**4. NRTI in the pipeline**

68 Pharmacology

glucuronosyltransferase

The acyclic nucleoside phosphonate tenofovir (*R-*9-(2-phosphonylmethoxypropyl)-adenine) has no sugar ring structure but contains an acyclic methoxypropyl linker between the base N9 atom and a non-hydrolyzable C-P phosphonate bond. Thus tenofovir represents the only currently approved *nucleotide* HIV inhibitor. Tenofovir is poorly absorbed by the oral route and is therefore administered as a lipophilic orally bioavailable prodrug tenofovir disoproxil fumerate (TDF), a fumaric acid salt of the bis-isopropoxycarbonyloxymethyl ester of tenofovir (Figure 5). TDF is readily absorbed by the gastrointestinal epithelial cells with an oral bioavailability of 25% (Barditch-Crovo et al., 2001). Administration with a high fat meal increases absorption to 40%. Degradation of TDF to its monoester and subsequently to tenofovir occurs readily in the intestinal mucosa by the action of carboxylesterases and phosphodiesterases, respectively. The mono- or bis-ester forms of tenofovir are not observed in plasma suggesting efficient release of tenofovir following oral administration of TDF (Naesens et al., 1998). Following oral administration tenofovir has a long terminal half-life of 17 hours. The phosphonic acid linkage is chemically and metabolically stable and phosphorolysis back to the nucleoside does not occur (Naesens et al., 1998). Tenofovir is rapidly converted intracellularly to tenofovir-monophosphate and the active tenofovirdiphosphate forms by adenylate monophosphate kinase and 5'-nucleoside diphosphate kinase, respectively (Robbins et al., 1998). Tenofovir is not subject to intracellular deamination or deglycosylation. This stability results in a very long intracellular half-life for tenofovir-diphosphate of 15 hours in activated lymphocytes and 50 hours in resting lymphocytes (Robbins et al., 1998). Tenofovir is eliminated by glomerular filtration and active tubular secretion by organic anion transporter mediated uptake and MRP4 mediated efflux (Ray et al., 2006). At 72 hours post oral administration 70 - 80 % is recovered from urine as unchanged tenofovir. Tenofovir does not inhibit cytochrome P450 enzymes.

Fig. 4. Metabolic pathways for abacavir

**3.6 Tenofovir and tenofovir disoproxil fumerate** 

Despite the widespread clinical success of NRTI-containing therapy, the currently FDA approved NRTIs display important limitations including the selection of drug resistance mutations that display cross-resistance to other NRTI, toxicity-related adverse events, and drug-drug interactions (for review see Cihlar & Ray, 2010). Thus, there is a need for novel NRTI that overcome these limitations. Here we will discuss the pharmacology of several novel drug candidates.

#### **4.1 Apricitabine**

Apricitabine (ATC) is the (-)-enantiomer of 2'-deoxy-3'-oxa-4'-thiocytidine, a deoxycytidine analog that is currently in phase II/III clinical trials (Figure 6). Both the (+) and (-) enantiomers of apricitabine demonstrate potent inhibition of HIV-1 replication, however the (+)-enantiomer demonstrated significant mitochondrial and cellular toxicity in pre-clinical studies that was not observed with the (-) enantiomer (de Muys et al., 1999; Taylor et al., 2000). Racemic conversion of (-)-apricitabine to (+)-apricitabine is not observed in vivo (Holdich et al., 2006). Orally administered ATC is absorbed quickly, reaching maximal plasma levels within 2 hours with a plasma half-life of 3 hours. Maximal peripheral blood mononuclear cell (PBMC) intracellular concentrations of apricitabine -TP are achieved 3.5 – 4 hours after oral administration in healthy and HIV-infected patients. The intracellular halflife is 6 – 7 hours (Sawyer & Struthers-Semple, 2006; Cahn et al., 2008; Holdich et al., 2007). Apricitabine is not metabolized by hepatocytes *in vitro*, however a deaminated metabolite was observed likely due to gastrointestinal metabolism (Nakatani-Freshwater et al., 2006). This metabolite is excreted renally and does not demonstrate antiviral or pharmacologic effects. Apricitabine had no effect on cytochrome P450 or glucouronidase but was a weak inhibitor of P-glycoprotein (Sawyer & Cox, 2006). The first phosphorylation of apricitabine is

Molecular Pharmacology of Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors 71

The purine nucleoside analog 1-β-D-dioxolane guanosine (DXG) has potent activity against HIV and hepatitis B virus (Kim et al., 1993). However, it demonstrates poor solubility and limited oral bioavailability in monkeys (Chen et al., 1996). The analog 1-β-D-2,6 diaminopurine dioxolane (amdoxovir; Figure 8) also exhibits antiviral activity and is more water soluble and orally bioavailable (Chen et al., 1999; Kim et al., 1993)). Amdoxovir serves as a prodrug for DXG by deamination at the 6-position by adenosine deaminase (Gu et al., 1999). *In vitro*, amdoxovir bound adenosine deaminase as efficiently as adenosine, however amdoxovir was deaminated 540-fold slower than adenosine (Furman et al., 2001). Only DXG-triphosphate was detected in PBMC and CEM cells following exposure to DXG or amdoxovir (Rajagopalan et al., 1994; Rajagopalan et al., 1996). DXG is phosphorylated to DXG-MP by 5'-nucleotidase using IMP as a phosphate donor (Feng et al., 2004). DXGdiphosphate is then generated by guanosine monophosphate kinase (GMP kinase). DXG-DP acts as substrate for phosphorylation to the active DXG-TP for several enzymes including nucleotide diphosphate kinase (NDP kinase), 3-phosphoglycerate kinase (3-PG kinase, creatine kinase, and pyruvate kinase. Amdoxovir is rapidly converted to DXG in monkeys,

woodchucks, and rats with approximately 61 % of the dose converted to DXG (Chen et al., 1996; Chen et al., 1999; Rajagopalan et al., 1996). The oral bioavailability of amdoxovir is estimated to be 30% (Chen et al., 1999). Following oral administration of amdoxovir to HIVinfected patients, peak plasma levels of amdoxivir and DXG were reached within 2 hours

Fig. 7. Metabolic pathway of festinavir

Fig. 8. Metabolic pathway of amdoxovir

**4.3 Amdoxovir** 

mediated by deoxycytidine kinase, the enzyme also responsible for the initial phosphorylation of lamivudine and emtricitabine (de Muys et al., 1999). The possibility of competition for deoxycytidine kinase was examined in PBMC. Co-administration of apricitabine with lamivudine or emtricitabine leads to a dose-dependent decrease in apricitabine phosphorylation, whereas lamivudine and emtricitabine phosphorylation was not affected by apricitabine (Bethell et al., 2007). In healthy volunteers given apricitabine and lamivudine, the intracellular PBMC levels of apricitabine-TP were decreased 75% compared to apricitabine alone (Holdich et al., 2006). Consequently, administration of apricitabine in combination with lamivudine or emtricitabine is not recommended. Similarly, lamivudine and emtricitabine co-administration is also contraindicated. Apricitabine-MP is sequentially phosphorylated to the di- and tri-phosphate forms by cytidine or deoxycytidine monophosphate kinase and 5'-nucleotide diphosphate kinase, respectively.

