In Vivo Bioavailability and Metabolism of Topical Diclofenac Lotion in Human Volunteers

Purpose. The primary objective of this study was to determine the rate and extent of transdermal absorption for systemic delivery of diclofenac from Pennsaid (Dimethaid Research, Inc.) topical lotion into the systemic circulation after the lotion was applied to human volunteers, in an open treatment, non-blinded, non-vehicle controlled study. In addition, the in vivo metabolism of this topical diclofenac lotion has also been studied. Methods. Human volunteers were dosed with topical [¹⁴C]-diclofenac sodium 1.5% lotion on the knee for 24 h. Sequential time blood and urine samples were taken to determine pharmacokinetics, bioavailability and metabolism. Results. Topical absorption was 6.6% of applied dose. Peak plasma ¹⁴C occurred at 30 h after dosing, and peak urinary ¹⁴C excretion was at 24–48 h. The urinary ¹⁴C excretion pattern exhibits more elimination towards 24 h and beyond, as opposed to early urinary ¹⁴C excretion. This suggests a continuous delivery of [¹⁴C]-diclofenac sodium from the lotion into and through skin which only ceased when the dosing site was washed. Skin surface residue at 24 h was 26 ± 9.5% dose (remainder assumed lost to clothing and bedding). Extraction of metabolites from urine amounted to 7.4–22.7% in untreated urine, suggesting substantial diclofenac metabolism to more water soluble metabolites, probably conjugates, which could not be extracted by the method employed. Two Dimensional TLC analysis of untreated urine showed minimal or no diclofenac, again emphasizing the extensive in vivo metabolism of this drug. Treatment of the same urine samples with the enzymes sulfatase and (-glucuronidase showed a substantial increase in the extractable material. Three spots were consistently present in each sample run, namely diclofenac, 3hydroxy diclofenac and an intermediate polar metabolite (probably a hydroxylated metabolite). Therefore, there was significant sulfation and glucuronidation of both diclofenac and numerous hydroxy metabolites of diclofenac, but many of the metabolites/conjugates remain unidentified. Conclusions. There was a continuous delivery of diclofenac sodium from the lotion into and through the skin, which ceased after the dosing site was washed. The majority of the material excreted in the urine were conjugates of hydroxylated metabolites, and not the parent chemical, although further identification is required.     

Fungal Metabolism of 4-Methylprimaquine

4-Methylprimaquine (4-MPQ), an 8-aminoquinoline antimalarial drug, was subjected to microbial metabolism studies in an effort to isolate, identify, and predict the probable mammalian metabolite(s). Preliminary screening with a number of strains of Aspergillus indicated that several species were capable of metabolizing 4-MPQ to one major metabolite based on thin-layer chromatographic analysis. Preparative scale conversion of 4-MPQ using whole-cell suspensions of Aspergillus ochraceus afforded one major metabolite (4-MPQ-I). Based on spectroscopic and chemical evidence, the structure of the metabolite is proposed as 6-methoxy-8-(3-carboxyl-l-methylpropylamino)-lepidine.     

白藜芦醇PLGA长效注射微球的制备及工艺考察

目的采用乳化溶剂挥发法制备白藜芦醇聚乳酸羟基乙酸[poly(lactic-co-glycolic acid),PL-GA]长效微球,评价各因素对微球性质的影响。方法以微球的包封率、载药量、突释和粒径作为微球的质量评价指标,研究分散相与连续相的体积比、PLGA浓度、聚乙烯醇(polyvinyl alcohol,PVA)浓度、搅拌速度对微球性质的影响,并优化白藜芦醇PLGA微球的制备工艺。结果分散相与连续相的体积比为1∶50时,包封率高,但4 h突释量达到76%,当分散相与连续相体积比由1∶50提升到1∶150时,突释降低了22%;随着聚合物浓度的增加粒径明显增大,突释显著降低;理论载药量对粒径影响不大,在高载药量时突释显著减少;搅拌速度的增加使粒径减小,突释增加;PVA浓度的增加对粒径没有明显的影响,但当PVA的质量浓度从1 g.L-1增加到5 g.L-1时,包封率从93.57%降低到80.31%。结论分散相与连续相的体积比、PLGA浓度、PVA浓度、搅拌速度对微球性质有很大的影响。优化条件下制备的微球形态完整,载药量为(27.86±1.00)%,包封率为(93.57±2.87)%,平均粒径约为21.12μm。白藜芦醇PLGA微球体外释放25 d的累积释药率达(94.04±4.94)%,有望研制成1个月给药1次的给药系统。     

