Reverse transcriptase; NNRTI; dTTP- and dGTP-dependent DNA polymerase (Ki = 0.20 μM); RNA polymerase (Ki = 0.01 μM)

Emivirine (EMV) has no effect on HIV-2 and is also unique to HIV-1 RT[2]. For human healthy cells, emvironment (EMV) shows no discernible toxicity. [2]

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Bone marrow toxicity has been associated with certain nucleoside analogs. Since EMV contains a substituted nucleobase, we examined the effect of the compound on bone marrow progenitor cells (Table 3). The results of these experiments demonstrated that EMV did not cause significant cytotoxicity compared with AZT (positive control). EMV was determined to have 50% cytotoxic concentrations of 30 and 50 μM for the erythroid (BFU-E) and granulocyte macrophage (CFU-GM) progenitor cells, respectively. In these experiments AZT gave 50% cytotoxic concentrations of <0.1 μM for the BFU-E progenitor cells and 7 μM for the CFU-GM progenitor cells.[2]

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Mitochondrial toxicity.[2]
The effect of EMV on mitochondrial functions was examined in exponentially growing HepG2 cells (Table 4). After the HepG2 cells were incubated with EMV at concentrations of 0.1 to 10 μM, no effect on cell growth, lactic acid production, mitochondrial DNA synthesis, or mitochondrial structure was seen compared to what occurred with untreated HepG2 cells.

Emivirine (EMV) had an approximate fatal oral dose of ≥3 g/kg for male rats and 2.5 g/kg for female rats[2].

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When rats were given EMV by gavage, by intrarectal infusion, intravenously, and by infusion into the hepatic portal vein, the oral absorption was 68%, but the data (Table 6) indicated that first-pass hepatic metabolism accounted for the lower oral bioavailability of 18%. The experiment with rats given EMV for 14 days showed that there was induction of hepatic microsomal drug-metabolizing enzymes (Table 7). At 150 mg kg−1 day−1, the relative weight of the liver was significantly increased (4.3 g/100 g of body weight, compared to 3.9 g for controls and 5.4 g for rats given 80 mg of phenobarbitol kg−1 day−1). Cytochrome P450 content increased with increasing doses of EMV, starting with 0.9 ± 0.1 nmol/mg of protein (control), and reached significance at the high dose, 1.4 ± 0.2 nmol/mg of protein (P < 0.01 relative to the value for the control). Significant increases in the activity of 7-ethoxycoumarin O-deethylase occurred at all doses of EMV (Table 7) compared to the activity in the control. Phenobarbital had positive responses for the parameters measured, as expected (Table 7) [2].

In vitro metabolism. [2]
Three experiments designed to determine species-related differences in the microsomal oxidative metabolism of Emivirine (EMV) and to identify the principal (human) cytochrome P450 enzymes involved were performed in vitro. First, liver microsomes from four humans, four cynomolgus monkeys, and four Wistar rats, all males, were isolated and incubated for 25 min at 37°C with seven concentrations of EMV, ranging from 3 to 300 μM. Reactions were stopped by adding acetonitrile to the mixture. The samples were then extracted and analyzed by HPLC for EMV and its dealkylated product. Km values and maximum rates of metabolism (Vmax) were determined by the Gauss Newton method from Lineweaver-Burk plots. Second, pooled liver microsomes (0.2 mg of protein) isolated from male rats, male cynomolgus monkeys, and male humans were incubated for 0 to 60 min at 37°C with 100 μM EMV. The incubation times were selected to be higher and lower than the 25 min used in the first experiment. Reactions were stopped with cold perchloric acid (30%), and protein was precipitated by centrifugation. The supernatants were then analyzed by the HPLC-mass spectrometry (MS) method described below. Third, liver microsomes from 10 to 14 individual human donors were incubated for 0 or 15 min at 37°C with EMV at concentrations of 10 and 100 μM. Samples were prepared and analyzed by HPLC-MS. Some of the human samples were incubated with troleandomycin (20 or 100 μM) or nifedipine (20 μM), inhibitors of many cytochrome P450 3A-catalyzed reactions, or 5 μM furafylline, a cytochrome P450 1A2 inhibitor, to obtain chemical inhibition data for correlation analyses. The experiments were controlled by using microsomes with certified activity, by running samples in duplicate or triplicate, by using zero protein blanks, by comparing interindividual variation in the rates of Emivirine (EMV) metabolism among samples of liver microsomes from 14 humans, and by using a Pearson product-moment correlation to calculate regression coefficients between the metabolic data for EMV and several standard probe drugs.