Fig. 6. Metabolic pathway of apricitabine

#### **4.2 Festinavir**

Festinavir (2',3'-didehydro-3'-deoxy-4'-ethynylthymidine; 4'-Ed4T) is a 4'-ethynyl analog of stavudine that is 5-10 fold more potent (Figure 7) (Haraguchi et al., 2003; Nitanda et al., 2005). Festinavir shows decreased cellular toxicity compared to stavudine, with little or no inhibition of host polymerases (Yang et al., 2007; Dutschman et al., 2004). Stepwise phosphorylation of festinavir occurs via the same enzymes as stavudine. Thymidine kinase 1 phosphorylates festinavir to festinavir-MP with 4-fold greater efficiency than stavudine (Hsu et al., 2007). The efficiency of festinavir-MP phosphorylation by thymidinylate monophosphate kinase is approximately 10 % of that seen for stavudine-MP or zidovudine-MP. Conversion from festinavir-DP to festinavir-TP appears to be catalyzed by multiple enzymes including nucleoside diphosphate kinase, pyruvate kinase, creatine kinase, and 3 phosphoglycerate kinase (Hsu et al., 2007). In contrast to other thymidine analogs which are readily catabolized by thymidine phosphorylase, festinavir catabolism cannot be detected. Furthermore, festinavir efflux from the cell is much less efficient than that of zidovudine. The festinavir nucleoside form alone is effluxed by a yet to be identified cellular transporter, while zidovudine and zidovudine-MP are effluxed from the cell. A Phase 1a study investigated the pharmacokinetic profile of a single oral dose between 10 and 900 mg and found a linear dose response in plasma with no apparent effects from food (Paintsil et al., 2009). A Phase 1b/2a study of festinavir oral monotherapy in 32 patients was recently completed. The results indicated that festinavir was safe (few festinavir related adverse events), well tolerated, and demonstrated dose dependent decreases in viral load between 0.87 and 1.36 logs (Cotte et al., 2010).

Fig. 7. Metabolic pathway of festinavir

#### **4.3 Amdoxovir**

70 Pharmacology

mediated by deoxycytidine kinase, the enzyme also responsible for the initial phosphorylation of lamivudine and emtricitabine (de Muys et al., 1999). The possibility of competition for deoxycytidine kinase was examined in PBMC. Co-administration of apricitabine with lamivudine or emtricitabine leads to a dose-dependent decrease in apricitabine phosphorylation, whereas lamivudine and emtricitabine phosphorylation was not affected by apricitabine (Bethell et al., 2007). In healthy volunteers given apricitabine and lamivudine, the intracellular PBMC levels of apricitabine-TP were decreased 75% compared to apricitabine alone (Holdich et al., 2006). Consequently, administration of apricitabine in combination with lamivudine or emtricitabine is not recommended. Similarly, lamivudine and emtricitabine co-administration is also contraindicated. Apricitabine-MP is sequentially phosphorylated to the di- and tri-phosphate forms by cytidine or deoxycytidine monophosphate kinase and 5'-nucleotide diphosphate kinase,

Festinavir (2',3'-didehydro-3'-deoxy-4'-ethynylthymidine; 4'-Ed4T) is a 4'-ethynyl analog of stavudine that is 5-10 fold more potent (Figure 7) (Haraguchi et al., 2003; Nitanda et al., 2005). Festinavir shows decreased cellular toxicity compared to stavudine, with little or no inhibition of host polymerases (Yang et al., 2007; Dutschman et al., 2004). Stepwise phosphorylation of festinavir occurs via the same enzymes as stavudine. Thymidine kinase 1 phosphorylates festinavir to festinavir-MP with 4-fold greater efficiency than stavudine (Hsu et al., 2007). The efficiency of festinavir-MP phosphorylation by thymidinylate monophosphate kinase is approximately 10 % of that seen for stavudine-MP or zidovudine-MP. Conversion from festinavir-DP to festinavir-TP appears to be catalyzed by multiple enzymes including nucleoside diphosphate kinase, pyruvate kinase, creatine kinase, and 3 phosphoglycerate kinase (Hsu et al., 2007). In contrast to other thymidine analogs which are readily catabolized by thymidine phosphorylase, festinavir catabolism cannot be detected. Furthermore, festinavir efflux from the cell is much less efficient than that of zidovudine. The festinavir nucleoside form alone is effluxed by a yet to be identified cellular transporter, while zidovudine and zidovudine-MP are effluxed from the cell. A Phase 1a study investigated the pharmacokinetic profile of a single oral dose between 10 and 900 mg and found a linear dose response in plasma with no apparent effects from food (Paintsil et al., 2009). A Phase 1b/2a study of festinavir oral monotherapy in 32 patients was recently completed. The results indicated that festinavir was safe (few festinavir related adverse events), well tolerated, and demonstrated dose dependent decreases in viral load between

respectively.

**4.2 Festinavir** 

Fig. 6. Metabolic pathway of apricitabine

0.87 and 1.36 logs (Cotte et al., 2010).

