In vitro Studies: Inhibition of Nevirapine Metabolism by Nortriptyline in Hepatic Microsomes

Materials and Reagents

1. 10 ml polypropylene tube 16 x 95 mm (Deltalab, catalog number: 400900 )
   
2. 5 ml ultracentrifuge tube 13 x 51 mm (Beckman Coulter, catalog number: 326819 )
   
3. 1.5 ml screw cap vial 32 x 11.6 mm
   
4. 7.5 ml screw cap tube 13 x 100 mm
   
5. Wistar rat livers
   
6. Saline solution (Laboratorios ERN, catalog number: 999790 )
   
7. Protein Standard (2 mg BSA) (BD Biosciences, Falcon^®, catalog number: P5619 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: P5619”.
   
8. Na₂CO₃ (EMD Millipore Corporation, catalog number: 106392 )
   
9. Copper(II) sulfate pentahydrate (CuSO₄^.5H₂O) (Sigma-Aldrich, catalog number: 209198 )
   
10. Potassium sodium tartrate tetrahydrate (Sigma-Aldrich, catalog number: 217255 )
    
11. Folin-Ciocalteu’s phenol reagent (EMD Millipore Corporation, catalog number: 109001 )
    
12. Potassium phosphate monobasic (KH₂PO₄) (BD Biosciences, Falcon^®, catalog number: 60229 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 60229”.
    
13. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, Falcon^®, catalog number: 56814 )
    
14. KCl (EMD Millipore Corporation, catalog number: 104936 )
    
15. Ethylendiaminetetraacetic acid disodium salt-2-hydrate (EDTA) (Riedel-de Haën, catalog number: 34549 )
    Note: Currently, it is “Sigma-Aldrich, FLUKA, catalog number: 34549 ”.
    
16. Sucrose (BD Biosciences, Falcon^®, catalog number: 84100 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 84100”.
    
17. K₂HPO₄^.3H₂O (AppliChem GmbH, catalog number: 122333 )
    
18. Nevirapine (Viramune) (Boehringer Ingelheim)
    
19. Methanol, HiPerSolv CHROMANORM for HPLC-Gradient Grade (VWR International, Prolabo, catalog number: 20864.320 )
    
20. Dimethyl sulfoxide (DMSO) (minimum 99.5% GC, plant cell culture tested) (Sigma -Aldrich, catalog number: D4540 )
    
21. Nortriptyline hydrochloride, minimum 98% TLC (Sigma-Aldrich, catalog number: N7261 )
    
22. D-Glucose 6-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G7250 )
    
23. β-Nicotinamide adenine dinucleotide phosphate disodium salt (β-NADP) (Sigma-Aldrich, catalog number: 93205 )
    
24. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma-Aldrich, catalog number: G5760 )
    
25. MgCl₂^.6H₂O, BioReagent, suitable for cell culture, suitable for insect cell culture (Sigma-Aldrich, catalog number: M2393 )
    
26. Acetonitrile (VWR, Prolabo, catalog number: 83639.320 )
    
27. Human liver microsomes pooled from 50 different individual donors (Thermo Fisher Scientific, Invitrogen^TM, catalog number: HMMCPL )
    
28. Sodium hydroxide (extra pure, pellets) (Scharlab,S.L., catalog number: SO0420005P )
    
29. Ethyl acetate (extra pure) (Scharlab,S.L., catalog number: AC01431000 )
    
30. NaH₂PO₄^.2H₂O (Guinama, catalog number: 90790 )
    
31. Triethylamine (Sigma-Aldrich, catalog number: T0886 )
    
32. 50 mM sodium-potassium phosphate buffer (see Recipes)
    
33. 0.1 M potassium phosphate buffer (see Recipes)
    
34. Lowry reagent (see Recipes)
    
35. 50 mM sodium phosphate monobasic buffer (see Recipes)
    

Equipment

1. Surgical tools
   
2. Scissors
   
3. Spectrophotometer (Hitachi High-Technologies Corporation, model: U-2900 )
   
4. 1 cm polystyrene spectrophotometer cuvette (Sigma-Aldrich, catalog number: C5219 )
   
5. Thermostatic water bath
   
6. Refrigerated centrifuge (Sigma Laborzentrifugen, model: 2K15 )
   
7. Refrigerated ultracentrifuge (130,000 rpm) (Beckman Coulter, model: Optima^TM MAX ultracentrifuge )
   
8. Tissue Grinder, Potter-Elvehjem type, 30 ml Glass Vessel and Plain Plunger (VWR, Prolabo, catalog number: 432-0204 and 89026-400 , respectively)
   

Procedure