用液相质谱法鉴定盐酸关附甲素在大鼠胆汁中Ⅰ相和Ⅱ相代谢产物(英文)

目的:研究盐酸关附甲素在大鼠胆汁中的代谢产物.方法:建立了液相质谱和串联质谱法(LC-MS)对关附甲素及其代谢产物鉴定的方法.大鼠静脉注射盐酸关附甲素后采集胆汁,通过与对照化合物的色谱保留时间、分子离子峰、碎片离子峰和紫外图谱对照从而鉴定Ⅰ相代谢物.胆汁经过葡萄糖醛酸酶或硫酸酯酶水解,鉴定其水解产物(苷元),从而确定Ⅱ相结合物,再通过LC-MS分离和确定分子离子峰,最后利用MS-MS寻找特征子离子和母离子的方法进行验证.结果:大鼠胆汁中存在Ⅰ相代谢物关附壬素;Ⅱ相结合物经葡萄糖醛酸酶和硫酸酯酶酶解后,出现关附甲素和关附壬素;LC-MS检测发现胆汁中m/z 606和 510两个准分子离子峰,推测分别为关附甲素葡萄糖醛酸苷和关附甲素硫酸酯:经MS-MS鉴定出m/z 606特征子离子m/z 177和m/z 430,进一步确证大鼠胆汁中存在关附甲素葡萄糖醛酸苷.结论:大鼠胆汁中存在Ⅰ相代谢产物关附壬素,以及Ⅱ相结合物关附甲素葡萄糖醛酸和硫酸结合物、关附壬素葡萄糖醛酸和硫酸结合物.     

Metabolism and hepatotoxicity of N,N-dimethylformamide,N-hydroxymethyl-N-methylformamide,and N-methylformamide in the rat

The metabolism and hepatotoxicity ofN,N-dimethylformamide (DMF) and two of its metabolites,N-hydroxymethyl-N-methylformamide (HMMF) andN-methylformamide (NMF) were evaluated over a 4-day period in rats. DMF toxicity was dose dependent and delayed toxicity after the administration of a high DMF dose (13.7 mmol/kg) in comparison to a lower dose (4.1 mmol/kg) was observed. Treatment of rats with 13.7 mmol/kg DMF, HMMF, or NMF showed i) that DMF is more toxic than HMMF or NMR, and ii) that hepatotoxicity occurs later for DMF than for HMMF or NMF. Analysis of serum and urine samples demonstrated that DMF is first metabolized to HMMF, which is then partially converted to NMF. After HMMF administration, NMF was found both in serum and in urine. The time course of DMF and HMMF toxicity in relation to NMF formation fitted the hypothesis that the hepatotoxicity of DMF and HMMF is mediated via NMF. The degree of hepatotoxicity after HMMF and NMF treatment is similar. However, the degree of DMF hepatotoxicity is much higher than in the case of NMF or HMMF. The role of NMF as an obligatory intermediate in DMF and HMMF hepatotoxicity is discussed.     