The purine nucleoside analog 1-β-D-dioxolane guanosine (DXG) has potent activity against HIV and hepatitis B virus (Kim et al., 1993). However, it demonstrates poor solubility and limited oral bioavailability in monkeys (Chen et al., 1996). The analog 1-β-D-2,6 diaminopurine dioxolane (amdoxovir; Figure 8) also exhibits antiviral activity and is more water soluble and orally bioavailable (Chen et al., 1999; Kim et al., 1993)). Amdoxovir serves as a prodrug for DXG by deamination at the 6-position by adenosine deaminase (Gu et al., 1999). *In vitro*, amdoxovir bound adenosine deaminase as efficiently as adenosine, however amdoxovir was deaminated 540-fold slower than adenosine (Furman et al., 2001). Only DXG-triphosphate was detected in PBMC and CEM cells following exposure to DXG or amdoxovir (Rajagopalan et al., 1994; Rajagopalan et al., 1996). DXG is phosphorylated to DXG-MP by 5'-nucleotidase using IMP as a phosphate donor (Feng et al., 2004). DXGdiphosphate is then generated by guanosine monophosphate kinase (GMP kinase). DXG-DP acts as substrate for phosphorylation to the active DXG-TP for several enzymes including nucleotide diphosphate kinase (NDP kinase), 3-phosphoglycerate kinase (3-PG kinase, creatine kinase, and pyruvate kinase. Amdoxovir is rapidly converted to DXG in monkeys,

Fig. 8. Metabolic pathway of amdoxovir

woodchucks, and rats with approximately 61 % of the dose converted to DXG (Chen et al., 1996; Chen et al., 1999; Rajagopalan et al., 1996). The oral bioavailability of amdoxovir is estimated to be 30% (Chen et al., 1999). Following oral administration of amdoxovir to HIVinfected patients, peak plasma levels of amdoxivir and DXG were reached within 2 hours

Molecular Pharmacology of Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors 73

concentrations of CMX-157 provide >300-fold greater activity against clinical isolates than tenofovir with EC50 values < 1 nM (Lanier et al., 2010). It has additionally been proposed that CMX-157 may bind cell free virions by direct lipid insertion into the viral envelope resulting in facilitated delivery to target cells (Painter et al., 2007). CMX-157 recently completed a Phase I clinical trial to evaluate safety, tolerability and pharmacokinetics. CMX-157 was well tolerated with no drug-related adverse events. Plasma levels increased linearly with dose and active TFV-DP was detected up to six days post administration of a 400 mg

dose suggesting the possibility of a once weekly dosing regimen.

Fig. 9. Intracellular metabolism of GS-7340 and CMX-157

Nucleoside and nucleotide reverse transcriptase inhibitors have remained the backbone of antiretroviral therapy. The absolute dependence of NRTI on host cellular enzymes for activation is a unique property of this drug class. The eight approved NRTI and numerous experimental NRTI display great diversity for all of these factors, thus presenting pharmacological advantages and challenges that are unique to the NRTI class. The complex relationships between NRTIs and host cell enzymes have necessitated detailed studies of the *in vitro* and *in vivo* pharmacologic properties of novel NRTIs in pre-clinical development. Current drug discovery efforts increasingly utilize NRTI prodrugs in order to accelerate NRTI phosphorylation or otherwise improve pharmacologic properties. Further understanding of the cellular pharmacology of NRTI is crucial for the development of novel drugs for increased potency, improved safety and tolerability, and decreased resistance.

**5. Conclusions** 

(Thompson et al., 2005). Amdoxovir was eliminated from plasma with half-life of 1 - 2 hours by conversion to DXG, whereas DXG demonstrated a longer half-life of 4 - 7 hours. In animal studies amdoxovir toxicities included obstructive nephropathy, uremia, islet cell atrophy, hyperglycemia, and lens opacities (Rajagopalan et al., 1996). In a phase I/II clinical study 4 of 18 patients developed nongradeable lens opacities (Thompson et al., 2005). In other studies most adverse events were minor and included nausea, headache, and diarrhea (Gripshover et al., 2006; Murphy et al., 2008).

#### **4.4 GS-7340**

GS-7340 (9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl] methoxy]propyl]adenine) is a novel isopropylalaninyl phenyl ester prodrug of tenofovir designed to increase intracellular delivery of the active tenofovir-DP metabolite by masking the charged phosphonate (Figure 9; Eisenberg et al 2001). Preclinical studies demonstrated 200-fold improved plasma stability and 400-fold increased accumulation of tenofovir and active tenofovir-DP in lymphatic tissues and peripheral blood mononuclear cells (PBMC) compared to tenofovir (Lee et al., 2005; Eisenberg et al., 2001). GS-7340 has 1000-fold improved potency *in vitro* over tenofovir. Following rapid target cell uptake, GS-7340 is hydrolyzed at the carboxy ester bond in lysozomes by the serine protease cathepsin A and other serine and cysteine proteases (Birkus et al., 2007; 2008). The resulting partially stable product spontaneously releases phenol by intramolecular cyclization and hydrolysis to a negatively charged, cell impermeable tenofovir-alanine intermediate (Balzarini et al., 1996). Formation of tenofovir-alanine is faster in resting PBMC compared to activated PBMC, while metabolism to parent tenofovir by a phosphoamidase and downstream phosphorylation to tenofovir-MP and tenofovir-DP is much faster in activated PBMC. A recent clinical study comparing 50 mg and 150 mg doses of GS-7340 with 300 mg TDF was conducted to determine the efficacy, safety and pharmacokinetics over 14 days (Markowitz et al., 2011). Viral loads were reduced -1.71-log and -1.57-log for 150 mg and 50 mg doses, respectively, compared to 0.94-log for TDF. PBMC levels of tenofovir were 4 – 33- times greater with GS-7340 than those for TDF at day 14 while plasma levels of tenofovir were decreased up to 88% at 24 hours with administration of GS-7340 compared to TDF. No serious adverse events were reported while the most frequent complaint was mild to moderate headache and nausea.

#### **4.5 CMX-157**

Like GS-7340, CMX-157 is an alternative prodrug of tenofovir designed to increase cell penetration by the natural lipid uptake pathways (Figure 9; Hostetler et al., 1997; Painter et al., 2004). CMX-157 contains a hexadecyloxypropyl (HDP) lipid conjugation which mimics lysophosphatidylcholine. CMX-157, unlike TDF is not cleaved to free tenofovir in the intestinal mucosa and thus circulates in plasma as the tenofovir-HDP lipid conjugate (Painter et al., 2007). Tenofovir-HDP is not a substrate for human organic anion transporters and therefore is subject to decreased renal excretion and increased intracellular drug exposure compared to TDF (Tippin et al., 2010). Free tenofovir is liberated intracellularly by hydrolytic removal of the HDP lipid by phospholipases. Intracellular activation to the active tenofovir-DP form is achieved in the same manner as TDF. CMX-157 delivers > 30-fold increased active metabolite tenofovir-DP in PBMC than tenofovir. Higher intracellular concentrations of CMX-157 provide >300-fold greater activity against clinical isolates than tenofovir with EC50 values < 1 nM (Lanier et al., 2010). It has additionally been proposed that CMX-157 may bind cell free virions by direct lipid insertion into the viral envelope resulting in facilitated delivery to target cells (Painter et al., 2007). CMX-157 recently completed a Phase I clinical trial to evaluate safety, tolerability and pharmacokinetics. CMX-157 was well tolerated with no drug-related adverse events. Plasma levels increased linearly with dose and active TFV-DP was detected up to six days post administration of a 400 mg dose suggesting the possibility of a once weekly dosing regimen.

