Side selection for cochlear implantation in a case of Cogan's syndrome.

Cogan's syndrome is a rare clinical entity that is characterised by non-syphilitic interstitial keratitis and audiovestibular symptoms. The cause of Cogan's syndrome is considered to be autoimmune disease, which is supported by the resolution of hearing loss after steroid treatment, and the association with other autoimmune diseases. The sensorineural hearing loss of Cogan's syndrome is progressive over a few months, and sudden, bilateral deafness often occurs, which may be an indication for cochlear implantation. This paper presents the case of a young woman suffering from Cogan's syndrome and sudden, bilateral deafness. With reference to this case, we describe problems regarding cochlear implantation for Cogan's syndrome and radiological findings aiding selection of cochlear implantation side.

Identification of cytochrome P450 enzymes responsible for N -dealkylation of a new oral erectogenic, mirodenafil.

The purpose of this paper is to characterize the cytochrome P450 (CYP) enzymes involved in the metabolism of a new oral erectogenic, mirodenafil, to a major circulating active metabolite, N-dehydroxyethyl-mirodenafil, and to investigate the inhibitory potential of mirodenafil on seven CYP enzymes in human liver microsomes. CYP3A4 was identified as the major enzyme and CYP2C8 as a minor enzyme responsible for mirodenafil N-dealkylation based on correlation analysis, inhibition studies, and cDNA-expressed CYP enzyme activities. Plasma concentrations of mirodenafil and its N-dealkylated metabolite could therefore change with co-administration of known CYP3A4 inducers or inhibitors. Mirodenafil inhibited CYP3A4, CYP2C19 and CYP2D6 activities with IC50 values of 15.6, 38.2 and 77.0 microM, respectively, in human liver microsomes. However, it is very unlikely that mirodenafil will significantly alter the clearance of other compounds metabolized by CYPs 1A2, 2A6, 2C8, 2C9, 2C19, 2D6 and 3A4 because the maximum plasma concentration of mirodenafil is 0.55 microM after oral dosing of mirodenafil (100 mg) in male volunteers.

Synthesis and phosphodiesterase inhibitory activity of new sildenafil analogues containing a carboxylic acid group in the 5'-sulfonamide moiety of a phenyl ring.

New sildenafil analogues possessing a carboxylic acid group in the 5'-sulfonamide of the phenyl ring, 9a-l, were prepared from the readily available starting compounds 6a-b and cyclic amines 3-5 in a three-step sequence. In the enzyme assays, it has been shown that all the target compounds 9a-l proved to be more potent in inhibiting phosphodiesterase type 5 (PDE5) than sildenafil by 4-38-fold. The effects on the IC(50) values were investigated by varying the alkoxy group (R) of the phenyl ring, the sulfonamide type (X), and the length of the methylene chain linking the carboxylic acid, and the results were discussed in detail. From this study, we have clearly demonstrated that introduction of a carboxylic acid group to the 5'-sulfonamide moiety of the phenyl ring greatly enhanced PDE5 inhibitory activity, probably by mimicking the phosphate group of cGMP. The piperidinyl propionic acid derivative 9i, which showed the highest PDE5 inhibitory activity and comparable to better selectivity over PDE isozymes in comparison with sildenafil, has been selected for more detailed biological investigations.

Synthesis and phosphodiesterase 5 inhibitory activity of novel phenyl ring modified sildenafil analogues.

New sildenafil analogues containing an ether ring fused into the phenyl moiety, 6a--d and 7a--d, were efficiently synthesized from the readily available starting materials, 1a--d and 2, in five steps. Ab initio calculations indicated that introduction of a cyclic ether to the phenyl group might enhance the co-planarity of the molecule. The torsional angles were calculated to be 2--3 degrees for the 5-membered cyclic ether derivatives, 6a, 6c, 7a, and 7c, and 12--16 degrees for the 6-membered ones, 6b, 6d, 7b, and 7d. On the other hand, sildenafil showed the least co-planarity with the torsional angle of 23 degrees compared with the target compounds, 6a--d and 7a--d. In the enzyme assay, however, the in vitro PDE 5 inhibitory activity was found out to be inversely related to the degree of co-planarity. In other words, the least planar sildenafil showed the highest activity, and the most planar 5-membered cyclic ether derivatives were least active by 100--200-fold compared with sildenafil. Our study clearly demonstrated that the open chain 2'-alkoxy group of the phenyl ring, although less effective for inducing the co-planarity, seemed to act as a much better lipophilic requirement than the cyclic alkoxy moiety.