Microbial Models of Mammalian Metabolism: Stereoselective Metabolism of Warfarin in the Fungus Cunninghamella elegans

Biotransformation stereoselectivity of warfarin was studied in the fungus Cunninghamella elegans (ATCC 36112) as a model of mammalian metabolism. This organism was previously shown to produce all known phenolic mammalian metabolites of warfarin, including 6-, 7-, 8-, and 4-hydroxywarfarin, and the previously unreported 3-hydroxywarfarin, as well as the diastereomeric warfarin alcohols, warfarin diketone, and aliphatic hydroxywarfarins. Using S-warfarin and R-warfarin as substrates, and an HPLC assay with fluorescence detection to analyze metabolite profiles, the biotransformation of warfarin was found to be highly substrate and product stereoselective. Both aromatic hydroxylation and ketone reduction were found to be stereoselective for R-warfarin. Ketone reduction with the warfarin enantiomers exhibited a high level of product stereoselectivity in that R-warfarin was predominantly reduced to its S-alcohol, while S-warfarin was reduced primarily to the corresponding R-alcohol.     

<Emphasis Type="Italic">In Vitro</Emphasis> Metabolism of a New Neuroprotective Agent,KR-31543 in the Human Liver Microsomes: Identification of Human Cytochrome P450

KR-31543, (2S,3R,4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amino]-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran, is a new neuroprotective agent for preventing ischemia-reperfusion damage. This study was performed to identify the metabolic pathway of KR-31543 in human liver microsomes and to characterize cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-31543. Human liver microsomal incubation of KR-31543 in the presence of NADPH resulted in the formation of two metabolites, M1 and M2. M1 was identified as N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amine on the basis of LC/MS/MS analysis with a synthesized authentic standard, and M2 was suggested to be hydroxy-KR-31543. Correlation analysis between the known CYP enzyme activities and the rates of the formation of M1 and M2 in the 12 human liver microsomes have showed significant correlations with testosterone 6beta-hydroxylase activity (a marker of CYP3A4). Ketoconazole, a selective inhibitor of CYP3A4, and anti-CYP3A4 monoclonal antibodies potently inhibited both N-hydrolysis and hydroxylation of KR-31543 in human liver microsomes. These results provide evidence that CYP3A4 is the major isozyme responsible for the metabolism of KR-31543 to M1 and M2.     

Resveratrol, a dietary polyphenolic phytoalexin, is a functional scavenger of peroxynitrite

Oxidant damage from reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major contributor to the cellular damage seen in numerous types of renal injury. Resveratrol (trans-3,4′,5-trihydroxystilbene) is a phytoalexin found naturally in many common food sources. The anti-oxidant properties of resveratrol are of particular interest because of the fundamental role that oxidant damage plays in numerous forms of kidney injury. To examine whether resveratrol could block damage to the renal epithelial cell line, mIMCD-3, cells were exposed to the peroxynitrite donor 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1). Resveratrol produced a concentration-dependent inhibition of cytotoxicity induced by SIN-1. To examine the mechanism of protection, resveratrol was incubated with authentic peroxynitrite and found to block nitration of bovine serum albumin with an EC₅₀ value of 22.7 μM, in contrast to the known RNS scavenger, N-acetyl-l-cysteine, which inhibited nitration with an EC₅₀ value of 439 μM. These data suggested that resveratrol could provide functional protection by directly scavenging peroxynitrite. To examine whether resveratrol was a substrate for peroxynitrite oxidation, resveratrol was reacted with authentic peroxynitrite. Resveratrol nitration products and dimers were detected using liquid chromatograph with tandem electrospray mass spectrometry. Similar products were detected in the media of cells treated with SIN-1 and resveratrol. Taken collectively, the data suggest that resveratrol is able to provide functional protection of renal tubular cells, at least in part, by directly scavenging the RNS peroxynitrite. This property of resveratrol may contribute to the understanding of its anti-oxidant activities.     