Emivirine (EMV) and its major oxidative metabolites were separated with a Zorbax XDB-C8 column (5.0 cm by 2.1 mm, 5-μm particle size) on a Hewlett-Packard model 1090 HPLC. The solvent system was a binary mobile phase consisting of 0.2% aqueous acetic acid (phase A) and 80/20 (vol/vol) methanol–0.2% aqueous acetic acid (phase B) and run with the following gradient: 50 to 100% phase B from 0.0 to 6.0 min, followed by 50% phase B from 6.1 to 7.5 min. The flow rate was 0.5 ml/min, and the sample injection volume was 25 μl. The peaks were eluted onto an API 300 triple-quadrupole MS equipped with an APCI source operating in the positive ion mode. The heated nebulizer was set at 350°C with a pressure of 80 lb/in2 and an auxiliary flow of 1 liter/min. Perkin-Elmer/Sciex software was used for data analysis and integration. Selected ion monitoring (dwell time, 100 ms) of eight characteristic ions (m/z 243, 245, 257, 261, 273, 289, 303, and 319) was used to determine relative percentages of EMV and putative metabolites formed.

Cell Viability Assay[2]
Cell Types: Human bone marrow cells collected from normal healthy volunteers.
Tested Concentrations: 0, 0.1, 1, 10, or 100 μM.
Incubation Duration: 14 days.
Experimental Results: At concentrations of 0.1 to 10 μM, no effect on cell growth, lactic acid production, mitochondrial DNA synthesis, or mitochondrial structure was seen compared to what occurred with untreated HepG2 cells.

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Cytotoxicity. [2]
Human bone marrow cells collected from normal healthy volunteers were used to assess bone marrow stem cell cytotoxicity. The mononuclear cells were isolated from whole heparinized marrow by Ficoll-Hypaque gradient centrifugation as described previously. Cells were washed twice with Hanks' balanced salt solution and counted with a hemocytometer, and viability was assessed by Trypan blue dye exclusion. The cells were then plated in a bilayer of soft agar or methylcellulose (105/plate), and treated with a 0, 0.1, 1, 10, or 100 μM concentration of either Emivirine (EMV) or zidovudine (AZT). After 14 days of incubation at 37°C in a humidified atmosphere of 5% CO2, colonies (≥50 cells) were counted with an inverted microscope.

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Mitochondrial toxicity. [2]
Toxicity towards mitochondria was evaluated with HepG2 cells by measuring cell growth, the production of lactic acid in extracellular medium, mitochondrial DNA content, and structural changes in the mitochondria as previously described. HepG2 cells (2.5 × 104 cells/ml), grown in minimal medium with nonessential amino acids and supplemented with 10% serum, 1% sodium pyruvate, and 1% penicillin–streptomycin, were plated in 12-well culture dishes and treated with various concentrations (0, 0.1, 1, and 10 μM) of Emivirine (EMV). After 4 days of incubation, cell growth was assessed by counting the number of cells. The medium was collected, and lactic acid was measured with an assay kit.

To determine the effect of Emivirine (EMV) on mitochondrial DNA synthesis, HepG2 cells (5 × 104 cells) were treated with the above-named concentrations of Emivirine (EMV) and incubated at 37°C in a humidified 5% CO2 atmosphere for 14 days. The cells were then collected and heated at 100°C for 10 min in 0.4 M NaOH–10 mM EDTA. The extracted DNA was immobilized on a Zeta-Probe membrane with a slot blot apparatus. To detect mitochondrial DNA, an [α-32P]dATP-labeled specific human oligonucleotide mitochondrial probe, spanning nucleotides 4212 to 4242, was used at 2.5 × 106 dpm/ml. After autoradiography, the mitochondrial probe was removed by washing the membrane twice in 0.1 × SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and then in 0.1% sodium dodecyl sulfate for 15 min. The total cellular DNA loaded on the membrane was standardized with a 625-bp fragment of a human β-actin cDNA plasmid probe labeled with [α-32P]dCTP (5 × 106 dpm/ml). Autoradiograms were scanned with a model CS9000U dual-wavelength flying-spot densitometer. The amount of mitochondrial DNA in each sample was expressed as a ratio of the mitochondrial oligonucleotide probe radioactive signal and the β-actin probe radioactive signal that was independent of DNA load.