Pharmacokinetic studies of 2-amino-9-(3-acetoxymethyl-4-isopropoxycarbonyl-oxybut-1-yl)purine, an oral prodrug for the antiviral agent penciclovir.

2-Amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)- purine (SK1899) was tested as an oral prodrug for penciclovir. SK1899 was administered orally to rats and dogs at doses up to 2 and 0.68 mmol/kg, respectively. SK1899 was well absorbed, and the major metabolites detected in plasma and urine were penciclovir, the active antiviral compound, and 6-deoxypenciclovir (M4) in both species. In rats, SK1899 was rapidly and extensively metabolized to penciclovir, which reached the peak plasma concentration (C(max)) of 39.5 microM at 0.5 h after 0.2-mmol/kg dosing. The area under the plasma concentration-time curve (AUC) for penciclovir was 57.5 microM x h. After an oral dose of 0.034 mmol/kg to dogs, extensive conversion of SK1899 to penciclovir also occurred with slower rate of formation of penciclovir from M4 than in rats. The mean C(max) and AUC for penciclovir were 4.5 microM at 2.7 h and 28.2 microM x h, respectively. The 0- to 24-h urinary recovery of penciclovir represented 36.1 and 36.3% of dose to rats and dogs, respectively. Radioactivity was found in fetuses following an oral administration of [(14)C]SK1899 to pregnant rats, but no significant accumulation was observed. Although substantial milk transfer of [(14)C]SK1899 occurred in rats, the radioactivity in milk was rapidly cleared. The values of C(max), AUC, and urinary recovery of penciclovir after dosing with SK1899 to rats and dogs were similar or slightly higher than those from famciclovir. These data indicate that introduction of an isopropoxy carbonate group into one of the two hydroxyl groups of M4 did not significantly alter the oral bioavailability of penciclovir compared with famciclovir.

Synthesis and phosphodiesterase 5 inhibitory activity of new 5-phenyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one derivatives containing an N-acylamido group phenyl ring.

New sildenafil analogues with an N-acylamido group at the 5'-position of the phenyl ring, 6a--e, were prepared from the readily available starting compound 2 in four straightforward steps. Enzyme assays demonstrated that all the target compounds 6a-e showed higher PDE5 inhibitory activities than sildenafil. It was observed that the PDE5 inhibitory activity was enhanced as the chain length of R group increased, but introduction of the branched alkyl groups such as isopropyl (6d) and cyclohexyl (6e) resulted in the drop of potency compared with 6c. In particular the N-butyrylamido derivative 6c exhibited the highest PDE5 inhibitory activity, and was about 6-fold more potent than sildenafil. However, all the compounds exhibited somewhat weak selectivity (1--3-fold) over PDE6, indicating that the compounds 6a--e have intrinsically lower selectivity than sildenafil.

Synthesis and biological evaluation of novel A-ring modified hexacyclic camptothecin analogues.

Eleven A-ring modified hexacyclic analogues of camptothecin (CPT) containing a 1,4-oxazine ring were synthesized from 10-hydroxycamptothecin (11a) and 7-ethyl-10-hydroxycamptothecin (3) (SN-38) in four to five steps and were subjected to the biological tests such as cytotoxicity, topoisomerase I (Topo I) inhibitory activity, acetylcholinesterase (AChE) inhibition, and stability in human plasma. Four compounds 15a, 15b, 16a, and 16c were about 2-fold more potent than topotecan and as potent as CPT toward human cancer cell lines A549, H128, WiDr, MKN45, SK-OV-3, and SK-BR-3 in vitro, even though the most active compound 15b was slightly less potent than SN-38. The potency of Topo I inhibition of these compounds showed relatively good correlation with their cytotoxicity. Most of the compounds exhibited AChE inhibitory activity weaker (9 +/- 2 to 20 +/- 3%) than CPT (23 +/- 5%) or topotecan (20 +/- 4%) and similar to SN-38 (13 +/- 2%), indicating that they might have little effect on causing early diarrhea. The stability of lactone forms of these compounds in human plasma seemed to be much higher than that of CPT and similar to that of topotecan but lower than that of SN-38. Among the new hexacyclic CPT analogues, compound 15b showed higher antitumor activity against human tumor xenograft, WiDr, in nude mice compared to that of SN-38. The most promising compound 15b has been selected for further development.

