Interleukin-2 (NIBSC repository reference ARP901) was obtained from the Centre for AIDS reagents, National Institute of Biological Standards and Control (NIBSC), United Kingdom. Raltegravir was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Raltegravir (Cat # 11680) from Merck & Company, Inc. Efavirenz was also obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Efavirenz.
Efavirenz
Manufactured by Merck Group
Sourced in Switzerland, United States
Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used as a component of antiretroviral therapy for the treatment of HIV-1 infection. It functions by binding to and inhibiting the HIV-1 reverse transcriptase enzyme, thereby preventing the replication of the virus.
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Lab products found in correlation
Nevirapine, by Merck Group (4 mentions)
Raltegravir, by Merck Group (3 mentions)
Formic acid, by Merck Group (3 mentions)
Midazolam d6, by Lipomed (2 mentions)
1 hydroxymidazolam, by Lipomed (2 mentions)
Midazolam, by Roche (2 mentions)
High performance liquid chromatography grade methanol, by Merck Group (1 mentions)
Paraxanthine, by Merck Group (1 mentions)
Metoprolol, by Merck Group (1 mentions)
Omeprazole, by LGC (1 mentions)
Losartan, by LGC (1 mentions)
β glucuronidase, by Merck Group (1 mentions)
Fluconazole, by LGC (1 mentions)
Caffeine d9, by LGC (1 mentions)
8 hydroxyefavirenz, by LGC (1 mentions)
8 14 dihydroxyefavirenz, by LGC (1 mentions)
Metoprolol d7, by LGC (1 mentions)
5 hydroxy omeprazole, by LGC (1 mentions)
α hydroxymetoprolol, by LGC (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
16 protocols using efavirenz
1
Interleukin-2 and Antiretroviral Agents Protocol
2
Quantitative Bioanalysis of Pharmaceutical Compounds
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Caffeine-d9, ciprofloxacin, efavirenz-d4, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, fluconazole, losartan, losartan carboxylic acid (E3174), losartan-d4, α-hydroxyMetoprolol, Metoprolol-d7, omeprazole, 5-hydroxyomeprazole, omeprazole-d3, paroxetine, and rifampicin were purchased from Toronto Research Chemicals (Toronto, Canada). 1’-HydroxyMidazolam and Midazolam-d6 were purchased from Lipomed (Lipomed AG, Arlesheim, Switzerland). Metoprolol, paraxanthine, and β-glucuronidase were obtained from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Midazolam (Hoffmann-La Roche, Basel, Switzerland) and efavirenz (Merck, NJ, USA) were kindly provided by the producers.
Formic acid, high-performance liquid chromatography (HPLC)-grade methanol, and water were purchased from Merck (Merck, Darmstadt, Germany). Stock solutions, calibration spiking solutions, and quality controls were prepared in dimethyl sulfoxide. Calibration standards were prepared by enriching caffeine-free blank human serum using the corresponding spiking solutions. Internal standard solutions containing the deuterated compounds were prepared in methanol.
Formic acid, high-performance liquid chromatography (HPLC)-grade methanol, and water were purchased from Merck (Merck, Darmstadt, Germany). Stock solutions, calibration spiking solutions, and quality controls were prepared in dimethyl sulfoxide. Calibration standards were prepared by enriching caffeine-free blank human serum using the corresponding spiking solutions. Internal standard solutions containing the deuterated compounds were prepared in methanol.
3
Quantitative Analysis of Probe Drugs
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8′-hydroxyefavirenz, efavirenz-d4, losartan, losartan-carboxylic acid (E3174), losartan-d4, omeprazole, 5′-hydroxyomeprazole, omeprazole-d3, metoprolol, α-hydroxymetoprolol, and metoprolol-d7 were purchased from TRC (Toronto, Canada). 1′-hydroxyMidazolam and Midazolam-d6 were acquired from Lipomed (Arlesheim, Switzerland), whereas rifampicin and β-glucuronidase (type HP-2 from Helix pomatia) were obtained from Sigma-Aldrich (Sigma- Aldrich Chemie GmbH, Buchs, Switzerland). Midazolam (F. Hoffmann-La Roche, Basel, Switzerland) and efavirenz (Merck, NJ, USA) were kindly provided by the respective manufacturers. The chemical structures of the probe drugs and their phase I metabolites are provided in Supplementary Figure 2 . Formic acid, HPLC grade methanol, and HPLC grade water were purchased from Merck (Darmstadt, Germany). Media used were purchased from GIBCO (Lucerne, Switzerland).
Stock solutions, calibration, and quality control spiking solutions were prepared in DMSO. Calibration standards and quality controls were prepared by enriching the respective medium with the corresponding spiking solutions. Internal standard solutions containing the deuterated cocktail probe drugs were prepared in methanol.
Stock solutions, calibration, and quality control spiking solutions were prepared in DMSO. Calibration standards and quality controls were prepared by enriching the respective medium with the corresponding spiking solutions. Internal standard solutions containing the deuterated cocktail probe drugs were prepared in methanol.
4
Multidrug Tuberculosis Treatment Protocol
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Adults not previously treated for TB were given an initial dose of oral INH of 5mg/kg (maximum dose, 300mg/day), RIF 10mg/kg (maximum dose, 600mg/kg), PZA 25mg/kg (maximum dose, 2g/d), and ETB 20mg/kg (maximum dose, 1.2g/d), once daily for 3 months. After the initial three months PZA and ETB were stopped and RIF and INH maintenance therapy was continued for a further six months. Streptomycin, administered intramuscularly (20mg/kg; maximum dose, 1g/d) was added to the initial treatment regime of patients previously treated for TB. All patients received dexamethasone at an initial dose of 300-400 µg/kg/d unless it was contraindicated. ART or placebo tablets were given as soon as possible and no later than one week after randomisation. The ART regime constituted of oral zidovudine (GSK) at a twice daily dose of 300mg, lamivudine (GSK) at a twice daily dose of 150mg, and efavirenz (Merck) at a once daily oral dose of 800mg (if receiving rifampicin) or 600mg (if not receiving rifampicin). After two months all patients received ART until the end of the study (12 months).