The morphology of the HepG2 mitochondria was assessed by electron microscopy, as described previously. Briefly, HepG2 cells (2.5 × 104 cells/ml) were grown on 35-mm-diameter culture dishes in the presence of 0, 0.1, 1, or 10 μM Emivirine (EMV). Following a 4-day incubation period, the medium (with and without compound) was changed every other day. At day 8, the medium was removed and cells were fixed with 1% glutaraldehyde for 1 h, rinsed in sodium phosphate buffer, and fast-fixed in 1% osmium tetroxide for 1 h. The cells were then gradually dehydrated with graded concentrations of ethanol (from 50 through 100%) to propylene oxide. The cells were then slowly infiltrated and embedded in epon. Thin sections were prepared with a Reichter-Jung ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Hitachi model 7000 electron microscope.

Animal/Disease Models: Male SD (Sprague-Dawley) rats[2].
Doses: 50 mg/kg.
Route of Administration: Gavage.
Experimental Results: The oral absorption was 68%.

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Absorption, distribution, metabolism, and excretion. [2]
To guide the selection of an appropriate nonrodent animal model for Emivirine (EMV) toxicology experiments, male Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys were given oral doses of the compound. EMV was suspended in 0.5% tragacanth gum, and a single dose of 50 mg/kg of body weight was given by gavage to seven groups of five male rats (body weight, 128 to 200 g) and to a group of four fasted male cynomolgus monkeys weighing 3.1 to 3.9 kg. Blood samples were collected from the rats at 0.25, 0.5, 1, 2, 4, 6, and 8 h postdose and from the monkeys at 0.5, 1, 4, 8, 24, and 48 h postdose. For the dogs, EMV was placed in gelatin capsules and a single dose was given to four fasted males. Blood samples were collected at 5 min and 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h postdose. The blood samples were spun to separate the plasma, which was then analyzed for concentrations of EMV by the HPLC method described above. In this experiment, additional groups of male rats were given a single dose of EMV by injection into the caudal vein (0.88 mg/kg), by gavage (5 mg/kg), by intrarectal infusion (5 mg/kg) while the rats were anesthetized with urethane, and by infusion into the portal vein (0.25 mg/kg), again while the rats were anesthetized. The vehicles were 0.5% tragacanth gum in the cases of the oral and intrarectal doses and plasma (filtrate collected from untreated animals) in the cases of the intravenous and intraportal vein doses. Blood samples were collected at 5 min and 0.25, 0.5, 1, 2, 4, 6, and 8 h after the intravenous dose; at 5 min and 0.25, 0.5, and 1 h after the intraportal vein dose; and at 0.25, 0.5, 1, 2, 4, 6, and 8 h after the oral or intrarectal dose. Concentrations of EMV in plasma harvested from the blood samples were measured as described above to study absorption and first-pass metabolism in rats.

Absorption, tissue distribution, and excretion of Emivirine (EMV) in male Sprague-Dawley rats were studied with [14C]EMV prepared by Mitsubishi Chemical Corp. Emivirine (EMV) was labeled with 14C at the benzylic position attached to C-6. The specific activity was 2,029 MBq/mmol, and the radiochemical purity was 99.5%. A group of three rats, weighing 250 to 330 g, were given a single oral dose of [14C]EMV suspended in 0.5% tragacanth gum at a dose of 4,430 kBq 10 mg−1 kg−1. Blood samples were collected at 19 intervals from 5 min to 120 h postdose and analyzed for radioactivity. Seven male rats given [14C]EMV as described above were scanned for whole-body autoradiograms. One rat was used for an autoradiogram at 0.5, 1, 4, 8, 24, 96, and 240 h postdose. We studied excretion of EMV into urine and feces in five male rats given an oral dose of [14C]EMV of 364 kBq 10 mg−1 kg−1. They were placed in individual metabolism cages after the dose was administered. Urine was collected at 0 to 8 and 8 to 24 h postdose and every 24 h thereafter until 10 days postdose. Feces were collected every 24 h for 10 days postdose, lyophilized, weighed, and pulverized. 14CO2 in expired air was collected in a 20% solution of monoethanolamine for 24 h postdose. The radioactivity in urine was measured with a liquid scintillation counter. Carbo-Sorb was used to absorb the expired air collected, and the samples were counted after a liquid scintillator was added. The blood and feces samples were weighed and placed into a Combusto-cone and combusted in a Packard 360 sample oxidizer. Radioactivity was then determined by scintillation spectrometry.

