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Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues
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Kinetics of glyburide metabolism by humans and baboons using liver and placental tissues

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  • 1. • Primary objectives: – To determine if the in vitro metabolism of glyburide in human and baboon placenta microsomes is similar – Do these results support the use of the baboon as an appropriate in vivo model for testing the effects of glyburide on the fetus? 1
  • 2. • Humannutrients from the mother Supplies the fetus with oxygen and Placenta – Feto-placental-maternal circulation becomes established at 10 weeks – Until then, chemicals pass by diffusion and embryos are most susceptible• Active endocrine organ vital to the homeostasis of pregnancy – Synthesizes hormones and other bioactive molecules• Acts as a protective barrier by metabolizing foreign chemicals• Xenobiotics pass mainly by passive diffusion – Dependent on MW, polarity, pH, lipophilicity, and protein binding – Metabolism by placenta • Oxidation, reduction, conjugation, hydrolysis • Usually far less active than maternal and fetal liver • Thought to be involved in steroidogenesis• Specific transport mechanisms transfer: – Xenobiotics, nutrients, and metabolic waste products 2
  • 3. Anatomy and Physiology 3
  • 4. Placentophagy• Most mammals eat the placenta (except most humans)• Tom Cruise in GQ magazine: – ""Im going to eat the placenta. I thought that would be good. Very nutritious. Im going to eat the cord and the placenta right there."• No real danger in eating one’s placenta only someone else’s – HIV, hepatitis, etc.• Believed to help with: – Postpartum depression – Postpartum hemorrhaging • High in progesterone and oxytocin• Lots of recipes for placenta tartare, pizza, spaghetti, jerky, cocktails• Some people bury it and plant a tree over it• Some instances of art projects  placental prints 4
  • 5. Teratogenicity• Classes of drugs commonly taken during pregnancy – Analgesics, anti-emetics, anti-bacterials, tranquilizers, anti- histamines – Drugs to treat maternal conditions: epilepsy, diabetes, HIV – Drugs to stimulate or inhibit uterine contractions• Approximately 200,000 babies/year born with a birth defect – Approximately 1% are chemically induced• Teratogens and consequences (FDA has a rating system) – Cigarette smoke  inhibits fetal growth – Thalidomide  severe limb malformations – Alcohol  “fetal alcohol syndrome” = mental and growth retardation, etc. – Cocaine  intrauterine death – Retinoic acid (Accutane)  malformations, low IQ 5
  • 6. Warfarin (Coumadin)• Anticoagulant O Warfarin (650 µM): Liver vs. Placenta OH (S) = 30X more turnover (R) = 58X more turnover Rettie, et. al. O O• Teratogenic effect of warfarin is dependent on time of dosage – Early pregnancy (1st trimester)  fetal warfarin syndrome • Hypoplastic nose, eye abnormalities, mental retardation, bone abnormalities – Late stages of pregnancy (2nd and 3rd trimesters)  • CNS abnormalities: mental and growth retardation, etc.• Teratogenicity is thought to be tied to its mechanism of action (VKER inhibition) 6
  • 7. DrugTransfer• Fetal blood pH is 0.1 units less than maternal blood pH• Fetal albumin and α1-acid glycoprotein concentration compared to maternal is 10% and 30%, respectively, at 12 weeks and increases to 30- 40% and 120%, respectively, at term 7
  • 8. Drug Transport and Metabolism• Levels of these enzymes are regulated by foreign chemicals• Transporters – > 10 identified: • Pgp, MRP1-3, BCRP, SERT, NET, OCT3, OCTN, MCTs, NaDC3, SMVT – Vary in their location in the placenta and substrate selectivity• CYPs – approximately 10-30% of maternal liver CYP amount – Mostly steroid metabolizing CYPs, xenobiotic metabolizing CYPs are minor – > 15 identified • 1A1, 1A2, 1B1, 2A6, 2A7, 2A13, 2B6, 2B7, 2C, 2D6, 2E1, 2F1, 3A3-7, 4B1 – Type and amount vary with gestation and maternal health • More CYP isoforms are expressed in the first trimester than at term• Conjugating enzymes – UDP-GTs – high interindividual variability – GSTs – very active – Epoxide hydrolase and sulfotransferases are also present 8
  • 9. 9
  • 10. 10
  • 11. Esterases in Placenta• Presence of cholinesterases in placenta – Cocaine can be hydrolyzed at term – Localization varies with gestation – Plasma BuChE is reduced in maternal and fetal plasma• Carboxylesterases are present in placenta – Cholinesterase inhibitors only inhibit half of the esterase activity – Multiple CEs have been identified • Sequence similarity to hCE-1 and hCE-2 – Interindividual variability can be as high as 3-fold 11