Comparison Between Permeability Coefficients in Rat and Human Jejunum

Purpose. Our main aim is to determine the effective intestinal permeability (P_eff) in the rat jejunum in situ for 10 compounds with different absorption mechanisms and a broad range of physico chemical properties, and then compare them with corresponding historical human in vivo P_eff values. Methods. The rat P_eff coefficients are determined using an in situ perfusion model in anaesthetized animals. The perfusion flow rate used is 0.2 ml/min, which is 10 times lower than that used in humans. The viability of the method is assessed by testing the physiological function of the rat intestine during perfusions. Results. The P_eff for passively absorbed compounds is on average 3.6 times higher in humans compared to rats (P_eff,man = 3.6 · P_eff,rat + 0.03·10^–4; R^2 = 1.00). Solutes with carrier-mediated absorption deviate from this relationship, which indicates that an absolute scaling of active processes from animal to man is difficult, and therefore needs further investigation. The fraction absorbed of drugs after oral administration in humans (fa) can be estimated from 1 – e^–(2·P _eff,man ^·t _res ^/r·2.8). Conclusions. Rat and human jejunum P_eff estimates of passively absorbed solutes correlate highly, and both can be used with precision to predict in vivo oral absorption in man. The carrier-mediated transport requires scaling between the models, since the transport maximum and/or substrate specificity might differ. Finally, we emphasize the absolute necessity of including marker compounds for continuous monitoring of intestinal viability.     

HPLC Assay for FK 506 and Two Metabolites in Isolated Rat Hepatocytes and Rat Liver Microsomes

Despite the current use of a standard two-step enzyme immunoassay in the clinical monitoring of the immunosuppressant FK 506, the lack of specificity for the parent drug in this assay renders it unsuitable for drug metabolism studies. An HPLC assay has been developed for studying the metabolism of FK 506 in isolated hepatocytes and microsomal mixtures. This assay allows simultaneous measurement of the parent drug and two of its time dependent metabolites. Metabolism of this drug was studied in intact rat liver cells and rat liver microsomes. We have shown that the metabolites observed are products of phase 1 oxidation reactions. Correlation of the 6-testosterone hydroxylase activity with the FK 506 metabolite (Ml) initial formation rate is consistent with the belief that CYP 3A isozymes are involved in FK 506 metabolism in male rats.     

Transdermal Delivery of Bioactive Peptides: The Effect of n-Decylmethyl Sulfoxide,pH, and Inhibitors on Enkephalin Metabolism and Transport

We investigated the effects of the nonionic surfactant, n-decylmethyl sulfoxide (NDMS), pH, and inhibitors on the metabolism and the permeation of amino acids, dipeptides, and the pentapeptide enkephalin, through hairless mouse skin. An HPLC gradient method was developed to identify the possible peptide and amino acid metabolites of leucine-enkephalin. NDMS increased the permeability of all amino acids and peptides tested. At neural pH, the enzyme activity within the skin was such that no flux of leucine-enkephalin (YGGFL) was observed and the donor cell concentration of YGGFL decreased rapidly. The major cleavage occurred at the Tyr-Gly bond. At pH 5.0 the metabolic activity was reduced significantly and a substantial flux of YGGFL was observed. Enzymatically stable YGGFL analogues, Tyr-D-Ala-Gly-Phe-Leu (YDAGFL) and its amide, exhibited significant fluxes even at neutral pH in the presence of NDMS, but with substantial metabolism. YDAGFL amide was more stable to metabolism than YDAGFL. The rates of metabolism of the peptides in the skin homogenates were in the order: FL.>>YGGFL > GFL > GGFL >> YG, YGG >> YDAGFL amide. In the skin homogenates puromycin and amastatin showed the highest inhibitory effects, while FL and GFL were only slightly active. However, in the skin diffusion experiments, FL allowed the highest amount of intact parent compound to permeate, making it the most potent inhibitor. These results show that the complex proteolytic enzyme activities occurring during skin permeation are different from those in skin homogenates and that a combination of enhancer, pH adjustment, and inhibitors can increase the transdermal delivery of peptides.     