We studied the biliary excretion of Emivirine (EMV) in eight male Sprague-Dawley rats weighing 229 to 244 g and having a surgically implanted bile duct cannula. [14C]EMV (814 MBq/mmol) was suspended in 0.5% tragacanth gum, and a single dose was administered by gavage at 10 mg/kg of body weight (6.4 MBq or 173 μCi/kg). Bile samples were collected at 1, 2, 4, 8, 24, and 48 h postdose. Urine samples were collected at 8, 24, and 48 h postdose, and feces samples were collected at 24 and 48 h postdose. Radioactivity in these samples and in water used to wash the individual cages was measured by scintillation spectrometry as described above. The cumulative excretion of radioactivity was expressed as a percentage of the dose administered.

For the distribution of Emivirine (EMV) into brain, the compound was suspended in 0.5% tragacanth gum and given orally to male Sprague-Dawley rats (four per time point) at a dose of 250 mg/kg of body weight. The rats were anesthetized, and blood and brain samples were collected at 5 and 10 min and at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h postdose. Plasma and brain, the latter homogenized in a fourfold volume of 1.15% KCl, were extracted and analyzed for concentrations of EMV by HPLC.

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In vivo metabolism. [2]
To study the effects of Emivirine (EMV) on hepatic drug-metabolizing enzymes, groups of five male Sprague-Dawley rats were given 0 (tragacanth vehicle), 15, 50, or 150 mg of EMV kg−1 day−1 by gavage for 14 days. The daily dose was divided and given in two equal installments separated by approximately 6 h, similar to the procedure in the rat toxicology experiments described below. Another group of four rats was given phenobarbital once a day at 80 mg kg−1 day−1 for 14 days as a positive control. Following necropsy, livers from the rats were perfused with 10 ml of saline via the portal vein, removed, and weighed. An aliquot (2.5 g) from the left lateral lobe was homogenized in 0.25 M sucrose on ice, and microsomes were isolated. Protein content, cytochrome b5 and P450 levels, and the activities of NADPH-cytochrome c reductase, aniline hydroxylase, aminopyrine N-demethylase, UDP-glucuronyltransferase, and 7-ethoxycoumarin O-deethylase were measured by standard spectrophotometric assays. Statistical differences were calculated by one-way analysis of variance and Dunnett's critical difference test or, in the case of phenobarbital, Student's t test.

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Safety pharmacology experiments. [2]
Experiments performed to detect potential pharmacologic effects of Emivirine (EMV) are listed in Table 1. EMV was suspended in 0.5% tragacanth gum and administered orally to mice and rats (3 to 10/group) at the concentrations indicated in Table 1. Male and female beagle dogs (five/group), anesthetized with pentobarbital, were given EMV intraduodenally to study a range of cardiovascular parameters. Isolated strips of ileum collected from male Hartley guinea pigs (five/group) were exposed in vitro to concentrations of EMV as large as 106 μM in an effort to detect potential pharmacologic effects on smooth muscle. Other parameters studied are listed in Table 1.

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Toxicology experiments. [2]
The acute toxicities of Emivirine (EMV) and one of its putative metabolites, 6-benzyl-5-isopropyl-uracil (BIU), were assessed in CD male and female rats. Briefly, EMV and BIU were synthesized, suspended in 0.5% tragacanth gum, and administered to groups of five male and five female rats as single doses of 0, 2,083, 2,500, and 3,000 mg/kg. Animals were observed for 14 days, and body weights were recorded. In addition, preclinical safety evaluation experiments were performed with mice, rats, and cynomolgus monkeys as listed in Table 2. In all the experiments, EMV was delivered orally. The vehicle in the 1-month experiments was 0.5% tragacanth gum, and that in the subchronic and chronic experiments was 0.5% methylcellulose. The daily doses shown in Table 2 were given in two equal portions with 6 to 12 h between doses. The animals were bled at five to seven timed intervals to provide toxicokinetic data. In each subchronic experiment, the bleeding was performed on dose day 1 or 2 and repeated at the end of the dose period. In the chronic (6-month) rat study, bleeding was at dose day 1 and weeks 13 and 26. Monkeys in the 1-year experiment were bled at dose day 1 and weeks 4, 13, 26, and 52. Plasma was separated from the blood, extracted, and analyzed by HPLC for concentrations of EMV.

