Potential herb-drug interaction risk of thymoquinone and phenytoin.

Thymoquinone is a main bioactive compound of Nigella sativa L. (N.sativa), which has been used for clinical studies in the treatment of seizures due to its beneficial neuroprotective activity and antiepileptic effects. It has been evidenced that thymoquinone may inhibit the activity of cytochrome P450 2C9 (CYP2C9). However, little is known about the effect of thymoquinone or N.sativa on the pharmacokinetic behavior of phenytoin, a second-line drug widely used in the management of status epilepticus. In this study, we systematically investigated the risk of the potential pharmacokinetic drug interaction between thymoquinone and phenytoin. The inhibitory effect of thymoquinone on phenytoin hydroxylation activity by CYP2C9 was determined using UPLC-MS/MS by measuring the formation rates for p-hydroxyphenytoin (p-HPPH). The potential for drug-interaction between thymoquinone and phenytoin was quantitatively predicted by using in vitro-in vivo extrapolation (IVIVE). Our data demonstrated that thymoquinone displayed effective inhibition against phenytoin hydroxylation activity. Enzyme kinetic studies showed that thymoquinone exerted a competitive inhibition against phenytoin hydroxylation with a Ki value of 4.45 ± 0.51 µM. The quantitative prediction from IVIVE suggested that the co-administration of thymoquinone (>18 mg/day) or thymoquinone-containing herbs (N.sativa > 1 g/day or N.sativa oil >1 g/day) might result in a clinically significant herb-drug interactions. Additional caution should be taken when thymoquinone or thymoquinone-containing herbs are co-administered with phenytoin, which may induce unexpected potential herb-drug interactions via the inhibition of CYP2C9.

Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and ß of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

Risk prediction of drug-drug interaction potential of phenytoin and miconazole topical formulations.

The drug-drug interaction (DDI) risk of phenytoin with several topical formulations of miconazole is still unclear. The present investigation conducted in vitro-in vivo extrapolation to predict the potential risks. Our data indicated that miconazole potently inhibited phenytoin hydroxylation in both pooled human liver microsomes (HLMs) and recombinant cytochrome P450 2C9 (CYP2C9) with the Ki values of 125 ± 7 nM and 30 ± 2 nM, respectively. Quantitative prediction of DDI risk suggests that, beside intravenous administration or swallowed tablet, combination of phenytoin and miconazole high dose oral gel or buccal tablet may also result in a clinically significant increase of phenytoin AUC (>53%) by the inhibition of miconazole against phenytoin hydroxylation, consequently a higher frequency of adverse events, while the coadministration of miconazole vaginal formulation and phenytoin will be safe.

Midazolam as a Probe for Drug-Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation.

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F-F' loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug-drug interactions in human P450 CYP3A4.

Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies.

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6ß-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (Km) for progesterone 6ß-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6ß-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6ß-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6ß-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6ß-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6ß-hydroxylation mediated by all CYP3A subfamily members. The inhibitory/stimulatory pattern of steroid-steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated.

Minoxidil Cannot Be Used To Target Lysyl Hydroxylases during Postnatal Mouse Lung Development: A Cautionary Note.

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking Plod1 exhibit 15% mortality, and mice lacking Plod2 or Plod3 exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen toxicity-based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg⋅kg-1⋅day-1 as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg⋅kg-1⋅day-1 to newborn mouse pups, minoxidil was detected in the lungs but did not impact lysine hydroxylation, collagen crosslinking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg⋅kg⋅day-1, pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. SIGNIFICANCE STATEMENT: Extracellular matrix crosslinking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen crosslinking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl hydroxylases as pathogenic mediators of disease and potential "druggable" targets. Minoxidil, administered at the maximum possible dose, did not inhibit lysyl hydroxylation in newborn mouse lungs, suggesting that minoxidil was unlikely to be of use in studies that pharmacologically target lysyl hydroxylation in vivo.

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism.

BACKGROUND: Styrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions. PURPOSE: This study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered. STUDY DESIGN: The inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated. METHODS: The inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis. RESULTS: In vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6ß-hydroxylation. CONCLUSION: Styrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.

Inhibitory effects of sesamin on CYP2C9-dependent 7-hydroxylation of S-warfarin.

