HIV-1
HIV-1 Reverse Transcriptase (RT)
Ki = 0.08 μM for HIV-1 RT [1]
DNA polymerase α, β, γ (cellular polymerases). Ki values: 300 μM for DNA polymerase α, 2.2 μM for DNA polymerase β, and 11 μM for DNA polymerase γ [1]

With IC50 values of 0.2 μM, 1.45 μM, 2.2 μM, and 2.4 μM for HIV-1RF, Wild type, 3TC resistant, 3TC, and AZT resistant, respectively, apricitabine (SPD754; AVX754) is effective against clinical isolates of HIV-1 in cultured PBMCs[1].
In MT-4 cells, apricitabine (SPD754; AVX754) exhibits antiviral activities against HIV-1 clinical isolates resistant to nucleoside reverse transcriptase inhibitors. Its mean IC50 values for HIV-1IIIB, wild-type (control), zidovudine-resistant, lamivudine-resistant, zidovudine-resistant/lamivudine-resistant, Abacavir-resistant, and stavudine-resistant viruses are, respectively, 20 μM, 25 μM, 30 μM, 21 μM, 55 μM, 32 μM, and 71 μM[2].

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The 5'-triphosphate (TP) derivative of (-)-dOTC inhibited HIV-1 RT in vitro with a Ki of 0.08 μM. [1]
(-)-dOTC-TP was evaluated against human DNA polymerases α, β, and γ, with Ki values of 300 μM, 2.2 μM, and 11 μM, respectively. [1]
In cell culture, (-)-dOTC was a potent inhibitor of primary HIV-1 isolates from antiretroviral drug-naive patients, with a mean 50% inhibitory concentration (IC50) of 1.45 ± 0.74 μM. [1]
The mean IC50 of (-)-dOTC for viruses resistant to 3TC (with M184V mutation) was 2.2 ± 0.86 μM, and for viruses resistant to both 3TC and AZT (with multiple mutations like M184V, L41, Y215) was 2.4 ± 0.45 μM. [1]
In a study of resistance development, HIV-1RF was cultured in the presence of increasing concentrations of (-)-dOTC. After 12 passages, no phenotypic resistance was observed. [1]

The 50% cytotoxic concentration (CC50) of (-)-dOTC in various cell lines, determined by [³H]thymidine uptake, was >100 μM in MOLT-4, DU-145, HSF, HT-1080, and HepG2 cells. [1]
For murine bone marrow progenitor cells, the CC50 of dOTC (racemate) was >500 μM. [1]

In a 14-day continuous culture of HepG2 cells, (-)-dOTC at concentrations up to 10 μM showed no measurable toxic effect on mitochondrial DNA replication. The mtDNA/28S rDNA signal ratio remained similar to the control. [1]

Rats administered apricitabine (SPD754; AVX754) orally once daily at a dose of 10 mg/kg show good oral bioavailability, with 68% of male rats and 69.4% of female rats exhibiting this trait. Furthermore, in female rats, the T1/2, AUC0–∞, Tmax, and Cmax are 62.2 min, 157.4 μg/min/ml, and 37.3 min, 1.16 μg/ml, respectively[1]. When administered intravenously at a dose of 10 mg/kg once daily, apricitabine (SPD754; AVX754) displays T1/2 and AUC0–∞ values of 12.7 min and 226.9 μg/min/ml in female rats[1].

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In a repeat-dose oral toxicity study in rats, dOTC (the racemate) was administered at doses of 50, 250, and 500 mg/kg/day for 14 days. No compound-related deaths, clinical signs, body weight changes, food consumption changes, or effects on clinical pathology, organ weights, or histopathology were observed at any dose. [1]

The effect of nucleoside triphosphates on DNA polymerase activity was determined. For HIV-1 RT assays, a primed 16S rRNA from E. coli was used in a 50-μl reaction mixture containing Tris buffer (pH 8.0), KCl, MgCl2, β-mercaptoethanol, glycerol, bovine serum albumin, RT enzyme, dATP, dGTP, dTTP, and various concentrations of [³H]dCTP at the measured Km. After incubation at 37°C for 60 min, DNA was precipitated onto glass fiber filters using trichloroacetic acid solution with pyrophosphate. The filters were washed, and radioactivity was counted. IC50 values were determined by fitting the data (percentage of solvent control versus log concentration) to a straight line. Kinetic constants were determined at 37°C. [1]
DNA polymerase α and β activities were measured similarly to HIV-1 RT, except that the reaction mixture for DNA polymerase α and β contained 100 mM KCl instead of 20 mM potassium phosphate. [1]
DNA polymerase γ was purified from the mitochondria of CEM cells by affinity chromatography (single-stranded DNA cellulose column). Its activity was measured similarly to DNA polymerase α and β. [1]