Preclinical pharmacokinetics and toxicokinetics. [2]
Pharmacokinetic experiments identified rats and monkeys as appropriate for toxicology experiments (Table 5). In dogs, levels of Emivirine (EMV) in plasma declined rapidly and were undetectable by 1 h postdose. Autoradiography demonstrated that [14C]EMV was widely distributed to tissues of rats given 10 mg/kg by gavage, and at 0.5 h postdose, radioactivity was noted in all tissues, including brain and spinal cord. At 96 h postdose, radioactivity was detected only in the contents of the gastrointestinal tract. The total excretion of EMV was 99% of the administered dose, with 38% of the radioactivity being excreted into urine and 61% being excreted into feces. In the rat biliary excretion experiment, 25% of the administered EMV was excreted into bile at 1 h postdose; 75% was excreted into bile after 8 h. At 48 h postdose, 87, 11, and 2% of the labeled compound was found in the bile, urine, and feces, respectively. In a separate experiment, concentrations of EMV in brains from rats given a dose of 250 mg/kg by gavage were the same as those in plasma over the interval of 0.5 to 12 h postdose (Fig. 2).

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In vitro metabolism. [2]
When liver microsomes from rats, cynomolgus monkeys, and humans were incubated with Emivirine (EMV), the relative affinities of the drug (at 3 μM) for the microsomes were greater for humans than rats but greater for monkeys than humans while the Km/Vmax values were greater for rats than monkeys but significantly greater for humans than rats (Table 8), suggesting a much slower metabolism of EMV in humans. This was confirmed with additional in vitro experiments where human liver microsomes formed only about a third (8 nmol) of the total EMV metabolites measured for microsomes from rats (24 nmol) and monkeys (26 nmol) after 60 min of incubation under identical conditions. These in vitro experiments also showed that three putative metabolites, detected with m/z of 319, 245, and 261 by MS, were produced (although their identities have not yet been confirmed) by the three species, but in differing proportions. In the rat and monkey, 65% of the total metabolites formed was that with the m/z of 245, tentatively identified as BIU. The other two metabolites were approximately equal in amount. In contrast, that with an m/z of 319 was the predominant metabolite in human microsomes, accounting for 54% of the total metabolites measured, followed by the putative metabolite BIU (37%). The three species formed the m/z-319 and -261 metabolites at similar rates, but at 60 min, the rat and monkey microsomes had produced five times more BIU than the human samples. Comparison with results of standard cytochrome P450-associated reactions, such as testosterone 6β-hydroxylation for cytochrome P450 3A4 or -5, and inhibition experiments with nifedipine and troleandomycin showed that, in humans, EMV was metabolized by the cytochrome P450 enzymes 3A4 and 3A5. However, at low, pharmacologic concentrations (10 μM) of EMV, 40% of the total BIU formed in human microsomes was not abolished by troleandomycin, a cytochrome P450 3A4- and P3A5-specific inhibitor. Coupled with the results of the correlation analysis of cytochrome P450 1A2 activity (7-ethoxyresorufin O-dealkylase) in the individual human samples, this result suggested that in humans, the formation of BIU was catalyzed, in part, by cytochrome P450 1A2. We then observed that furafylline, a cytochrome P450 1A2-specific inhibitor, inhibited BIU formation in human microsomes treated with 10 μM EMV by approximately 50%, without affecting the formation of the other two major metabolites.

rat LDLo oral 2500 mg/kg Antimicrobial Agents and Chemotherapy., 44(123), 2000 [PMID:10602732]

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Safety pharmacology experiments.[2]
There were no important or consistent pharmacologic effects of Emivirine (EMV) in the wide variety of safety pharmacology experiments performed. A significant increase in the duration of anesthesia produced by hexobarbital was observed in mice given an oral dose at 300 mg/kg but not at 100 mg/kg. Similarly, EMV accelerated intestinal transit in mice at 300 mg/kg per os but not at 100 mg/kg. There was no effect on respiration rate, blood pressure, heart rate, or electrocardiographs in anesthetized dogs given EMV at 300 mg/kg of body weight.

