Human UDP-Glucuronosyltransferase (UGT) 2B10: Validation of Cotinine as a Selective Probe Substrate, Inhibition by UGT Enzyme-Selective Inhibitors and Antidepressant and Antipsychotic Drugs, and Structural Determinants of Enzyme Inhibition.

Although there is evidence for an important role of UGT2B10 in the N-glucuronidation of drugs and other xenobiotics, the inhibitor selectivity of this enzyme is poorly understood. This study sought primarily to characterize the inhibition selectivity of UGT2B10 by UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors used for reaction phenotyping, and 34 antidepressant and antipsychotic drugs that contain an amine functional group. Initial studies demonstrated that cotinine is a highly selective substrate of human liver microsomal UGT2B10. The kinetics of cotinine N-glucuronidation by recombinant UGT and human liver microsomes (± bovine serum albumin) were consistent with the involvement of a single enzyme. Of the UGT enzyme-selective inhibitors employed for reaction phenotyping, only the UGT2B4/7 inhibitor fluconazole reduced recombinant UGT2B10 activity to an appreciable extent. The majority of antidepressant and antipsychotic drugs screened for effects on UGT2B10 inhibited enzyme activity with IC50 values <100 µM. The most potent inhibition was observed with the tricyclic antidepressants amitriptyline and doxepin and the tetracyclic antidepressant mianserin, and the structurally related compounds desloratadine and loratadine. Molecular modeling using a ligand-based approach indicated that hydrophobic and charge interactions are involved in inhibitor binding, whereas spatial features influence the potency of UGT2B10 inhibition. Respective mean Ki,u (± S.D.) values for amitriptyline, doxepin, and mianserin inhibition of human liver microsomal UGT2B10 were 0.61 ± 0.05, 0.95 ± 0.18, and 0.43 ± 0.01 µM. In vitro-in vivo extrapolation indicates that these drugs may perpetrate inhibitory drug-drug interactions when coadministered with compounds that are cleared predominantly by UGT2B10.

Inhibition of Human UDP-Glucuronosyltransferase Enzymes by Canagliflozin and Dapagliflozin: Implications for Drug-Drug Interactions.

Canagliflozin (CNF) and dapagliflozin (DPF) are the first sodium-glucose cotransporter 2 inhibitors to be approved for clinical use. Although available evidence excludes clinically significant inhibition of cytochromes P450, the effects of CNF and DPF on human UDP-glucuronosyltransferase (UGT) enzymes are unknown. Here, we report the inhibition of human recombinant UGTs by CNF and DPF, along with the Ki values for selected recombinant and human liver microsomal UGTs. CNF inhibited all UGT1A subfamily enzymes, but the greatest inhibition was observed with UGT1A1, UGT1A9, and UGT1A10 (IC50 values ≤ 10 µM). DPF similarly inhibited UGT1A1, UGT1A9, and UGT1A10, with IC50 values ranging from 39 to 66 µM. In subsequent kinetic studies, CNF inhibited recombinant and human liver microsomal UGT1A9; Ki values ranged from 1.4 to 3.0 µM, depending on the substrate (propofol/4-methylumbelliferone) enzyme combination. Ki values for CNF inhibition of UGT1A1 were approximately 3-fold higher. Consistent with the activity screening data, DPF was a less potent inhibitor of UGT1A1 and UGT1A9. The Ki for DPF inhibition of UGT1A1 was 81 µM, whereas the Ki values for inhibition of UGT1A9 ranged from 12 to 15 µM. Based on the in vitro Ki values and plasma concentrations reported in the literature, DPF may be excluded as a perpetrator of DDIs arising from inhibition of UGT enzymes, but CNF inhibition of UGT1A1 and UGT1A9 in vivo cannot be discounted. Since the sodium-glucose cotransporter 2 inhibitors share common structural features, notably a glycoside moiety, investigation of drugs in this class for effects on UGT to identify (or exclude) potential drug-drug interactions is warranted.

Albendazole stimulates the excretion of strongyloides stercoralis Larvae in stool specimens and enhances sensitivity for diagnosis of strongyloidiasis.