Fig. 9. Intracellular metabolism of GS-7340 and CMX-157

#### **5. Conclusions**

72 Pharmacology

(Thompson et al., 2005). Amdoxovir was eliminated from plasma with half-life of 1 - 2 hours by conversion to DXG, whereas DXG demonstrated a longer half-life of 4 - 7 hours. In animal studies amdoxovir toxicities included obstructive nephropathy, uremia, islet cell atrophy, hyperglycemia, and lens opacities (Rajagopalan et al., 1996). In a phase I/II clinical study 4 of 18 patients developed nongradeable lens opacities (Thompson et al., 2005). In other studies most adverse events were minor and included nausea, headache, and diarrhea

GS-7340 (9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl] methoxy]propyl]adenine) is a novel isopropylalaninyl phenyl ester prodrug of tenofovir designed to increase intracellular delivery of the active tenofovir-DP metabolite by masking the charged phosphonate (Figure 9; Eisenberg et al 2001). Preclinical studies demonstrated 200-fold improved plasma stability and 400-fold increased accumulation of tenofovir and active tenofovir-DP in lymphatic tissues and peripheral blood mononuclear cells (PBMC) compared to tenofovir (Lee et al., 2005; Eisenberg et al., 2001). GS-7340 has 1000-fold improved potency *in vitro* over tenofovir. Following rapid target cell uptake, GS-7340 is hydrolyzed at the carboxy ester bond in lysozomes by the serine protease cathepsin A and other serine and cysteine proteases (Birkus et al., 2007; 2008). The resulting partially stable product spontaneously releases phenol by intramolecular cyclization and hydrolysis to a negatively charged, cell impermeable tenofovir-alanine intermediate (Balzarini et al., 1996). Formation of tenofovir-alanine is faster in resting PBMC compared to activated PBMC, while metabolism to parent tenofovir by a phosphoamidase and downstream phosphorylation to tenofovir-MP and tenofovir-DP is much faster in activated PBMC. A recent clinical study comparing 50 mg and 150 mg doses of GS-7340 with 300 mg TDF was conducted to determine the efficacy, safety and pharmacokinetics over 14 days (Markowitz et al., 2011). Viral loads were reduced -1.71-log and -1.57-log for 150 mg and 50 mg doses, respectively, compared to 0.94-log for TDF. PBMC levels of tenofovir were 4 – 33- times greater with GS-7340 than those for TDF at day 14 while plasma levels of tenofovir were decreased up to 88% at 24 hours with administration of GS-7340 compared to TDF. No serious adverse events were reported while the most frequent complaint was mild to

Like GS-7340, CMX-157 is an alternative prodrug of tenofovir designed to increase cell penetration by the natural lipid uptake pathways (Figure 9; Hostetler et al., 1997; Painter et al., 2004). CMX-157 contains a hexadecyloxypropyl (HDP) lipid conjugation which mimics lysophosphatidylcholine. CMX-157, unlike TDF is not cleaved to free tenofovir in the intestinal mucosa and thus circulates in plasma as the tenofovir-HDP lipid conjugate (Painter et al., 2007). Tenofovir-HDP is not a substrate for human organic anion transporters and therefore is subject to decreased renal excretion and increased intracellular drug exposure compared to TDF (Tippin et al., 2010). Free tenofovir is liberated intracellularly by hydrolytic removal of the HDP lipid by phospholipases. Intracellular activation to the active tenofovir-DP form is achieved in the same manner as TDF. CMX-157 delivers > 30-fold increased active metabolite tenofovir-DP in PBMC than tenofovir. Higher intracellular

(Gripshover et al., 2006; Murphy et al., 2008).

moderate headache and nausea.

**4.5 CMX-157** 

**4.4 GS-7340** 

Nucleoside and nucleotide reverse transcriptase inhibitors have remained the backbone of antiretroviral therapy. The absolute dependence of NRTI on host cellular enzymes for activation is a unique property of this drug class. The eight approved NRTI and numerous experimental NRTI display great diversity for all of these factors, thus presenting pharmacological advantages and challenges that are unique to the NRTI class. The complex relationships between NRTIs and host cell enzymes have necessitated detailed studies of the *in vitro* and *in vivo* pharmacologic properties of novel NRTIs in pre-clinical development. Current drug discovery efforts increasingly utilize NRTI prodrugs in order to accelerate NRTI phosphorylation or otherwise improve pharmacologic properties. Further understanding of the cellular pharmacology of NRTI is crucial for the development of novel drugs for increased potency, improved safety and tolerability, and decreased resistance.

Molecular Pharmacology of Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors 75

Cahn, P., Rolon, M., Cassetti, I., Shiveley, L., Holdich, T. & Sawyer, J. (2008). Multiple-dose

Cammack, N., Rouse, P., Marr, C.L., Reid, P.J., Boehme, R.E., Coates, J.A., Penn, C.R. &

Chang, C.N., Skalski, V., Zhou, J.H. & Cheng, Y.C. (1992). Biochemical pharmacology of (+)-

Chen, H., Boudinot, F.D., Chu, C.K., Mcclure, H.M. & Schinazi, R.F. (1996).

guanine in rhesus monkeys. *Antimicrob. Agents Chemother.* 40(10): 2332-6. Chen, H., Schinazi, R.F., Rajagopalan, P., Gao, Z., Chu, C.K., McClure, H.M. & Boudinot,

Chittick, G.E., Gillotin, C., McDowell, J.A., Lou, Y., Edwards, K.D., Prince, W.T. & Stein, D.S.

Cihlar, T. & Ray, A.S. (2010). Nucleoside and nucleotide HIV reverse transcriptase

Cirno, N.M., Cameron, C.E., Smith, J.S., Rausch, J.W., Roth, M.J., Benkovic, S.J. & Le Grice,

immunodeficiency virus reverse transcriptase. *Biochemistry* 34: 9936-9943. Cooney, D.A., Ahiuwalia, G., Mitsuya, H., Fridland, A., Johnson, M., Hao, Z., Dalal, M.,

Cotte, L., Dellamonica, P., Raffi, F., Yazdanpanah, L.Y., Molina, J. M., Boue, F., & Urata, Y.