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Toxicology experiments.[2]
In single-dose experiments, the approximate lethal oral dose of Emivirine (EMV) for rats was ≥3 g/kg for males and 2.5 g/kg for females. BIU, a putative metabolite of EMV, did not produce death in rats given a single oral dose of 3 g/kg, indicating that it was no more toxic than EMV. The 1-month experiment with rats identified 50 mg/kg/day as a no-effect dose where average peak levels of EMV in plasma were 3,090 ng/ml and the average AUC∞ was 5,064 ng · h/ml, as determined in the pharmacokinetic experiment. At the next dose level (150 mg/kg/day 4), decreased body weights, increased blood urea nitrogen (BUN), vacuoles in kidney tubules, increased serum alanine aminotransferase activity, and hepatocellular hypertrophy were observed in the animals. The livers were normal by histopathology. The 1-month experiment with monkeys identified a no-effect dose of 40 mg/kg/day where average peak values for plasma EMV ranged from 5 to 67 ng/ml and the AUC ranged from 30 to 412 ng · h/ml. Inconsistent emesis, mild diarrhea, and liver and kidney effects similar to those in rats were also observed in the monkeys at the next-highest dose, 200 mg/kg/day. At this dose, average peak values for EMV in plasma ranged from 30 to 170 ng/ml and the AUC0–24 ranged from 324 to 1,912 ng · h/ml.

The no-effect doses in the 3-month and chronic toxicity experiments were the same as those in the 1-month experiments with rats and monkeys, as shown in Table 2. Again, signs of toxicity at the higher doses were essentially limited to effects on the kidney as noted above. The kidneys were histologically normal, as were related laboratory values, at 4 weeks postdose in all of the toxicologic experiments. In the 1-year monkey experiment, emesis and diarrhea occurred at the high dose (180 mg/kg/day), but were mostly limited to the first five weeks of dosing. There were also increased values for BUN and creatinine, in addition to minor increases in alanine aminotransferase activities and insignificant decreases in erythrocyte counts. In the 1-year experiment, one of six high-dose male monkeys was necropsied when it was moribund at week 5. This animal had protracted diarrhea that was unresponsive to treatment. Histopathology defined moderate to severe enteritis as the cause of the diarrhea. Toxicity was sufficient in several of the animals in the high-dose group and in one monkey in the mid-dose group to require brief (1-week) interruptions of dosing in the second to fourth dose months. However, there was no further indication of toxicity at any dose after the effects described above resolved. Sperm counts and motility were normal in rats given EMV for 3 months in the chronic rat experiment. Nerve conduction velocities were measured in the chronic monkey experiment at 6 months and at 1 year and were unaffected by treatment with EMV.

Toxicokinetic analyses were consistent with the expected induction of hepatic drug-metabolizing enzymes in both rats and monkeys because the level of exposure to the drug at 1 week was greater than those measured at later time points. However, in all experiments, exposures were proportional to dose. In the 6-month rat experiment, exposures to EMV were comparable at both weeks 13 and 26, suggesting that autoinduction of drug-metabolizing enzymes had reached a plateau. In the case of rats at the high dose (160 mg of EMV/kg/day), the AUC0–24 for males averaged 2.6 μg · h/ml at week 13 while the corresponding value for females was 16.6 μg · h/ml. A sex difference was not noted in the three-month monkey experiment or in the 1-year monkey experiment. In those experiments, the 180-mg/kg/day EMV dose on day 2 resulted in an AUC0–24 value of 1.1 μg · h/ml for male monkeys and 0.9 μg · h/ml for females. Corresponding values for week 13 averaged 0.2 μg · h/ml for both male and female monkeys, again indicating enzyme induction and first-pass metabolism of EMV.