A recent report demonstrated that sesamin strongly and non-competitively inhibits S-warfarin 7-hydroxylation activity in human liver microsomes with a Ki value of 0.2 µM. This finding suggests that sesamin predominantly binds to CYP2C9 at another site for which it has a higher affinity than its affinity for the active site, thereby inhibiting the activity of CYP2C9 non-competitively. In this study, we found that sesamin competitively inhibited the 7-hydroxylation activity of S-warfarin in human liver microsomes with a Ki value of 15.7 µM. In addition, the recombinant CYP2C9-dependent 7-hydroxylation activity of S-warfarin was competitively inhibited by sesamin with a Ki value of 13.1 µM. These results are consistent with the fact that sesamin is a good substrate of CYP2C9, and its activity follows Michaelis-Menten kinetics. As the plasma concentration of sesamin after its administration is usually lower than 0.01 µM, the inhibition of S-warfarin metabolism by sesamin does not appear to be severe.

Aspartate/asparagine-ß-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors.

The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-ß-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z'-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.

Metabolic targeting of HIF-1α potentiates the therapeutic efficacy of oxaliplatin in colorectal cancer.

Drug resistance is a major problem limiting the efficacy of chemotherapy in cancer treatment, and the hypoxia-induced stabilization of HIF-1α plays a role in this process. HIF-1α overexpression has been observed in a variety of human cancers, including colorectal cancer (CRC). Therefore, targeting HIF-1α is a promising strategy for overcoming chemoresistance to enhance the efficacy of chemotherapies in CRC. Here, we show that DNMT inhibitors can induce HIF-1α degradation to overcome oxaliplatin resistance and enhance anti-CRC therapy. We found that a low-toxicity DNMT inhibitor, zebularine, could downregulate HIF-1α expression and overcome hypoxia-induced oxaliplatin resistance in HCT116 cells and showed efficacy in HCT116 xenograft models and AOM/DSS-induced CRC mouse models. Zebularine could induce the degradation of HIF-1α protein through hydroxylation. LC-MS analysis showed a decrease in succinate in various CRC cells under hypoxia and in colon tissues of AOM/DSS-induced CRC mice. The decrease was reversed by zebularine. Tumor angiogenesis was also reduced by zebularine. Furthermore, zebularine potentiated the anticancer effect of oxaliplatin in AOM/DSS-induced CRC models. This finding provides a new strategy in which an increase in HIF-1α hydroxylation could overcome oxaliplatin resistance to enhance anti-CRC therapy.

Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450.

Benzalkonium chlorides (BACs) are widely used as disinfectants in cleaning products, medical products, and the food processing industry. Despite a wide range of reported toxicities, limited studies have been conducted on the metabolism of these compounds in animal models and none in human-derived cells or tissues. In this work, we report on the metabolism of BACs in human liver microsomes (HLM) and by recombinant human hepatic cytochrome P450 (CYP) enzymes. BAC metabolism in HLM was NADPH-dependent and displayed apparent half-lives that increased with BAC alkyl chain length (C10 < C12 < C14 < C16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. Tandem mass spectrometry fragmentation of BAC metabolites confirmed that oxidation occurs on the alkyl chain region. Major metabolites of C10-BAC were identified as ω-hydroxy-, (ω-1)-hydroxy-, (ω, ω-1)-diol-, (ω-1)-ketone-, and ω-carboxylic acid-C10-BAC by liquid chromatography-mass spectrometry comparison with synthetic standards. In a screen of hepatic CYP isoforms, recombinant CYP2D6, CYP4F2, and CYP4F12 consumed substantial quantities of BAC substrates and produced the major microsomal metabolites. The use of potent pan-CYP4 inhibitor HET0016, the specific CYP2D6 inhibitor quinidine, or both confirmed major contributions of CYP4- and CYP2D6-mediated metabolism in the microsomal disappearance of BACs. Kinetic characterization of C10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ω-hydroxylation (Vmax = 380 pmol/min/mg, Km = 0.69 µM) and (ω-1)-hydroxylation (Vmax = 126 pmol/min/mg, Km = 0.13 µM) reactions. This work illustrates important roles for CYP4-mediated ω-hydroxylation and CYP2D6/CYP4-mediated (ω-1)-hydroxylation during the hepatic elimination of BACs, an environmental contaminant of emerging concern. Furthermore, we demonstrate that CYP-mediated oxidation of C10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.

Vitamin D regulates prostate cell metabolism via genomic and non-genomic mitochondrial redox-dependent mechanisms.