For antiviral assays, the sensitivities of clinical isolates to (-)-dOTC were determined by measuring the reduction in p24 antigen levels in the cell culture fluid after growth in PBMCs. [1]

For intracellular metabolism studies, cells (dCK⁺ CEM, dCK⁻ CEM, or PBMCs) were plated in culture medium with ³H-labeled (-)-dOTC. After incubation, cells were harvested, washed, and the nucleosides and nucleotides were extracted using trichloroacetic acid. The extracts were neutralized and analyzed by HPLC with a C18 reversed-phase column to identify phosphorylated metabolites (MP, DP, TP). The half-life (t1/2) of metabolites was determined by incubating cells with radiolabeled drug for 24 h, then removing the drug and culturing for additional time. [1]
For toxicity evaluation, cell proliferation was assessed by [³H]thymidine uptake. Cells in the mid-logarithmic phase were treated with various concentrations of test compounds for 4 days. [³H]thymidine was added 18 h before harvesting. Cells were collected onto glass fibers, and intracellular radioactivity was measured with a liquid scintillation counter. [1]
For mitochondrial DNA (mtDNA) analysis, HepG2 cells were treated with compounds for 14 days. Genomic DNA was extracted, digested with SacI, and separated on an agarose gel. After transfer to nitrocellulose membranes, the DNA was hybridized with molecular probes for a 6-kb mtDNA fragment and a 1.5-kb 28S rDNA fragment. The amount of mtDNA was determined as a ratio of the 6-kb mtDNA signal to the 1.5-kb rDNA signal. [1]

For pharmacokinetic studies, male and female Sprague-Dawley rats (15 per sex per group) received a single dose of 10 mg/kg of (-)-dOTC. For oral administration, the test compound was suspended in 0.5% (wt/vol) carboxymethyl cellulose and administered by gavage. For intravenous (i.v.) administration, the test compound (2 mg/ml) was suspended in 0.9% sodium chloride and administered by bolus injection. Blood samples were collected from five animals per sex per time point at predetermined intervals (e.g., 5 min, 15 min, 30 min, 1, 2, 4, 8, 12, 24 h post-dose). [1]

For central nervous system (CNS) penetration studies, male Sprague-Dawley rats were fitted with cisterna magna catheters and tail vein catheters. The test compounds were administered orally by gavage at a dose of 5 mg/kg with radiolabeled tracer. Blood and cerebrospinal fluid (CSF) samples were simultaneously collected over a 5-hour period (0, 5, 15, 30, 60, 90, 120, 180, 240, 300 min post-administration). Radioactivity was determined by liquid scintillation counting. [1]

For repeat-dose oral toxicity study, Sprague-Dawley rats (24 males and 24 females) were divided into four groups (six rats of each sex per group). The control group received 0.5% (wt/vol) carboxymethyl cellulose, while treatment groups received dOTC at 50, 250, or 500 mg/kg/day in 0.5% carboxymethyl cellulose. Dosing was once daily for 14 days. Parameters monitored included clinical signs, body weight, food consumption, and ophthalmological condition. On day 15, animals were killed for necropsy. Blood samples were collected for toxicokinetic determinations at 0.5, 1, and 4 h after the 1st and 14th administrations. Organ weights were recorded, and histopathological examinations were performed. [1]

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For pharmacokinetic studies, male and female Sprague-Dawley rats (15 per sex per group) received a single dose of 10 mg/kg of (-)-dOTC. For oral administration, the test compound was suspended in 0.5% (wt/vol) carboxymethyl cellulose and administered by gavage. For intravenous (i.v.) administration, the test compound (2 mg/ml) was suspended in 0.9% sodium chloride and administered by bolus injection. Blood samples were collected from five animals per sex per time point at predetermined intervals (e.g., 5 min, 15 min, 30 min, 1, 2, 4, 8, 12, 24 h post-dose). [1]

For central nervous system (CNS) penetration studies, male Sprague-Dawley rats were fitted with cisterna magna catheters and tail vein catheters. The test compounds were administered orally by gavage at a dose of 5 mg/kg with radiolabeled tracer. Blood and cerebrospinal fluid (CSF) samples were simultaneously collected over a 5-hour period (0, 5, 15, 30, 60, 90, 120, 180, 240, 300 min post-administration). Radioactivity was determined by liquid scintillation counting. [1]

For repeat-dose oral toxicity study, Sprague-Dawley rats (24 males and 24 females) were divided into four groups (six rats of each sex per group). The control group received 0.5% (wt/vol) carboxymethyl cellulose, while treatment groups received dOTC at 50, 250, or 500 mg/kg/day in 0.5% carboxymethyl cellulose. Dosing was once daily for 14 days. Parameters monitored included clinical signs, body weight, food consumption, and ophthalmological condition. On day 15, animals were killed for necropsy. Blood samples were collected for toxicokinetic determinations at 0.5, 1, and 4 h after the 1st and 14th administrations. Organ weights were recorded, and histopathological examinations were performed. [1]