Daluge, S.M., Good, S.S., Faletto, M.B., Miller, W.H., St Clair, M.H., Boone, L.R., Tisdale, M.,

de Muys, J.M., Gourdeau, H., Nguyen-Ba, N., Taylor, D.L., Ahmed, P.S., Mansour, T., Locas,

formulations, and effect of food. *Pharmacotherapy.* 19(8): 932-42.

inhibitors: 25 years after zidovudine. *Antiviral Res.* 85(1):39-58.

in patients with HIV-1 infection. *Clin. Drug. Invest.* 28: 129 -38.

thiacytidine. *Biochem. Pharmacol.* 43: 2059-2064.

267(31): 22414-20.

15(18): 1625-30.

*Biochem. Pharmacol.* 36: 1765-1768.

U.S.A. September 12-15, 2010.

*Antimicrob. Agents Chemother*. 43: 2245–2250.

pharmacokinetics of apricitabine, a novel nucleoside reverse transcriptase inhibitor,

Cameron, J.M. (1992). Cellular metabolism of (-) enantiomeric 2'-deoxy-3'-

and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents. *J. Biol. Chem.*

Pharmacokinetics of (-)-beta-D-2-aminopurine dioxolane and (-)-beta-D-2-amino-6 chloropurine dioxolane and their antiviral metabolite (-)-beta-D-dioxolane

F.D. (1999). Pharmacokinetics of (-)-beta-D-dioxolane guanine and prodrug (-)-beta-D-2,6-diaminopurine dioxolane in rats and monkeys. *AIDS Res. Hum. Retroviruses.* 

(1999). Abacavir: absolute bioavailability, bioequivalence of three oral

S.F.J. (1995) Divalent cation modulation of the Ribonuclease h functions of human

Balzarini, J., Broder, S. & Johns, D.G. (1987). Initial studies on the cellular pharmacology of 2',3'-dideoxyadenosine, an inhibitor of HTLV-III infectivity.

(2010). A Phase-Ib/IIa Dose-Escalation Study of OBP-601 (4'-ethynyl-d4T, Festinavir) in Treatment-Experienced, HIV-1-Infected Patients. *50th Interscience Conference on Antimicrobial Agents and Chemotherapy* (ICAAC 2010). Boston, MA.

Parry, N.R., Reardon, J.E., Dornsife, R.E., Averett, D.R. & Krenitsky, T.A. (1997). 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity. *Antimicrob. Agents Chemother.* 41(5): 1082-93. Darque, A., Valette, G., Rousseau, F., Wang, L.H., Sommadossi, J.P. & Zhou, X.J. (1999).

Quantitation of intracellular triphosphate of emtricitabine in peripheral blood mononuclear cells from human immunodeficiency virus-infected patients.

C., Richard, N., Wainberg, M.A. & Rando, R.F. (1999). Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and

#### **6. Acknowledgements**

Research in the Sluis-Cremer laboratory was supported by grants AI081571, GM068406 and AI071846 from the National Institutes of Health (NIH), United States of America. Brian Herman was supported by an NIH training grant (T32 AAI 49820).

#### **7. References**


Research in the Sluis-Cremer laboratory was supported by grants AI081571, GM068406 and AI071846 from the National Institutes of Health (NIH), United States of America. Brian

Ahluwalia, G., Cooney, D.A., Mitsuya, H., Fridland, A., Flora, K.P., Hao, Z., Dalal, M.,

August, E.M., Marongiu, M.E., Lin, T.,S. & Prusoff, W.H. (1988). Initial studies on the

Balzarini, J., Herdewijn, P. & De Clercq, E. (1989). Differential patterns of intracellular

Balzarini, J., Karlsson, A., Aquaro, S., Perno, C.F., Cahard, D., Naesens, L., De Clercq, E. &

Bang, L. & Scott, L.J. (2003). Emtricitabine: an antiretroviral agent for HIV infection. *Drugs*.

Barbier, O., Turgeon, D., Girard, C., Green, M.D., Tephly, T.R., Hum, D.W. & Bélanger, A.

glucuronosyltransferase 2B7 (UGT2B7). *Drug Metab. Dispos.* 28(5): 497-502. Barditch-Crovo, P., Deeks, S.G., Collier, A., Safrin, S., Coakley, D.F., Miller, M., Kearney,

Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet,

Birkus, G., Kutty, N., He, G.X., Mulato, A., Lee, W., McDermott, M. & Cihlar, T.

prodrugs GS-7340 and GS-9131. *Antimicrob. Agents Chemother.* 51(2): 543-50.

acquired immune deficiency syndrome (AIDS). *Science* 220: 868–871. Bethell, R., de Muys, J., Lippens, J., Richard, A., Hamelin, B., Ren, C. & Collins, P. (2007). In

Baltimore, D. (1970) Viral RNA-dependent DNA polymerase. *Nature* 226: 1209-1211.

derivatives. *Proc. Natl. Acad. Sci. U.S.A.* 93: 7295–7299.

*Antimicrob Agents Chemother.* 2001; 45: 2733-9.

*Antimicrob. Agents Chemother.* 51: 2948 -53.

Broder, S. & Johns, D.G. (1987). Initial studies on the cellular pharmacology of 2',3' dideoxyinosine, an inhibitor of HIV infectivity. *Biochem. Pharmacol.* 36(22): 3797-800.

cellular pharmacology of 3'-deoxythymidin-2'-ene (d4T): a potent and selective inhibitor of human immunodeficiency virus. *Biochem. Pharmacol*., 37(23): 4419-22.

metabolism of 2',3'-didehydro-2',3'-dideoxy-thymidine (D4T) and 3'-azido-2',3' dideoxythymidine (AZT), two potent anti-HIV compounds. *J. Biol. Chem.* 264: 6127-

McGuigan, C. (1996). Mechanism of anti-HIV action of masked alaninyl d4T-MP

(2000). 3'-azido-3'-deoxythimidine (AZT) is glucuronidated by human UDP-

B.P., Coleman, R.L., Lamy, P.D., Kahn, J.O., McGowan, I. & Lietman, P.S. (2001). Phase I/II trial of the pharmacokinetics, safety and antiretroviral activity of tenofovir disoproxil fumarate in human immunodeficiency virus-infected adults.

C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum,W., & Montagnier, L., (1983) Isolation of a T-lymphotropic retrovirus from a patient at risk for

vitro interactions between apricitabine and other deoxycytidine analogues.