General pharmacology of the new antiviral agent SK 1899.

The general pharmacological properties of 2-amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine (CAS 247081-81-8, SK 1899), a new potential antiviral agent, were investigated in mice, rats, guinea pigs, rabbits, and dogs. The oral administration of 50, 150, and 500 mg/kg of SK 1899 had no effects on the central nervous system except that it slightly increased the spontaneous locomotor activity in mice at a dose of 500 mg/kg. SK 1899 did not disturb either the spontaneous motility or contractor-induced contraction of the isolated organs such as guinea pig ileum, rat uterus, guinea pig vas deferens, and guinea pig trachea at concentrations up to 10(-4) mol/l. It slightly increased the contractile force in the isolated guinea pig atrium at a concentration of 10(-4) mol/l. Following intravenous infusion of 5, 15, and 50 mg/kg of SK 1899 to anesthetized dogs, it did not change the mean arterial pressure, heart rate, left ventricular systolic pressure (LVSP), and respiratory rate, while it slightly increased the left ventricular positive dP/dtmax (LV + dP/dtmax) at a dose of 50 mg/kg. SK 1899 did not induce any significant changes in the intestinal charcoal meal transit in mice, basal gastric juice secretion in rats, and renal function in rats. It did not affect the blood coagulation system and phenolsulfonphthalein secretion in rats. These findings suggest that SK 1899 has a very low potential to induce any adverse pharmacological effects at the doses showing antiviral activity.

Synthesis and evaluation of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purines and 2-amino-9-(3-alkoxycarbonyloxymethyl-4-alkoxycarbonyloxybut-1- yl)purines as potential prodrugs of penciclovir.

A series of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purin es (1-8) and 2-amino-9-(3-alkoxycarbonyl-oxymethyl-4-alkoxycarbonyloxybut -1-yl)purines (9-12) were synthesized as potential prodrugs of penciclovir. Treatment of 6-deoxypenciclovir with trimethyl orthoacetate or triethyl orthopropionate (1.2 equiv) in DMF in the presence of p-TsOH.H2O (0.1 equiv) followed by quenching with excess H2O gave the corresponding mono-O-acetyl or mono-O-propionyl compound, 17 or 18, in excellent yields of 95 and 92%, respectively. Reactions of 17 or 18 with an appropriate alkyl (Me, Et, n-Pr, and i-Pr) 4-nitrophenyl carbonate (1.2 equiv) in pyridine in the presence of a catalytic amount of DMAP (0.1 equiv) at 80 degrees C afforded the monoacyl, monocarbonate derivatives of 6-deoxypenciclovir, 1-8, in 86 94% yields. Similar reactions of 6-deoxypenciclovir with 2.1 equiv of alkyl 4-nitrophenyl carbonate produced the dicarbonate derivatives 9 12 in 81-83% yields. Of the prodrugs tested in rats, 2-amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine (4) achieved the highest mean urinary recovery of penciclovir (36%), followed in order by compounds 2 (35%), 6 (35%), 7 (34%), 10 (34%), 8 (32%), 3 (32%), and famciclovir (31%). The mean urinary recovery of penciclovir and concentrations of penciclovir in the blood from 4 in mice were also slightly higher than those from famciclovir. The in vivo antiviral efficacy of 4 in HSV-1-infected normal BALB/c mice was higher than those of famciclovir and valaciclovir in terms of mortality (100, 80, and 40%) and mean survival time ( > 21, 13+/-5.0 (SEM), and 13+/-1.6 days). Compound 4 demonstrated an effective anti-hepadnaviral response with intrahepatic viral load being reduced by 90%, the viral supercoiled DNA levels reduced by 70% and Pre-S expression inhibited by 30% against duck hepatitis B virus (DHBV) in vivo, and did not cause any significant hepatotoxicity after 4 weeks of treatment.