Vehicle Effects on in Vitro Percutaneous Absorption Through Rat and Human Skin

We studied the effects of three vehicles (propylene glycol, octanol and ethyl decanoate) with differing polarity on the in vitro percutaneous absorption of three chemicals (fluazifop-butyl, dimethyl phthalate and fomesafen sodium salt) with a range of physico-chemical properties. Absorption rate measurements were made from high vehicle volume (200 µl/cm²) and low vehicle volume (<10 µLl/cm²) applications. For the lipophilic fluazifop-butyl absorption rate was highest from the more polar vehicle propylene glycol, but this effect was only significant under high-volume conditions. There was a variable vehicle effect on absorption of the intermediate chemical dimethyl phthalate. The largest vehicle effect was seen for the more hydrophilic fomesafen sodium salt where absorption was fastest from the least polar vehicle ethyl decanoate. These results support the hypothesis that the absorption process can in part be predicted from a knowledge of solute solubility. Vehicle effects were greater from high volume applications than from those more comparable to occupational exposure conditions.     

Metabolism of bupropion by baboon hepatic and placental microsomes

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (K_m 36 ± 6 μM, V_max 258 ± 32 pmol mg protein⁻¹ min⁻¹), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent K_m values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.     

Intestinal Metabolism of DNOC and DNBP in the Rat

Abstract The metabolism of the dinitrophenols DNOC and DNBP and their respective monoamino derivatives, 6-ANOC and 6-ANBP, was studied in rat caecal incubates. All of the compounds were reduced to their corresponding diamino metabolites. The results are discussed in relation to the comparative toxicology of the dinitrophenols with special regard to a possible significance of the intestinal metabolites.     

Disposition and Oxidative Metabolism of Antipyrine in the Rat

Abstract The main routes of excretion of antipyrine-N-methyl-¹⁴C and the role of oxidation in the metabolism of antipyrine were studied in the rat. After an intraperitoneal dose of 15 mg/kg ¹⁴C-antipyrine (1.0-1.5 uCi ¹⁴C), 74.2% of the radioactivity was found in the urine, 2.5% appeared in the faeces and 2.6% was recovered from expired CO₂ after 48 hrs. Most of the radioactivity (98%) was excreted during the first 24-hrs. The cumulative excretion of radioactivity in the bile amounted to 18% of the dose in 24 hrs. Only 1-2% of the radio activity in the urine represented unchanged antipyrine. Antipyrine was extensively metabolized by oxidation followed by conjugation. In urine 3-CH₂OH-antipyrine and 4-OH-antipyrine (unconjugated and conjugated) accounted for 35 and 18% of the radioactivity, respectively. 3-carboxy antipyrine represented 17% of the radioactivity. The corresponding figures for bile were 51,19 and 9%. In addition, the non-radioactive N-demethylated metabolite, nor-antipyrine, was found. The quantitation of N-demethylation based on radioactivity recovered from expired CO₂ suggested that at least 2.6% of antipyrine was metabolized by this pathway. The results show that antipyrine was extensively oxidized in the rat. Thus, in this species, antipyrine meets a fundamental requirement of a model compound for the study of factors influencing drug oxidation in vivo.     

Pharmacokinetics and First-pass Metabolism of Levomepromazine in the Rat

Abstract: The time course of the blood concentrations of levomepromazine and its two major non-polar metabolites in man were studied in the rat after single oral and intraarterial doses of levomepromazine hydrochloride. The blood levels of N-monodesmethyl levomepromazine were on average 179% of the levomepromazine levels after oral doses, but only 15% of the levomepromazine levels after intraarterial doses. The blood levels of levomepromazine sulfoxide, relative to the levomepromazine levels, were also generally higher after oral doses than after parenteral doses, on average 65% and 25%, respectively. Large interindividual variations were observed in the blood levels of levomepromazine and in the systemic availability of the drug after oral doses. The distribution phase lasted for about 8 hrs, the mean apparent volume of distribution was 16.6 l/kg, and the total body clearance was on average 12.3 ml/min. It is concluded that the rat provides a suitable model for the kinetics of levomepromazine in man, and that the sulfoxide and N-monodesmethyl metabolites of the drug are mainly formed by first-pass metabolism after oral doses in the rat, either in the liver or in the gut.     