Following oral administration of (-)-dOTC (10 mg/kg) to rats, the maximum concentration in plasma (Cmax) was 1.1 μg/mL for males and 1.3 μg/mL for females. The time to reach Cmax (Tmax) was 0.75 h for males and 1 h for females. The area under the curve (AUC0-∞) was 135.4 μg·min/mL for males and 157.4 μg·min/mL for females. The oral bioavailability of (-)-dOTC was 68.0% in males and 69.4% in females. [1]
The half-life (t1/2) of (-)-dOTC in plasma was 12.1 min in males and 12.7 min in females after i.v. administration. [1]
In CNS penetration studies, after a 5 mg/kg oral dose, the Cmax in blood for (-)-dOTC was 0.317 μg/mL, and in CSF it was 0.067 μg/mL. The AUC0-5h in blood was 61 μg·min/mL, and in CSF it was 15.6 μg·min/mL. [1]
In rats, dOTC (the racemate) was well absorbed after oral administration with a bioavailability of approximately 77%. A high proportion (approximately 16.5% of the levels in serum) of the compound was found in the cerebrospinal fluid. [1]

In cell proliferation studies using [³H]thymidine uptake, the 50% cytotoxic concentration (CC50) of (-)-dOTC was >100 μM in MOLT-4, DU-145, HSF, HT-1080, and HepG2 cells. [1]
For murine bone marrow progenitor cells, the CC50 of dOTC (racemate) was >500 μM. [1]
In a 14-day continuous culture, HepG2 cells treated with up to 10 μM dOTC showed no measurable toxic effect on cell confluence/morphology or on mitochondrial DNA replication. [1]

In a 14-day repeat-dose oral toxicity study in rats, dOTC (the racemate) at doses up to 500 mg/kg/day caused no compound-related deaths, clinical signs, body weight changes, food consumption changes, effects on clinical pathology, organ weights, histopathology, or bone marrow changes. Plasma drug concentrations were similar on days 1 and 14, indicating no drug accumulation. [1]

[1]. Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine.Antimicrob Agents Chemother. 1999 Aug;43(8):1835-44.

[2]. Efficacy and tolerability of 10-day monotherapy with apricitabine in antiretroviral-naive, HIV-infected patients.AIDS. 2006 Jun 12;20(9):1261-8.

Apricitabine has been used in clinical trials to study the treatment of HIV infection. Apricitabine is a cytidine analog and a nucleoside reverse transcriptase inhibitor (NRTI) with anti-HIV activity. Apricitabine is also effective against nucleoside-resistant HIV. Mechanism of Action: Apricitabine is a drug called a nucleoside reverse transcriptase inhibitor (NRTI). The mechanism of action of NRTIs is to block reverse transcriptase, a protein essential for HIV replication.

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The mechanism of action for dOTC is dependent upon its phosphorylation by cellular enzymes to the 5'-triphosphate (TP). The nucleoside TP analogue competes with the natural nucleoside TP for binding to the retroviral RT enzyme, and upon incorporation into the nascent DNA strand, acts as a terminator of chain elongation. [1]
(-)-dOTC has a sugar configuration more like that seen in natural deoxynucleoside triphosphate substrates and nucleoside analogues such as d4T, ddI, 1592U89, and ddC. [1]

The development of resistance to (-)-dOTC was slower compared to the (+)-enantiomer. No resistance was observed for virus cultured in the presence of (-)-dOTC after 12 passages. [1]

C8H11N3O3S

229.25624

229.052

C, 41.91; H, 4.84; N, 18.33; O, 20.94; S, 13.98

160707-69-7

143338-12-9 ((+/-)-);160707-68-6 (+/-);

455041

Solid powder

1.7±0.1 g/cm3

475.4±55.0 °C at 760 mmHg

241.3±31.5 °C

0.0±2.7 mmHg at 25°C

1.755

-1.35

2

4

2

15

331

2

OC[C@H]1S[C@H](CO1)N2C(NC(C=C2)=N)=O

RYMCFYKJDVMSIR-RNFRBKRXSA-N

InChI=1S/C8H11N3O3S/c9-5-1-2-11(8(13)10-5)6-4-14-7(3-12)15-6/h1-2,6-7,12H,3-4H2,(H2,9,10,13)/t6-,7-/m1/s1

4-amino-1-((2R,4R)-2-(hydroxymethyl)-1,3-oxathiolan-4-yl)pyrimidin-2(1H)-one

BCH-10618; SPD-754; (-)-BCH10652; AVX754; BCH10618; SPD754; (-)-BCH-10652; (-)-dOTC; AVX-754;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)