In the rat and rabbit developmental toxicology experiments, there was no indication of adverse effects on fetal development. However, at the high dose, 160 mg/kg/day, maternal toxicity was sufficient to produce abortion and death in rabbits. Fertility was normal at all doses of EMV (10, 40, and 160 mg/kg/day) in the rat fertility experiment. In the rat pre- and postnatal experiment, doses of 10 and 40 mg/kg/day were no-effect levels. At 160 mg/kg/day, maternal feed consumption and body weights were significantly decreased and body weights of the offspring were significantly lower than control values (P < 0.01 by Dunnett's test [8]) throughout the lactation period. However, all other parameters measured in the offspring such as activity, learning, memory, and reproductive function were unaffected by treatment of EMV regardless of the dose given. There was no indication of genotoxicity in any of the genetic toxicology experiments outlined above.

[1]. Theoretical studies on the molecular basis of HIV-1RT/NNRTIs interactions. J Enzyme Inhib Med Chem. 2011 Feb;26(1):29-36.

[2]. Safety assessment, in vitro and in vivo, and pharmacokinetics of emivirine, a potent and selective nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1. Antimicrob Agents Chemother. 2000 Jan;44(1):123-30.

Emivirine is a pyrimidone that is uracil which is substituted at positions 1, 5 and 6 by ethoxymethyl, isopropyl, and benzyl groups, respectively. A non-nucleoside inhibitor of HIV-1 reverse transcriptase, emivirine was an unsuccessful experimental agent for the treatment of HIV. It has a role as a HIV-1 reverse transcriptase inhibitor and an antiviral drug. It is functionally related to a uracil.
Emivirine has been used in trials studying the treatment of HIV Infections.
Emivirine is a non-nucleoside reverse transcriptase inhibitor. Emivirine has a structure typical of a nucleoside analog but has been shown to bind directly to the reverse transcriptase and act as an NNRTI. However, it is no longer in development due to the fact that it causes an increasingly rapid breakdown of other drugs metabolized by the cytochrome P450 enzyme.

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Emivirine (EMV), formerly known as MKC-442, is 6-benzyl-1-(ethoxymethyl)-5-isopropyl-uracil, a novel nonnucleoside reverse transcriptase inhibitor that displays potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity in vivo. EMV showed little or no toxicity towards human mitochondria or human bone marrow progenitor cells. Pharmacokinetics were linear for both rats and monkeys, and oral absorption was 68% in rats. Whole-body autoradiography showed widespread distribution in tissue 30 min after rats were given an oral dose of [(14)C]EMV at 10 mg/kg of body weight. In rats given an oral dose of 250 mg/kg, there were equal levels of EMV in the plasma and the brain. In vitro experiments using liver microsomes demonstrated that the metabolism of EMV by human microsomes is approximately a third of that encountered with rat and monkey microsomes. In 1-month, 3-month, and chronic toxicology experiments (6 months with rats and 1 year with cynomolgus monkeys), toxicity was limited to readily reversible effects on the kidney consisting of vacuolation of kidney tubular epithelial cells and mild increases in blood urea nitrogen. Liver weights increased at the higher doses in rats and monkeys and were attributed to the induction of drug-metabolizing enzymes. EMV tested negative for genotoxic activity, and except for decreased feed consumption at the high dose (160 mg/kg/day), with resultant decreases in maternal and fetal body weights, EMV produced no adverse effects in a complete range of reproductive toxicology experiments performed on rats and rabbits. These results support the clinical development of EMV as a treatment for HIV-1 infection in adult and pediatric patient populations.[2]

C17H22N2O3

302.374

302.163

C, 67.53; H, 7.33; N, 9.26; O, 15.87

149950-60-7

65013

White to off-white solid powder

2.244

1

3

6

22

451

0

MLILORUFDVLTSP-UHFFFAOYSA-N

InChI=1S/C17H22N2O3/c1-4-22-11-19-14(10-13-8-6-5-7-9-13)15(12(2)3)16(20)18-17(19)21/h5-9,12H,4,10-11H2,1-3H3,(H,18,20,21)

6-benzyl-1-(ethoxymethyl)-5-propan-2-ylpyrimidine-2,4-dione

MKC 442; DRG0302; MKC442; DRG 0302; MKC-442; 149950-60-7; Coactinon; 6-Benzyl-1-(ethoxymethyl)-5-isopropyluracil; 6-benzyl-1-(ethoxymethyl)-5-isopropylpyrimidine-2,4(1H,3H)-dione; DRG-0302; I-EBU

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)

DMSO : ~100 mg/mL (~330.72 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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