Vitamin D deficiency has been associated with increased risk for aggressive prostate cancer (PCa). Prostate epithelium has a unique metabolism compared to other tissues. Normal prostate exhibits low levels of mitochondrial respiration and there is a metabolic switch to increased oxidative phosphorylation in PCa. 25-hydroxyvitamin D (25(OH)D) is the major circulating form of vitamin D and is used clinically to determine vitamin D status. Activation of 25(OH)D to the transcriptionally active form, 1,25(OH)2D occurs via a reduction-oxidation (redox) reaction within the mitochondria that is catalyzed by the P450 enzyme, CYP27B1. We sought to determine if hydroxylation of 25(OH)D by CYP27B1 contributes to non-genomic activity of vitamin D by altering the redox-dependent state of the mitochondria in benign prostate epithelial cells. Exposure to 25(OH)D produced a transient pro-oxidant effect and change in mitochondrial membrane potential that was dependent on CYP27B1. Extended exposure ultimately suppressed mitochondrial respiration, consistent with a protective effect of 25(OH)D in supporting benign prostate metabolism. To model physiologically relevant changes in vitamin D, cells were cultured in constant 25(OH)D then changed to high or deficient concentrations. This model also incurred a biphasic effect with a pro-oxidant shift after short exposure followed by decreased respiration after 16 h. Several genes involved in redox cycling and Mitochondrial Health were regulated by 25(OH)D in these cells. These results indicate a secondary non-genomic mechanism for vitamin D to contribute to prostate cell health by supporting normal mitochondrial respiration.

In vitro and in vivo study of the toxicity of fullerenols С60, С70 and С120О obtained by an original two step method.

The toxicity of C60(OH)30, C70(OH)30, and C120O(OH)n fullerenols, prepared by a new original method, has been studied. This method allowed us to obtain high-purity fullerenols and eliminate the risks of synthesis of preparations containing insoluble fractions contaminated with impurities such as fullerenes not completely reacted by hydroxylation. All fullerenols were detected inside cultured cells. The MTT assay as well as the analysis of apoptosis and cell cycle showed that С60(ОН)30 and С70(OH)30 are non-toxic for cultured V79 и HeLa cells at concentrations exceeding physiological levels by an order of magnitude. С120O(OH)n caused low toxicity. Studies in Drosophila melanogaster showed that any preparations used did not result in a decreased lifespan or in behavior abnormalities in flies.

Drug Metabolite Cluster-Based Data-Mining Method for Comprehensive Metabolism Study of 5-hydroxy-6,7,3',4'-tetramethoxyflavone in Rats.

The screening of drug metabolites in biological matrixes and structural characterization based on product ion spectra is among the most important, but also the most challenging due to the significant interferences from endogenous species. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of prototype drug metabolites using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Although classical techniques are well-suited for achieving the partial characterization of prototype drug metabolites, there is a pressing need for a strategy to enable comprehensive drug metabolism depiction. Therefore, we present drug metabolite clusters (DMCs), different from, but complementary to, traditional approaches for mining the information regarding drugs and their metabolites on the basis of raw, processed, or identified tandem mass spectrometry (MS/MS) data. In this paper, we describe a DMC-based data-mining method for the metabolite identification of 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF), a typical hydroxylated-polymethoxyflavonoid (OH-PMF), which addressed the challenge of creating a thorough metabolic profile. Consequently, eight primary metabolism clusters, sixteen secondary metabolism clusters, and five tertiary metabolism clusters were proposed and 106 metabolites (19 potential metabolites included) were detected and identified positively and tentatively. These metabolites were presumed to generate through oxidation (mono-oxidation, di-oxidation), methylation, demethylation, methoxylation, glucuronidation, sulfation, ring cleavage, and their composite reactions. In conclusion, our study expounded drug metabolites in rats and provided a reference for further research on therapeutic material basis and the mechanism of drugs.

EglN3 hydroxylase stabilizes BIM-EL linking VHL type 2C mutations to pheochromocytoma pathogenesis and chemotherapy resistance.