Synthesis and evaluation of 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine mono- and diesters as potential prodrugs of penciclovir.

2-Amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (7), and its mono- and diesters 8-15 were prepared and evaluated for their potential as prodrugs of penciclovir. Treatment of 2-amino-6-chloro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (5) with trimethylamine in THF followed by a reaction of the resulting trimethylammonium chloride salt 6 with KF in DMF afforded 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (7) in 80% yield. Esterification of 7 with an appropriate acid anhydride [Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O] in DMF in the presence of a catalytic amount of DMAP produced the mono-esters 8-11 in 42-45% yields and diesters 12-15 in 87-99% yields. Of the prodrugs tested in rats, the monoisobutyrate 11 was the most efficiently absorbed and metabolized to 7, showing the mean maximum total concentration of penciclovir (5.5 microg/mL) and 7 (10.8 microg/mL) in the blood was much higher than the mean maximum concentration of penciclovir (11.5 microg/mL) from famciclovir. However, the mean concentrations of penciclovir from 11 were lower than those from famciclovir because of the limited conversion of a major metabolite 7 to penciclovir by adenosine deaminase.

Synthesis and evaluation of amino acid esters of 6-deoxypenciclovir as potential prodrugs of penciclovir.

The amino acid ester derivatives of 6-deoxypenciclovir, 11-20, were synthesized as potential prodrugs of penciclovir, and were evaluated for their oral penciclovir bioavailability in mice and rats. Esterification of 6-deoxypenciclovir with N-carbobenzyl-oxyglycine, -L-alanine, -L-valine, -L-leucine, or -L-isoleucine (3.75equiv.) using conventional coupling method (DCC/DMAP) afforded the mono-O-ester derivatives 1-5 in 47-55% yields as a mixture of two diastereomers along with the di-O-ester derivatives 6-10 in 20-29% yields. Reductive cleavage of carbobenzyloxy (Cbz) group (10% Pd/C, 1 atmosphere of H2, room temperature in methanol) followed by subsequent treatment of the resulting free amine with methanolic HCI solution provided the mono-O-ester derivatives 11-15 as di-HCl salt in 51-98% yields and the di-O-ester derivatives 16-20 as tri-HCl salt in 65 98% yields. Of the prodrugs tested in mice and rats, 6-deoxypenciclovir O-L-valinate (13), O-L-isoleucinate (15), and O,O-di-glycinate (16) showed significantly higher urinary recovery of penciclovir compared with that of penciclovir, but those are somewhat lower than that of famciclovir.

Synthesis and evaluation of 2-amino-9-(1, 3-dihydroxy-2-propoxymethyl)- 6-fluoropurine mono- and diesters as potential prodrugs of ganciclovir.

A series of 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)-6-fluoropurine mono- and diesters, 6a-h, were synthesized as potential prodrugs of ganciclovir and evaluated for their oral ganciclovir bioavailability in rats. Treatment of 2-amino-6-chloro-9-(1, 3-dihydroxy-2-propoxymethyl)purine (4) with Me3N in DMF/THF (1/4) followed by the reaction of the resulting trimethylammonium chloride salt 5 with KF in DMF gave 2-amino-9-(1, 3-dihydroxy-2-propoxymethyl)-6-fluoropurine (2) in 83% yield. Esterification of 2 with an appropriate acid anhydride (Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O) (6 equiv for 6a-d or 1 equiv for 6e-h) in DMF in the presence of a catalytic amount of DMAP produced the diesters 6a-d in 92-98% yields and the monoesters 6e-h in 37-44% yields. Of the prodrugs tested in rats, the monoisobutyrate 6h achieved the highest ganciclovir bioavailability (45%) that is 15-fold higher than that from ganciclovir (3%), followed in order by the diisobutyrate 6d (42%), the diacetate 6a (41%), the monobutyrate 6g (41%), the monopropionate 6f (39%), the dipropionate 6b (35%), the dibutyrate 6c (35%), and the monoacetate 6e (29%). The prodrugs 6e-h were found to be quite stable at pH 6.0 (t1/2 = >29 days), 7.4 (t1/2 = >7 days), and 8.0 (t1/2 = >2 days) but had relatively short half-lives at pH 1.2 (t1/2 = 60-83 min).