(2008). Activation of 9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-ethoxy]propyl]adenine (GS-7340) and other tenofovir phosphonoamidate prodrugs by human proteases. *Mol. Pharmacol.* 74(1): 92-100. Birkus, G., Wang, R., Liu, X., Kutty, N., MacArthur, H., Cihlar, T., Gibbs, C., Swaminathan,

S., Lee, W. & McDermott, M. (2007). Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate

Herman was supported by an NIH training grant (T32 AAI 49820).

**6. Acknowledgements** 

**7. References** 

33.

63: 2413–2424.


Molecular Pharmacology of Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors 77

Gripshover, B.M., Ribaudo, H., Santana, J., Gerber, J.G., Campbell, T.B., Hogg, E., Jarocki,

Haraguchi, K., Takeda, S., Tanaka, H., Nitanda, T., Baba, M., Dutschman, G.,E. & Cheng,

Ho, H.,T. & Hitchcock, M.,J. (1989). Cellular pharmacology of 2',3'-dideoxy-2',3'-

Holdich, T., Shiveley, L. & Sawyer, J. (2006). Pharmacokinetics of single oral doses of

Holdich, T., Shiveley, L.A. & Sawyer, J. (2007). Effect of lamivudine on the plasma and

Hostetler, K.Y., Beadle, J.R., Kini, G.D., Gardner, M.F., Wright, K.N., Wu, T.H. & Korba, B.A.

Hsu, C.H., Hu, R., Dutschman, G.E., Yang, G., Krishnan, P., Tanaka, H., Baba, M. & Cheng,

Johnson, M.A. & Fridland, A. (1989). Phosphorylation of 2',3'-dideoxyinosine by cytosolic 5'-

Johnson, M.A., Moore, K.H., Yuen, G.J., Bye, A. & Pakes, G.E. (1999). Clinical

Kim, H.O., Schinazi, R.F., Nampalli, S., Shanmuganathan, K., Cannon, D.L., Alves, A.J.,

Lanier, E.R., Ptak, R.G., Lampert, B.M., Keilholz, L., Hartman, T., Buckheit, Jr., R.W.,

Lee, W.A., He, G.X., Eisenberg, E., Cihlar, T., Swaminathan, S., Mulato, A. & Cundy, K.C.

nucleotidase of human lymphoid cells. *Mol. Pharmacol.* 36: 291-5.

pharmacokinetics of lamivudine. *Clin. Pharmacokinet.* 36(1):41-66.

virus type 1 variants. *Antimicrob. Agents Chemother.* 43(10): 2376-82.

deoxy-4'-ethynylthymidine. *Bioorg. Med. Chem. Lett.* 13(21): 3775-7.

virus. *Antimicrob. Agents Chemother*. 33(6): 844-9.

healthy volunteers. *Clin. Drug. Invest.* 26: 279 -86.

analogs. *Antimicrob. Agents Chemother.* 51(5): 1687-93.

*Biochem. Pharmacol.* 53: 1815–1822.

*Med. Chem.* 36(1): 30-37.

*Chemother.* 54(7): 2901-9.

*Chemother.* 49(5): 1898-906.

2943 -2947.

B., Hammer, S.M. & Kuritzkes, D.R.; A5118 Team. (2006). Amdoxovir versus placebo with enfuvirtide plus optimized background therapy for HIV-1-infected Gu, Z., Wainberg, M.A., Nguyen-Ba, N., L'Heureux, L., de Muys, J.M., Bowlin, T.L. & Rando,

R.F. (1999) Mechanism of action and in vitro activity of 1',3'-dioxolanylpurine nucleoside analogues against sensitive and drug-resistant human immunodeficiency

Y.C. (2003). Synthesis of a highly active new anti-HIV agent 2',3'-didehydro-3'-

didehydrothymidine, a nucleoside analog active against human immunodeficiency

apricitabine, a novel deoxycytidine analogue reverse transcriptase inhibitor, in

intracellular pharmacokinetics of apricitabine, a novel nucleoside reverse transcriptase inhibitor, in healthy volunteers. *Antimicrob Agents Chemother.* 51:

(1997). Enhanced oral absorption and antiviral activity of 1-O-octadecyl-sn-glycero-3-phospho-acyclovir and related compounds in hepatitis B virus infection, in vitro.

Y.C. (2007). Comparison of the phosphorylation of 4'-ethynyl 2',3'-dihydro-3' deoxythymidine with that of other anti-human immunodeficiency virus thymidine

Jeong, L.S., Beach, J.W. & Chu, C.K. (1993). 1,3-dioxolanylpurine nucleosides (2R,4R) and (2R,4S) with selective anti-HIV-1 activity in human lymphocytes. *J.* 

Mankowski, M.K., Osterling, M.C., Almond, M.R. & Painter, G.R. (2010). Development of hexadecyloxypropyl tenofovir (CMX157) for treatment of infection caused by wild-type and nucleoside/nucleotide-resistant HIV. *Antimicrob Agents* 

(2005). Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue. *Antimicrob. Agents* 

pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine. *Antimicrob. Agents. Chemother.* 43(8): 1835-44.


DeStefano, J.J., Bambara, R.A., & Fay, P.J. (1993) Parameters that influence the binding of

Dudley, M.N., Graham, K.K., Kaul, S., Geletko, S., Dunkle, L., Browne, M. & Mayer, K.

Dutschman, G.E., Grill, S.P., Gullen, E.A., Haraguchi, K., Takeda, S., Tanaka, H., Baba, M. &

Eisenberg, E.J., He, G.X. & Lee, W.A. (2001). Metabolism of GS-7340, a novel phenyl

Faletto, M.B., Miller, W.H., Garvey, E.P., St Clair, M.H., Daluge, S.M. & Good, S.S. (1997).

Feng, J.Y., Parker, W.B., Krajewski, M.L., Deville-Bonne, D., Veron, M., Krishnan, P., Cheng,

Furman, P.A., Fyfe, J.A., St Clair, M.H., Weinhold, K., Rideout, J.L., Freeman,

Furman, P.A., Jeffrey, J., Kiefer, L.L., Feng, J.Y., Anderson, K.S., Borroto-Esoda, K., Hill, E.,

Gallo, R.C., Salahuddin, S.Z., Popovic, M., Shearer, G.M., Kaplan, M., Haynes, B.F., Palker,

Ghosh M., Williams, J., Powell, M.G., Levin, J.G., & Le Grice, S.F.J. (1997) Mutating a

Goldschmidt, V. & Marquet, R. (2004). Primer unblocking by HIV-1 reverse transcriptase

Goody, R.S., Müller, B. & Restle, T. (1991). Factors contributing to the inhibition of HIV

agent 1592U89. *Antimicrob. Agents Chemother.* 41(5): 1099-107.

guanosine. *Antimicrob. Agents Chemother.* 45(1): 158-65.