Despite the discovery of the oxygen-sensitive regulation of HIFα by the von Hippel-Lindau (VHL) protein, the mechanisms underlying the complex genotype/phenotype correlations in VHL disease remain unknown. Some germline VHL mutations cause familial pheochromocytoma and encode proteins that preserve their ability to down-regulate HIFα. While type 1, 2A, and 2B VHL mutants are defective in regulating HIFα, type 2C mutants encode proteins that preserve their ability to down-regulate HIFα. Here, we identified an oxygen-sensitive function of VHL that is abolished by VHL type 2C mutations. We found that BIM-EL, a proapoptotic BH3-only protein, is hydroxylated by EglN3 and subsequently bound by VHL. VHL mutants fail to bind hydroxylated BIM-EL, regardless of whether they have the ability to bind hydroxylated HIFα or not. VHL binding inhibits BIM-EL phosphorylation by extracellular signal-related kinase (ERK) on serine 69. This causes BIM-EL to escape from proteasomal degradation, allowing it to enhance EglN3-induced apoptosis. BIM-EL was rapidly degraded in cells lacking wild-type VHL or in which EglN3 was inactivated genetically or by lack of oxygen, leading to enhanced cell survival and chemotherapy resistance. Combination therapy using ERK inhibitors, however, resensitizes VHL- and EglN3-deficient cells that are otherwise cisplatin-resistant.

Effect of IS01957, a para-coumaric acid derivative on pharmacokinetic modulation of diclofenac through oral route for augmented efficacy.

Diclofenac is one of the world's largest selling nonsteroidal anti-inflammatory drugs. The major concerns related to oral diclofenac therapy are gastrointestinal and cardiovascular side effects for which explicitly emphasis has been given to use it at lowest effective dose for the shortest duration. On the other hand, IS01957 has been designed under the purview of anti-inflammatory drug and bioavailability enhancer. IS01957 have dual action on inflammation and nociception with acceptable safety profile. In the quest for a suitable combination with improved therapeutic efficacy and better tolerability, pharmacodynamic and pharmacokinetic interaction studies were performed for diclofenac with or without IS01957 in mice model. Results showed that IS01957 enhanced both anti-inflammatory effect and plasma concentration of diclofenac upon concomitant oral administration. These interesting results steered to enumerate the possible role of IS01957 towards diclofenac pharmacokinetics through a panel of mechanistic investigations: (a) BCRP dependent ATPase activity was markedly interfered by IS01957; (b) IS01957 increased the intestinal permeability of diclofenac in the single pass in-situ perfusion model; (c) IS01957 inhibited the CYP2C9 catalyzed diclofenac 4-hydroxylation in human liver microsomes. Immunoblotting results suggest that diclofenac action was improved significantly in the presence of IS01957 involving MAPK pathways. Finally acute gastric damage study showed that IS01957 in combination with diclofenac was better to improve the desired PGE2 level as compare to alone. In nutshell, IS01957 have potential to augment the efficacy of diclofenac through pharmacokinetic modulation. Further investigations are required for dose reduction of diclofenac to combat its liabilities before going into clinical setting.

Pharmacokinetic interference of doxorubicin with tolbutamide due to reduced metabolic clearance with increased serum unbound fraction in rats.

The study examined the effect of doxorubicin (DOX) on the hepatic expression of CYP2C and its activity for metabolizing tolbutamide (TB), a specific CYP2C substrate, in rats and whether the pharmacokinetics of tolbutamide were altered by doxorubicin exposure. The expression level of hepatic CYP2C11 was depressed 1 day after doxorubicin administration (day 1), and this effect on CYP2C11 was augmented on day 4. However, the expression level of hepatic CYP2C6 remained unchanged. The activity of tolbutamide 4-hydroxylation in hepatic microsomes was decreased with time following doxorubicin administration. Regarding the enzyme kinetic parameters for tolbutamide 4-hydroxylation on day 4, the maximum velocity (Vmax ) was significantly lower in the DOX group than that in the control group, while the Michaelis constant (Km ) was unaffected. On pharmacokinetic examination, the total clearance (CLtot ) of tolbutamide on day 4 was increased, despite the decreased metabolic capacity. On the other hand, the serum unbound fraction (fu ) of tolbutamide was elevated with a reduced serum albumin concentration in the DOX group. Contrary to CLtot , CLtot /fu , a parameter approximated to the hepatic intrinsic clearance of unbound tolbutamide, was estimated to be significantly reduced in the DOX group. These findings indicate that the metabolic capacity of CYP2C11 in the liver is depressed time-dependently by down-regulation after doxorubicin exposure in rats, and that the decreased enzyme activity of TB 4-hydroxylation in hepatic microsomes reflects the pharmacokinetic change of unbound tolbutamide, not total tolbutamide, in serum.

Breast cancer cells rely on environmental pyruvate to shape the metastatic niche.