  • 12. Anatomy and Physiology• Placenta changes as gestation proceeds – Increase in placenta surface area and placental thinning – Blood flow increases from 50 mL/min (10 weeks)  600 mL/min (term) – Necessary for increased nutritional and energy requirements• 3 types of mammalian placenta – Classified by number of layers separating maternal and fetal blood • Haemochorial (rat, rabbit, guinea pig, HUMAN) – Fetal tissue is in direct contact with maternal blood • Endotheliochorial (cat, dog) • Epitheliochorial (sheep, pig, horse) – Structural differences affect function and drug transfer • Makes in vivo data from animals difficult to apply to the human situation 12
  • 13. Full Term Human Placentatransfer of:gases Early pregnancynutrients 50-100 µmwastexenobiotics Late pregnancy 4-5 µm 13
  • 14. Glyburide (glibenclamide) O H H• 2ndgeneration sulfonylurea S N N – used for treating type 2 diabetes O O O – 35 years Cl N H• Clinical trials: O – Gestational diabetes • 14% of pregnant women suffer from • 20-60% of those require therapy to control glucose levels • Glyburide ~ insulin for treatment – Cheaper and longer shelf-life• Want to understand role of placenta at different gestational ages in vivo – Can’t use humans – Baboon model • Non-human primate • Similar placental structure • Possible manifestation of diabetes • 95% DNA homology to humans 14
  • 15. Previous Studies• CYP2C9, 2C19, and 3A4 are involved in the metabolism of glyburide• Metabolic profile of glyburide is similar in rats, dogs, rabbits, and monkeys 15
  • 16. Tissue Material Used• Pool of 12 human placentae (term)  microsomes – Collected immediately after delivery• Pools of 11 baboon placentae and livers  microsomes – Placentae were collected by C-section• Pool of 15 HLMs purchased from CellzDirect 16
  • 17. Incubations• Total volume = 1 mL – 100 mM KPi (pH 7.4) – 0.5 or 1 mg PMs or LMs – 0-120 µM glyburide – Preincubated for 5 min at 37◦C – Reaction initiated by NADPH-GS and incubated for 15 min – Terminated by adding 10 µL of 10% (w/v) TCA and setting on ice – Add IS = estrone (20 µL) – Centrifuged – Metabolites were extracted with 3 mL CH2CL2-hexane (1:1, v/v) – Organic layer was dried, reconstituted in 150 µL mobile phase, and 100 µL was injected onto the HPLC 17
  • 18. Incubations and Analysis• Standard Incubations• HPLC-MS/MS – Wavelength = 203 nm – ACN:H2O (33:67) - pH 3.5 - 1.2 mL/min for 40 min - 1.2-1.5 mL/min 40-60 min - 1.5 mL/min 60-90 min - 1/3 of flow directed to the MS - SIM at m/z 510 for glyburide metabolites - SIM at m/z 272 for estrone (IS) 18
  • 19. Glyburide Metabolites 1 mg HPMs, 60 µM glyburide, NADPH-GS• Required NADPH-GS• Co-eluted with standards – M1-M4• M5 was identified by MS/MS pattern• M = mystery peak – < 1% of total metabolism 19
  • 20. Glyburide Metabolites All observed in vitro using HLMs only M1 and M2b observed in vivo active active • Compared to synthetic standards except M5 • in vivo M1:M2b = 4:1 • M5 was predicted based on MS/MS data 20
  • 21. Kinetics of Metabolism in HPMs ~ 10X higher• Saturation kinetics observed in all microsomes used 21
  • 22. Eadie-Hofstee Plots of HPM Metabolism V max -Km• Km calculations: – HPMs: 12 µM for all metabolites, except M4 (2 µM and 55 µM) – HLMs: 5 µM for all metabolites – BPMs: 10 µM for all metabolites, except M4 (43 µM) – BLMs: 6 µM for all metabolites 22
  • 23. Human vs. Baboon Comparison • HLM and BLM metabolic profiles are similar • % M1-M3 formed: PMs << LMs • % M4 formed: BPMs (65%) >> HPMs (5%) • % M5 formed: HPMs (87%) >> BPMs (16%) 23
  • 24. Summary • Glyburide metabolism: – Major metabolite was M1 for HLMs and M5 for all others – Rate of HLMs ~ BLMs– HLMs are 17X faster than HPMs, BLMs are 340X faster than BPMs – HPMs are 13X faster than BPMs 24
  • 25. Glyburide Metabolites Detected Major metabolite in HLMs Major metabolite in HPMs, BLMs, and BPMs 25
  • 26. Conclusions and Future Directions• M5 accounted for 22% of metabolites but was undetected in vivo – Not formed in vivo, undetected, or further metabolized• 2 CYPs (high and low Km) involved in formation of M4• PMs vs. LMs: same 7 metabolites formed but in different ratios – LMs are much more active than PMs – Different CYPs? Different enzyme expression levels? – Similar Kms  same CYPs, different levels – Different metabolite ratios  different CYPs• Different metabolite ratios were formed in HPMs vs. BPMs – Baboons are probably NOT a good in vivo model for glyburide• Future: – Need to look for new metabolites in vivo – Test their activity 26

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