We succeeded in stimulation of excretion of Strongyloides stercoralis larvae in stool by oral administration of a single dose of 400 mg albendazole to strongyloidiasis patients. This result overcame the false-negative results of stool examination due to low larval numbers. Stool samples were collected from 152 asymptomatic strongyloidiasis patients in the morning, prior to eating. After breakfast, they were given a dose of 400 mg albendazole, and stool samples were collected the following morning. Agar plate culture (APC), modified formalin-ether concentration technique (MFECT), and direct-smear (DS) methods were used to examine stool specimens within 3 h after defecation. The results before and after albendazole was taken were compared. All APCs that were positive became negative after albendazole administration, while MFECT showed a 1.4- to 18.0-fold increase in larval numbers in 97.4% (148/152) of the samples. The DSs were positive in 3 out of 3 smears at a larval number of ≥45 larvae per g (lpg) of stool, and in 1or 2 out of 3 smears at a larval number between 35 and 44 lpg. At a larval number of <35 lpg, the DS became negative. Interestingly 90.5% (19/21) of the samples that were negative by all methods before albendazole administration became positive by MFECT after the treatment. Thus, MFECT can be effectively used for diagnosis of strongyloidiasis with prior administration of albendazole to the subject.

Factors affecting recovery of Strongyloides stercoralis larvae: an approach to a newly modified formalin-ether concentration technique for diagnosis of strongyloidiasis.

To improve the diagnosis efficiency of human strongyloidiasis by using formalin-ether concentration technique (FECT), the effects of various factors on the recovery rates of Strongyloides stercoralis larvae were comparatively evaluated. Fresh stool and a short time exposure of larvae to formalin yielded significantly higher numbers of larvae than preserved stool and 10-min exposure. Likewise, straining through wire mesh yielded a significantly higher number of larvae recovered than straining through gauze did. In addition, centrifugation for 5 min for separation of larvae from debris yielded a significantly greater number of larvae recovered than centrifugation for 2 min did. The efficacies of the five versions of FECT with different factors--FECT 1, FECT 2, FECT 3, FECT 4, and FECT 5--were then compared. It was found that FECT 5 was 1.8, 2.0, 1.9, and 1.4 times more effective than FECT 1, FECT 2, FECT 3, and FECT 4, respectively. FECT 5 is a modified FECT method, whose modifications included using fresh stool without a preservative substance; a short-time rather than 10-min formalin exposure; and the use of wire mesh instead of gauze.

Effect of dilution of stool soluble component on growth and development of Strongyloides stercoralis.

Dispersion or dilution of stool by water from heavy rainfall may affect Strongyloides stercoralis free-living development producing infective filariform larvae (FL). This study examined effect of water dilution of stool on survival of S. stercoralis free-living development. One g of stool was prepared in water so that its soluble component was diluted sequentially from 1:2 to 1:480. Three dishes were used to compare FL production in three culture conditions: stool suspension, stool sediment deposited in soil, and isolated rhabditiform larvae (RhL) deposited in soil. The fourth dish was for developmental observation of RhL into free-living stages. Numerous FL were generated from undiluted or 1:2 diluted stool and stool sediment placed on soil. However, starting from dilution 1:5, FL production continuously decreased in both stool suspensions and stool sediments placed on soil. RhL isolated from stool dilutions placed on soil gave rise to few FL. Worm mating were seen at 24-30 hours in dilutions 1:20-1:120 only. Highest numbers of FL from indirect free-living cycle were 1/3 of those from control. FL production decreased as stool dilution increased, and reached zero production at 1:160 dilution. Rainfall may disperse or dilute stool so that nutritional supplement for S. stercoralis free-living development is insufficient.

Detrimental effect of water submersion of stools on development of Strongyloides stercoralis.

Strongyloidiasis is prevalent in Thailand, yet its prevalence in the south is lower than in other parts of the country. This might be due to the long rainy season in the south resulting in stool submersion in water inhibiting worm development. In this study, the effect of water submersion of fecal samples on development of Strongyloides stercoralis was investigated. Ten ml of a 1 ∶ 5 fecal suspension were placed in 15-ml tubes, 35-mm dishes, and 90-mm dishes producing the depths of 80 mm, 11 mm and 2 mm-suspensions, respectively. The worm development was followed at 1/6, 4, 6, 8, 10, 12, 14, 16, 24, and 36 h, by determining the number of filariform larva (FL) generated from agar-plate cultures (APC). Fecal suspensions kept in tubes and 35-mm dishes showed a decline in FL yield relative to incubation time and reached zero production 14 h after incubation. In contrast, the number of FL generated from the suspension kept in 90-mm dishes remained stable up to 36 h. Cumulatively, all tubes and 35-mm dishes became negative in APC after 14 h while 90-mm dishes remained APC-positive up to 36 h. Adding more water or stool suspension to dishes resulted in a decreased number of FL. Mechanical aeration of the suspensions in tubes restored an almost normal FL yield. It appears that the atmospheric air plays a significant role in growth and development of S. stercoralis in the environment and may be one of factors which contribute to a lower prevalence of human strongyloidiasis in the south of Thailand.