5
Integrin-Mediated Cell Adhesion Assay
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Gelatin (denatured collagen I, from bovine skin), vitronectin (from human plasma), bovine serum albumin (BSA, fraction V) and Efavirenz were obtained from Sigma-Aldrich (Milan, Italy). Human recombinant IL-1β, TNF-α, and IFN-γ, and fibronectin from human plasma were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). MAbs directed against the α5β1, αvβ3, αvβ5, or α6β4 integrins were obtained from Chemicon-Merck-Millipore (Darmstadt, Germany). The anti-CD31 (PECAM-1) mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Tat peptides (11–25), (46–60), and (66–80) were from UFP Service, University of Ferrara, Italy. Phosphate-buffered saline (PBS) solution, cell growth medium (RPMI 1640) and media supplements were obtained from Invitrogen-Life Technologies (Milan, Italy). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA).
6
Evaluating Anti-HIV and Anti-Alzheimer's Compounds
7
Synthesis and HIV-1 Inhibition Assays
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Compounds 13 and 21 were synthesized as previously reported [30 (link)]. The inhibitors: azidothymidine (AZT); efavirenz (EFV); nevirapine (NVP) and raltegravir (RAL) were purchased form Sigma-Aldrich (St. Louis, MO, USA). RNase H inhibitor was kindly provided by Prof. Roberto Di Santo. MT4 (ECACC 08081402) and HEK 293 T cells (ATCC® CRL-3216TM) were cultured in RPMI 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA), respectively. Both media were supplemented with 10% fetal calf serum (FCS) (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin. Stocks of HIV-1 NL4-3 strain were prepared by transfecting 293 T cells with the HIV-1 pNL4-3. The VSV-pseudotyped, NL4-3–GFP expressing virus (VSV/HIV-1GFP) was generated as reported [32 (link)].
8
Production and Infection of HIV-1 Variants
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The NL4-3-GFP wild type or modified to bind Vpx (NL4-3*GFP) were kindly provided by Dr. O. T. Keppler (Max von Pettenkofer Institute, Ludwig-Maximilians-Universität Munich, Germany). Wild-type NL4-3GFP or NL4-3*GFP31 (link),34 (link) were co-transfected with the Vpx expression construct SIVmac239- into HEK293T cells to produce viral stocks. Three days after transfection, the supernatants were collected, filtered, and concentrated using Lenti-X concentrator (Clontech, Catalog number 631232) and stored at −80 °C. Infections were performed using spinoculation (1200 g, 2 h at 37 °C) using 0.25 × 106 cells/well. After spinoculation, cells were kept in the incubator for 72 h prior to analysis by flow cytometry. Efavirenz (Sigma-Aldrich, Catalog number SML0536) was added when appropriate.
9
Efavirenz Nanoformulation Development
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Efavirenz was the gift sample from Jubilant Clinsys Noida, India, whereas Glyceryl monostearate (1-stearoyl-rac-glycerol), stearic acid (octadecanoic acid), and Tween 80 (polysorbate 80) along with all the other chemicals were of analytical grade and were purchased from Sigma-Aldrich (New Delhi, India). Compritol ATO 888 and Precirol were the gift sample from Asoj Soft Caps, Baroda, India. Commercial formulation was EFCURE oral solution (Emcure Pharmaceuticals Ltd.) containing Efavirenz (30 mg/60 mL).
10
Blocking Adhesion, HLA, and HIV Reverse Transcription
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For blockade of adhesion molecules, purified anti-human CD11a antibody (Genetex® cat: GTX26132 clone: 38), anti-human CD18 antibody (Biolegend® cat: 366302 clone CBR LFA-1/2), anti-human ICAM-2 antibody (Genetex® cat: GTX42521 clone: CBRIC2/2) and anti-human LFA3 antibody (Biolegend® cat: 330912 clone: TS2/9) were used.
For blockade of the HLA class I molecule, Ultra-LEAF™ purified anti-human HLA-A, B, C antibody (BioLegend® clone W6/32) was used. In order to block HIV reverse transcription, the retroviral drug Efavirenz (Sigma; SML0536) was used at a final concentration of 100nM. For the study of antigen processing pathways, the proteasome hydrolytic activities were blocked with epoxomicin (Enzo Life sciences-BML-PI127-0100), the aminopeptidase activities with Bestatin (Sigma-Aldrich B8385) and the cysteine proteases (cathepsins B, H and L) with E64 (Enzo Life Sciences ALX-260-007) at different final concentrations as indicated on the figures.
For blockade of the HLA class I molecule, Ultra-LEAF™ purified anti-human HLA-A, B, C antibody (BioLegend® clone W6/32) was used. In order to block HIV reverse transcription, the retroviral drug Efavirenz (Sigma; SML0536) was used at a final concentration of 100nM. For the study of antigen processing pathways, the proteasome hydrolytic activities were blocked with epoxomicin (Enzo Life sciences-BML-PI127-0100), the aminopeptidase activities with Bestatin (Sigma-Aldrich B8385) and the cysteine proteases (cathepsins B, H and L) with E64 (Enzo Life Sciences ALX-260-007) at different final concentrations as indicated on the figures.
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