The extracellular matrix is a major component of the local environment-that is, the niche-that determines cell behaviour1. During metastatic growth, cancer cells shape the extracellular matrix of the metastatic niche by hydroxylating collagen to promote their own metastatic growth2,3. However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen, because nutrients dictate which enzymatic reactions are active in cancer cells4,5. Here we show that breast cancer cells rely on the nutrient pyruvate to drive collagen-based remodelling of the extracellular matrix in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activates collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast-cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodelling and the nutrient environment in the metastatic niche.

Glutamate affects the CYP1B1- and CYP2U1-mediated hydroxylation of arachidonic acid metabolism via astrocytic mGlu5 receptor.

The extrahepatic CYP enzymes, CYP1B1 and CYP2U1, have been predominantly found in both astrocytes and brain microvessels. We investigated the alteration in the production of hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) mainly via CYP1B1 and CYP2U1 by glutamate. CYP1B1 and CYP2U1 mRNA levels were dose-dependently induced by glutamate in human U251 glioma cells and hCMEC/D3 blood-brain barrier cells. The increases in the CYP1B1 and CYP2U1 mRNA levels and the binding of CREB to CYP1B1 and CYP2U1 promoters following glutamate treatment were attenuated by mGlu5 receptor antagonist. The mRNA levels of CYP1B1 and CYP2U1 were increased in the cortex, hippocampus, and cerebellum from adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7 days of age; meanwhile, the protein levels of CYP1B1 and CYP2U1 in the astrocytes were induced by glutamate. Glutamate treatment significantly increased the production of 5-HETE, 8-HETE, 11-HETE, and 20-HETE in the cortex and cerebellum. These data suggested that the neuron-astrocyte reciprocal signaling can change the CYP-mediated AA metabolism (e.g. EETs and HETEs) in astrocytes via its specific receptor.

d-amino acid modification protects N-Acetyl-seryl-aspartyl-lysyl-proline from physiological hydroxylation and increases its antifibrotic effects on hepatic fibrosis.

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development in the liver. However, its extremely short half-life in vivo greatly compromises its potential applications. Here, we report an Ac-SDKP analog peptide with d-amino acid replacement (Ac-SDD KD P). The stability of Ac-SDD KD P and its prevention of liver fibrosis were investigated in vitro and in vivo. The stabilities of Ac-SDKP and Ac-SDD KD P exposed to angiotensin-1-converting enzyme (ACE) and their half-lives in rats and human sera were determined by high-performance liquid chromatography. The inhibitory effects of Ac-SDKP and Ac-SDD KD P on the proliferation and activation of hepatic stellate cells (HSC-T6) were evaluated using the Cell Counting Kit-8, Western blotting, reverse transcription quantitative polymerase chain reaction, and immunofluorescence assays. Finally, the protective effects of Ac-SDKP and Ac-SDD KD P on carbon tetrachloride (CCl4 )-induced liver fibrosis in rats were compared. d-Amino acid replacement significantly enhanced the stability of the peptide to ACE and prolonged the half-life of Ac-SDKP in rats and human sera. The Ac-SDKP-mediated inhibition of HSC-T6 cell proliferation was well preserved, and Ac-SDD KD P exerted inhibitory effects comparable to Ac-SDKP on α-smooth muscle actin (α-SMA), collagen I and III expression, and phosphorylated-Smad-2 expression. After intraperitoneal (i.p.) administration, Ac-SDD KD P exhibited significantly greater protection than Ac-SDKP against CCl4 -induced liver fibrosis in rats. The serum alanine aminotransferase, aspartate aminotransferase, albumin, and total protein levels of the Ac-SDD KD P-treated rats were significantly lower than those of the Ac-SDKP-treated rats. α-SMA, CD45, and collagen I and III expression, as well as Smad-2 phosphorylation were significantly attenuated in the livers of the Ac-SDD KD P-treated rats compared to those of the Ac-SDKP-treated rats. Furthermore, we showed that the Ac-SDD KD P concentration in the rat liver increased to a physiological level of 60 min after i.p. administration, although i.p. administration of Ac-SDKP failed to enhance the peptide concentration in the rat liver. Our findings indicate that d-amino acid replacement is a simple and effective method to enhance the stability of Ac-SDKP. Ac-SDD KD P represents potential application of Ac-SDKP in fibrosis treatment and provides a new potential treatment strategy for liver fibrosis. © 2019 IUBMB Life, 71(9):1302-1312, 2019.