Preclinical Drug Metabolism in the Age of High-Throughput Screening: An Industrial Perspective

With the advent of genomics, combinatorial paradigms and high-throughput screen (HTS)-based pharmacological testing, the number of compounds flowing through the discovery pipeline is likely to escalate. At the same time, with increased knowledge of the human drug-metabolizing enzymes and the availability of in vitro absorption-metabolism (AM) models, Preclinical Drug Metabolism is poised to meet the challenges of HTS. In order to be successful, however, a rational HTS strategy (vs. serendipitous HTS) has to be employed. Such a strategy is based on automation, validation and integration of in vitroAM models and database management (AVID). A generalized strategy for rational (AVID-based) HTS in Preclinical Drug Metabolism is described briefly.     

In Vivo Evaluation of Acyclovir Prodrug Penetration and Metabolism Through Rat Skin Using a Diffusion/Bioconversion Model

Purpose. In order to evaluate the in vivo penetration of prodrugs which undergo metabolism in skin, we analyzed thein vivo penetration profiles of acyclovir prodrugs based on a two-layer skin diffusion model in consideration of metabolic process. Methods. Acyclovir prodrugs (e.g., valerate, isovalerate and pivarate) were used as model prodrugs and the amounts excreted in urine were measured after percutaneous application. In vivo penetration profiles were then estimated by employing a deconvolution method and the penetration of acyclovir prodrugs was analyzed using a diffusion model. Subsequently, diffusion, partitioning and metabolic parameters were compared under in vitro and in vivo conditions. Results. Although total penetration amounts at the end of the experiment were similar for the three prodrugs, the ratio of intact prodrug to total penetration amount differed significantly. Moreover, the excretion and absorption profiles were also very different for each prodrug. Enzymatic hydrolysis rate constants calculated under in vivo conditions were considerably larger than those obtained in the skin homogenate and in vitro penetration experiments. Conclusions. The present skin diffusion/bioconversion model combined with computer analysis enables us to comprehensively account for diffusion, partitioning and metabolism during in vivo percutaneous absorption. Nevertheless, different enzymatic hydrolysis rate constants obtained under bothin vivo and in vitro conditions demonstrate the difficulty of obtaining accurate values for in vivo enzymatic activity from related in vitro experiments.     

Development of an in Vitro Rat Intestine Segmental Perfusion Model to Investigate Permeability and Predict Oral Fraction Absorbed

Purpose The aims of the study are to develop and evaluate an in vitro rat intestine segmental perfusion model for the prediction of the oral fraction absorbed of compounds and to assess the ability of the model to study intestinal metabolism. Methods The system consisted of a perfusion cell with a rat intestinal segment and three perfusion circulations (donor, receiver, and rinsing circulation). Lucifer yellow (LY) was applied as internal standard together with test compounds in the donor circulation. To validate the model, the permeability of eight noncongeneric passively absorbed drugs was determined. Intestinal N-demethylation of verapamil into norverapamil was followed in the donor and receiver circulations by high-performance liquid chromatography analysis. Results The in vitro model allowed ranking of the tested compounds according to their in vivo absorption potential. The Spearman's correlation coefficient between the oral fraction absorbed in humans and the ratio of permeation coefficient of test compound to the permeation coefficient of LY within the same experiment was 0.98 (P < 0.01). Moreover, intestinal N-demethylation of verapamil, its permeation, and the permeation of its metabolite norverapamil could be assessed in parallel. Conclusions Up to six permeation kinetics can be obtained per rat, and the method has shown to be a valuable tool to estimate human oral absorption.