Synthesis and evaluation of 2-amino-9-(3-hydroxymethyl-4-alkoxycarbonyloxybut-1-yl)purines as potential prodrugs of penciclovir.

A series of 2-amino-9-(3-hydroxymethyl-4-alkoxycarbonyloxybut-1-yl)purines (4-10) and 2-amino-9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (1) were synthesized as potential prodrugs of penciclovir and evaluated for their oral penciclovir bioavailability in mice and rats. Treatment of 2-(2-benzyloxyethyl)propane-1,3-diol (11) with 1,1'-carbonyldiimidazole in THF followed by hydrogenolytic removal of the benzyl group of the resulting cyclic carbonate 12 gave 5-(2-hydroxyethyl)-1,3-dioxan-2-one (13). Mesylation of the alcohol 13 and then a coupling reaction of the resulting mesylate 14 with 2-amino-6-chloropurine using anhydrous Cs2CO3 in DMF afforded 2-amino-6-chloro-9-(2-(2-oxo-1,3-dioxan-5-yl)ethyl)purine (16) after purification by flash column chromatography on silica gel using EtOAc/MeCN/Et3N as eluent. Hydrogenation of the 6-chloro cyclic carbonate 16 followed by a ring-opening reaction of the 6-deoxy cyclic carbonate 1 in a mixture of an appropriate alcohol and CHCl3 using activated SiO2 as a Lewis acid afforded the corresponding alkyl monocarbonate derivatives 3-10 in fair to good yields. Of the prodrugs tested in mice, the isopropyl monocarbonate 6 achieved the highest mean urinary recovery of penciclovir (53%), followed in order by the propyl monocarbonate 5 (51%), the isopentyl monocarbonate 10 (51%), the ethyl monocarbonate 4 (50%), and famciclovir (48%). In rats, the methyl monocarbonate 3, 4, 6, the n-butyl monocarbonate 7, and 10 (39-41%) showed levels of mean urinary recovery of penciclovir similar to that from famciclovir (40%). The alkyl monocarbonates 4-10 were found to be quite stable in the aqueous buffer solutions, and among them, 6 was the most stable with the half-lives (t1/2) of 88, >200, 61, and 26 days at pH 1.2, 6.0, 7.4, and 8.0, respectively. In addition, 6 was highly soluble in H2O (138.8 mg/mL, 20 degrees C).

Synthesis and evaluation of 2-amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine esters as potential prodrugs of acyclovir.

2-Amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine (2) and its ester derivatives 4a-d were synthesized as potential prodrugs of acyclovir, and were evaluated for their oral acyclovir bioavailability in rats and in vivo antiviral efficacy in HSV-1-infected mice. Treatment of 2-amino-6-chloro-9-(2-hydroxyethoxymethyl)purine (3) with trimethylamine in THF/DMF (4:1) followed by a reaction of the resulting trimethylammonium chloride salt 5 with KF in DMF gave 2 in 78% yield. Esterification of 2 with an appropriate acid anhydride (Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O) in DMF in the presence of a catalytic amount of DMAP at room temperature produced the esters 4a-d in 90-98% yields. Of the prodrugs tested in rats, the isobutyrate 4d achieved the highest mean urinary recovery of acyclovir (51%) that is 5.7-fold higher than that of acyclovir (9%) and comparable to that of valacyclovir (50%). The prodrug 4d protected dose-dependently the mortality of HSV-1-infected mice, and the group treated with 4d at a dose of 400 mg/kg showed the longest mean survival day (14.6+/-3.1 days) (mean+/-S.D.).

Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.

Action of uracil analogs on human immunodeficiency virus type 1 and its reverse transcriptase.

Three structural analogs of 5-ethyl-1-benzyloxymethyl-6-(phenylthio)uracil (E-BPU) inhibited human immunodeficiency virus type 1 (HIV-1) replication without cytotoxicity in vitro and were more potent than azidothymidine and were as potent as E-BPU. The target of these compounds is HIV-1 reverse transcriptase. Reverse transcriptases resistant to nevirapine (tyrosine at position 181 to cysteine) and TIBO R82150 (leucine at position 100 to isoleucine) are cross resistant to E-BPU analogs. Nevirapine- or TIBO R82150-resistant HIV-1 were cross resistant to E-BPU analogs but were inhibited at concentrations 11- to 135-fold lower than the cytotoxic doses.

