for the detection of SR9009 and SR9011 in equine plasma was developed and validated. Plasma samples were pretreated by protein precipitation with methanol and were loaded onto an ACQUITY ultra performance liquid chromatography high-strength silica C18 column (2.1 × 150 mm, 1.8 µm) for chromatographic separation. The mobile phase consisted of 5-mM ammonium formate (pH 3.0) in distilled water and 0.1% formic acid in acetonitrile, and a gradient elution was used at a flow rate of 0.25 ml/min. For the mass spectrometry detection, the selected reaction monitoring mode was used with transitions of 438.2 → 124.9 for SR9009, 479.2 → 125.1 for SR9011, and 292.2 → 109.1 for the internal standard (testosterone-d3) in the positive ionization mode. The linearity, lower limit of quantification, intra- and inter-day precision, accuracy, matrix effect, recovery, and stability were evaluated. The method was found to be accurate and reproducible for the quantitation of SR9009 and SR9011. The developed method was successfully applied to plasma samples of thoroughbreds injected intramuscularly with SR9009.

Tandem Mass Spectrometry Molecular Networking as a Powerful and Efficient Tool for Drug Metabolism Studies.

Molecular networking (MN) has become a popular data analysis method for untargeted mass spectrometry (MS)/MS-based metabolomics. Recently, MN has been suggested as a powerful tool for drug metabolite identification, but its effectiveness for drug metabolism studies has not yet been benchmarked against existing strategies. In this study, we compared the performance of MN, mass defect filtering, Agilent MassHunter Metabolite ID, and Agilent Mass Profiler Professional workflows to annotate metabolites of sildenafil generated in an in vitro liver microsome-based metabolism study. Totally, 28 previously known metabolites with 15 additional unknown isomers and 25 unknown metabolites were found in this study. The comparison demonstrated that MN exhibited performances comparable or superior to those of the existing tools in terms of the number of detected metabolites (27 known metabolites and 22 unknown metabolites), ratio of false positives, and the amount of time and effort required for human labor-based postprocessing, which provided evidence of the efficiency of MN as a drug metabolite identification tool.

Discovery of Mycobacterium tuberculosis Rv3364c-Derived Small Molecules as Potential Therapeutic Agents to Target SNX9 for Sepsis.

The serine protease inhibitor Rv3364c of Mycobacterium tuberculosis (MTB) is highly expressed in cells during MTB exposure. In this study, we showed that the 12WLVSKF17 motif of Rv3364c interacts with the BAR domain of SNX9 and inhibits endosome trafficking to interact with p47phox, thereby suppressing TLR4 inflammatory signaling in macrophages. Derived from the structure of this Rv3364c peptide motif, 2,4-diamino-6-(4-tert-butylphenyl)-1,3,5-trazine, DATPT as a 12WLVSKF17 peptide-mimetic small molecule has been identified. DATPT can block the SNX9-p47phox interaction in the endosome and suppress reactive oxygen species and inflammatory cytokine production; it demonstrated significant therapeutic effects in a mouse model of cecal ligation and puncture-induced sepsis. DATPT has considerably improved potency, with an IC50 500-fold (in vitro) or 2000-fold (in vivo) lower than that of the 12WLVSKF17 peptide. Furthermore, DATPT shows potent antibacterial activities by reduction in ATP production and leakage of intracellular ATP out of bacteria. These results provide evidence for peptide-derived small molecule DATPT with anti-inflammatory and antibacterial functions for the treatment of sepsis.

Study of cobalt doping control via various routes in thoroughbred horses.

Cobalt is a substance that has been abused for athletic performance enhancement and has thus been prohibited by human and animal sports doping control authorities. However, because cobalt is present in humans and animals as a trace element, a certain level of cobalt is naturally present in their excretions. In the racing industry, cobalt is a controlled substance with a threshold concentration specified by the International Agreement on Breeding, Racing and Wagering (IABRW) for international harmonization. Due to environmental and feed consumption differences among countries, regional cobalt concentration trends should be evaluated before cobalt testing is introduced. In this study, we conducted a preliminary evaluation of the urinary concentration of cobalt among a population of racehorses in Korea using inductively coupled plasma mass spectrometry (ICP-MS) analysis, followed by analysis of the urinary release of cobalt after the administration of cobalt chloride in various situations. The normal distribution for the Korea-based racehorses was used to determine a urine concentration limit (96.5 ng/ml, risk factor of 1 in 10,000). After the intravenous (IV) administration of CoCl2 , the initial elimination of cobalt was rapid. A high concentration (over 2,000 ng/ml) and a slow excretion pattern were observed during the final 2 weeks of the 3-week observation period. When CoCl2 was administered orally, maximum concentration (Cmax , 92-992 ng/ml) was observed at 6-8 h.