*Chemother.* 43(8): 1835-44.

*Biochemistry* 32: 6908-6915.

complex. *J. Infect. Dis*. 166: 480–485.

*Agents Chemother.* 48: 1640–1646.

*Nucleotides Nucleic Acids.* 20: 1091-8.

*Biochem. Pharmacol.* 68(9): 1879-88.

*Acad. Sci. U. S. A*. 83(21): 8333-7.

utilization*. Biochemistry* 36: 5758-5768.

*Science* 224, 500–503.

1687-705.

*Lett.* 291(1):1-5.

pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine. *Antimicrob. Agents.* 

human immunodeficiency virus reverse transcriptase to nucleic acid structures.

(1992). Pharmacokinetics of stavudine in patients with AIDS or AIDS-related

Cheng, Y.C. (2004). Novel 4'-substituted stavudine analog with improved antihumanimmunodeficiency virus activity and decreased cytotoxicity. *Antimicrob.* 

monophosphoramidate intracellular prodrug of PMPA, in blood. *Nucleosides* 

Unique intracellular activation of the potent anti-human immunodeficiency virus

Y.C. & Borroto-Esoda, K. (2004). Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and its 5'-phosphates by mammalian phosphotransferases.

G.A., Lehrman, S.N., Bolognesi, D.P., Broder, S., Mitsuya H, et al. (1986). Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5' triphosphate with human immunodeficiency virus reverse transcriptase. *Proc. Natl.* 

Copeland, W.C., Chu, C.K., Sommadossi, J.P., Liberman, I., Schinazi, R.F. & Painter, G.R. (2001). Mechanism of action of 1-beta-D-2,6-diaminopurine dioxolane, a prodrug of the human immunodeficiency virus type 1 inhibitor 1-beta-D-dioxolane

T.J., Redfield, R., Oleske, J., Safai, B., et al., (1984) Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS.

conserved motif of the HIV-1 reverse transcriptase palm subdomain alters primer

and resistance to nucleoside RT inhibitors (NRTIs). *Int. J. Biochem. Cell Biol.* (9):

reverse transcriptase by chain-terminating nucleotides in vitro and in vivo. *FEBS* 


Molecular Pharmacology of Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors 79

Painter, G.R., Almond, M.R., Trost, L.C., Lampert, B.M., Neyts, J., De Clercq, E., Korba, B.E.,

Painter, G.R. & Hostetler, K.Y. (2004). Design and development of oral drugs for the prophylaxis and treatment of smallpox infection. *Trends Biotechnol*. 22: 423–427. Paintsil, E., Mastuda, T., Ross, J., Schofield, J., Cheng, Y.C., Urata, Y., (2009). A Singledose

*Opportunistic Infections*, February 2009, Montreal, Canada. Abstract 568. Peliska, J.A. & Benkovic, S.J. (1992) Mechanism of DNA strand transfer reactions catalyzed

Rajagopalan, P., Boudinot, F. D., Chu, C.K., McClure, H.M., & Schinazi, R.F. (1994).

Rajagopalan, P., Boudinot, F.D., Chu, C.K., Tennant, B.C., Baldwin, B.H. & Schinazi, R.F.

Rana, K.Z. & Dudley, M.N. (1997). Clinical pharmacokinetics of stavudine. *Clin.* 

Ray, A. S., Cihlar, T., Robinson, K. L., Tong, L., Vela, J. E., Fuller, M. D., Wieman, L. M.,

Robbins, B.L., Srinivas, R.V., Kim, C., Bischofberger, N., Fridland, A. (1998). Anti-human

Ruane, .P.J, Richmond, G.J., DeJesus, E., Hill-Zabala, C.E., Danehower, S.C., Liao, Q.,

Sawyer, J. & Cox, S. (2006). In vitro pharmacology of Apricitabine, a new NRTI for HIV. *XVI International AIDS Conference,* August 2006, Toronto, Canada, abstract CDB0046. Sawyer, J. & Struthers-Semple, C. (2006). Pharmacokinetics of apricitabine in healthy

Starnes, M.C. & Cheng, Y.C. (1987). Cellular metabolism of 2',3'-dideoxycytidine, a

Stein, D.S. & Moore, K.H. (2001). Phosphorylation of nucleoside analog antiretrovirals: a

Taylor, D.L., Ahmed, P.S., Tyms, A.S., Wood, L.J., Kelly, L.A., Chambers, P., Clarke, J.,

infections. *Antimicrob. Agents Chemother.* 51: 3505–3509.

by HIV-1 reverse transcriptase. *Science* 258: 1112-1118.

tenofovir. *Antimicrob. Agents Chemother.* 50: 3297–3304.

immunodeficiency virus. *Pharmacotherapy.* 24(3): 307-12.

2006, Toronto, Canada, Abstract TUPE0077.

review for clinicians. *Pharmacotherapy.* (1):11-34.

7: 65–70.

612–617.

262(3): 988-91.

*Pharmacokinet*. 33:276–284.

guanosine in rhesus monkeys. *Pharm. Res.* 11(Suppl.): 381–386.

Aldern, K.A., Beadle, J.R. & Hostetler, K.Y. (2007). Evaluation of hexadecyloxypropyl-9-R-[2-(phosphonomethoxy)propyl]-adenine, CMX157, as a potential treatment for human immunodeciency virus type 1 and hepatitis B virus

escalation study to evaluate the safety, tolerability, and pharmacokinetics of OBP-601, a novel NRTI, in healthy subjects. In: *16th Conference on Retroviruses and* 

Pharmacokinetics of (2)-β-D-2,6-diaminopurine dioxolaneand its metabolite

(1996). Pharmacokinetics of (2)-β-D-2,6-diaminopurinedioxolane and its metabolite, dioxolane guanosine, in woodchucks (Marmotamonax). *Antivir. Chem. Chemother.*

Eisenberg, E. J. & Rhodes, G. R. (2006). Mechanism of active renal tubular efux of

immunodeficiency virus activity and cellular metabolism of a potential prodrug of the acyclic nucleoside phosphonate 9-*R*-(2-phosphonomethoxypropyl)adenine (PMPA), bis(isopropyloxymethylcarbonyl)PMPA. *Antimicrob. Agents Chemother.* 42:

Johnson, J. & Shaefer, M.S. (2004). Pharmacodynamic effects of zidovudine 600 mg once/day versus 300 mg twice/day in therapy-naïve patients infected with human

volunteers and HIV-infected individuals. *XVI International AIDS Conference,* August

compound active against human immunodeficiency virus in vitro. *J Biol. Chem.* 

Bedard, J., Bowlin, T.L., Rando, R.F. (2000). Drug resistance and drug combination


Markowitz, M., Zolopa, A., Ruane, P., Squires, K., Zhong, L., Kearney, B.P. & Lee, W. (2011).