Identification of the amino acid in the human immunodeficiency virus type 1 reverse transcriptase involved in the pyrophosphate binding of antiviral nucleoside triphosphate analogs and phosphonoformate. Implications for multiple drug resistance.

A recombinant clone of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with reduced sensitivity to 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) and phosphonoformate (PFA), a pyrophosphate analog, has been obtained from the RNA of HTLV-IIIB infected cells using the polymerase chain reaction. The mutant HIV-1 RT retained polymerase activity and was cross-resistant to triphosphate forms of other nucleoside analogs including 2',3'-dideoxycytidine 5'-triphosphate, 2',3'-dideoxyadenosine 5'-triphosphate, and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (D4TTP), but remained sensitive to the non-nucleoside HIV-1 RT inhibitors, such as nevirapine and TIBO R82150. Sequence analysis of the mutant HIV-1 RT revealed a single amino acid substitution (Val-->Ala) at amino acid 90. The substitution of amino acid 90 by the closely related amino acids, such as Thr and Gly, also showed decreased sensitivity to AZTTP, D4TTP, and PFA. All these mutations at amino acid 90 also caused an alteration of Km for thymidine triphosphate. These results suggest that Val at this site plays a role in determining the interaction of the HIV-1 RT enzyme with the pyrophosphate group of deoxynucleoside triphosphate (dNTP) and that the hydrophobicity of the amino acid at this position was the most important determinant in the binding of HIV-1 RT to dNTP.

A single conservative amino acid substitution in the reverse transcriptase of human immunodeficiency virus-1 confers resistance to (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5, 1- jk][1,4]benzodiazepin-2(1H)-thione (TIBO R82150).

Tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO) derivatives (e.g., R82150) are potent, human immunodeficiency virus-1 (HIV-1)-specific, inhibitors of reverse transcriptase (RT) that are undergoing initial evaluation in clinical trials. Because HIV-1 has become resistant to other RT inhibitors, we investigated the potential for viral resistance to TIBO R82150 by serial in vitro passage of HIV-1IIIB in the presence of drug. R82150-resistant variants (> 100-fold increase in IC50) dominated the replicating virus population after only three or four passages. R82150-resistant virus was partially cross-resistant to other HIV-1-specific RT inhibitors, including nevirapine (approximately 10-fold increase in IC50) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (approximately 3.5-fold increase) but remained susceptible to 2',3'-dideoxynucleosides and phosphonoformate. DNA sequencing of cloned resistant RT, combined with site-specific mutational analyses and construction of mutant recombinant proviruses, demonstrated that a single, conservative amino acid substitution (Leu100 to Ile) in HIV-1 RT is responsible for high level R82150 resistance and partial nevirapine resistance. These studies indicate that a subtle mutation in HIV-1 RT can dramatically affect viral susceptibility to an HIV-1-specific RT inhibitor. The clinical efficacy of TIBO derivatives and other HIV-1-specific RT inhibitors may be limited by the emergence of drug-resistant viral strains.

In vitro selection and molecular characterization of human immunodeficiency virus-1 resistant to non-nucleoside inhibitors of reverse transcriptase.

Several newly discovered potent and selective non-nucleoside inhibitors of human immunodeficiency virus-1 reverse transcriptase (RT) are undergoing evaluation in clinical trials. We studied the potential for development of viral resistance to one of the prototype compounds, BI-RG-587, a dipyridodiazepinone derivative. Human immunodeficiency virus-1 resistant to BI-RG-587 emerged after only one cycle of in vitro infection in the presence of the drug. Resistant virus was cross-resistant to the non-nucleoside tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione derivative R82150 but remained susceptible to 2',3'-dideoxynucleosides and phosphonoformate. Both native (virion-associated) and recombinant RT derived from resistant virus were insensitive to BI-RG-587 and R82150. Nucleotide sequence analysis of multiple drug-resistant and -sensitive recombinant RT clones identified a single predicted amino acid change common to all resistant clones (tyrosine-181----cysteine). These studies suggest that the viral resistance to non-nucleoside RT inhibitors may develop in vivo. This possibility should be carefully monitored in clinical trials of these compounds.