Masood, R.W., Ahluwalia, G.S., Cooney, D.A., Fridland, A., Marquez, V.E., Driscoll, J.S.,

McDowell, J.A., Lou, Y., Symonds, W.S. & Stein, D.S. (2000). Multiple-dose

Meyer, P.R., Matsuura, S.E., So, A.G. & Scott, W.A. (1998). Unblocking of chain-terminated

Mitsuya, H., & Broder, S. (1986) Inhibition of the in vitro infectivity and cytopathic effect of

agent 2',3'-dideoxyadenosine. *Mol. Pharmacol.* 37: 590-6.

Abstract # 152LB.

*Agents Chemother.* 44(8): 2061-7.

*Proc. Natl. Acad. Sci. U.S.A.* 95(23): 13471-6.

*Proc. Natl. Acad. Sci. U. S. A*. 82: 7096–7100.

February 3-6, 2008. Boston, MA. Abstract 794.

mice. *Antimicrob. Agents Chemother*. 42: 1568–1573.

*Pharmacol. Exper. Ther.* 319: 941 -947.

*Chemother.* 49: 3355–3360.

GS-7340 Demonstrates Greater Declines in HIV-1 RNA than Tenofovir Disoproxil Fumarate During 14 Days of Monotherapy in HIV-1 Infected Subjects. *18th Conference on Retroviruses and Opportunistic Infections* March 2, 2011 Boston, MA.,

Hao, Z., Mitsuya, H., Perno, C.F., Broder, S., et al. (1990). 2'-Fluoro-2',3' dideoxyarabinosyladenine: a metabolically stable analogue of the antiretroviral

pharmacokinetics and pharmacodynamics of abacavir alone and in combination with zidovudine in human immunodeficiency virus-infected adults. *Antimicrob.* 

primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism.

human T-lymphotrophic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleosides. *Proc. Natl. Acad. Sci. U.S. A*. 83: 1911–1915. Mitsuya, H., Jarrett, R.F., Matsukura, M., Veronese, F.D., DeVico, A.L., Sarngadharan, M.G.,

Johns, D.G., Reitz, M.S., & Broder, S. (1987) Long-term inhibition of human Tlymphotropic virus type III/lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2',3'-dideoxynucleosides in vitro. *Proc. Natl. Acad. Sci. U. S. A*. 84: 2033–2037. Mitsuya, H., Weinhold, K.J., Furman, P.A., St Clair, M.H., Nusinoff-Lehrman, S., Gallo, R.C.,

Bolognesi, D., Barry, D.W., & Broder, S. (1985) 3'-azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro.

S.J., Kivel, N.M., & Schinazi, R.F. (2008). Pharmacokinetics and potent anti-HIV-1 activity of amdoxovir plus zidovudine in a randomized double-blind placebocontrolled study. *15th Conference on Retroviruses and Opportunistic Infections.* 

Kim, C.U., De Clercq, E., (1998). Antiretroviral efficacy and pharmacokinetics of oral bis-(isopropyloxycarbonyloxymethyl)-9-(2-phosphonylmethoxypropyl)adenine in

trimethoprim on the clearance of apricitabine, a deoxycytidine analog reverse transcriptase inhibitor, and lamivudine in the isolated rat perfused kidney. *J.* 

(2005). Anti-human immunodeficiency virus type 1 activity and resistance profile of 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine in vitro. *Antimicrob. Agents* 

Murphy, R., Zala, C., Ochoa, C., Tharnish, P., Mathew, J., Fromentin, E., Asif, G., Hurwitz,

Naesens, L., Bischofberger, N., Augustijns, P., Annaert, P., Van den Mooter, G., Arimilli, M.N.,

Nakatani-Freshwater, T., Babayeva, M., Dontabhaktuni, A. & Taft, D.R. (2006). Effects of

Nitanda, T., Wang, X., Kumamoto, H., Haraguchi, K., Tanaka, H., Cheng, Y.C. & Baba, M.


**4** 

*Japan* 

**Opioid Kappa Receptor Selective** 

Hideaki Fujii, Shigeto Hirayama and Hiroshi Nagase

*School of Pharmacy, Kitasato University* 

**Agonist TRK-820 (Nalfurafine Hydrochloride)** 

TRK-820 (nalfurafine hydrochloride) is a selective opioid receptor agonist (Fig. 1) that was launched as an antipruritic for hemodialysis patients in Japan in 2009. In general, clinically used opioids, such as morphine, exhibit potent antinociceptive effects and simultaneous severe adverse effects, including drug dependence, derived from the opioid receptor. To develop analgesics without drug dependence, receptor agonists are investigated. However, conventional agonists, arylacetamide derivatives, showed aversive effects like psychotomimetic effects, and have not yet been used clinically. On the other hand, the novel agonist TRK-820 has no dependent or aversive properties. TRK-820, which has a structure different from arylacetamides, was first developed as an analgesic for postoperative pain, but the indication was changed to pruritus (Nakao & Mochizuki, 2009; Nagase & Fujii, 2011). The rational drug design and synthesis of the compound have been reported (Kawai et al., 2008; Nagase et al., 1998; Nagase & Fujii, 2011); therefore, in this chapter, we will focus

The binding affinities of TRK-820 were evaluated using various tritiated ligands and opioid receptors derived from various species (Table 1). The selectivity over the receptor (*K*<sup>i</sup> ratio /) tended to be higher than over the receptor (*K*i ratio /). Binding affinities for the L-type Ca2+ channel and 45 receptors, except the opioid receptors, were examined (Nakao & Mochizuki, 2009). Among the tested receptors, TRK-820 showed the strongest affinity for the muscarine M1 receptor, but its *K*i value was 1,700 nmol/L and approximately 7,000 times higher than that of the receptor. A comparison of the binding properties of TRK-820 and a conventional agonist, U-69,593, was noteworthy. In a competitive binding

**1. Introduction** 

on its pharmacological properties.

Fig. 1. Structure of nalfurafine hydrochloride (TRK-820)

**2. Opioid receptor type selectivity (***In vitro***)** 

features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2' deoxy-3'-oxa-4'-thiocytidine, dOTC]. *Antivir. Chem. Chemother*. 11(4): 291-301.

