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%.\n

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\n\nAbsorption, distribution, metabolism, and excretion. [2]
\nTo 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.\n

\nAbsorption, 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.\n

\nWe 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.\n

\nFor 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.\n

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\n\nIn vivo metabolism. [2]
\nTo 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.\n

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\n\n\nSafety pharmacology experiments. [2]
\nExperiments 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.\n

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\n\nToxicology experiments. [2]
\nThe 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 studies showed that rats and monkeys were suitable for toxicological studies (Table 5). In dogs, plasma concentrations of imivirine (EMV) decreased rapidly and were undetectable 1 hour after administration. Autoradiography showed that after gavage administration of 10 mg/kg of [14C]EMV, it was widely distributed in rat tissues, and radioactivity was detected in all tissues (including the brain and spinal cord) 0.5 hours after administration. At 96 hours after administration, radioactivity was detected only in the gastrointestinal contents. The total excretion of EMV was 99% of the administered dose, of which 38% of the radioactivity was excreted in the urine and 61% in the feces. In the rat bile excretion experiment, 25% of EMV was excreted in the bile 1 hour after administration and 75% in the bile 8 hours after administration. At 48 hours after administration, 87%, 11%, and 2% of the labeled compound were detected in the bile, urine, and feces, respectively. In another experiment, after rats were given a dose of 250 mg/kg of EMV by gavage, the concentration of EMV in their brain tissue was the same as that in plasma within 0.5 to 12 hours after administration (Figure 2). In vitro metabolism. [2] When liver microsomes of rats, cynomolgus monkeys and humans were incubated with imivirin (EMV), the relative affinity of the drug (at a concentration of 3 μM) to microsomes was higher in humans than in rats, and higher in monkeys than in humans; the Km/Vmax value was higher in rats than in monkeys, but significantly higher in humans than in rats (Table 8), indicating that EMV is metabolized much more slowly in humans. Further in vitro experiments confirmed this: after 60 minutes of incubation under the same conditions, the total EMV metabolites produced by human liver microsomes were only about one-third (8 nmol) of the total metabolites measured in rat (24 nmol) and monkey (26 nmol) microsomes. These in vitro experiments also showed that all three species produced three presumed metabolites (mass-to-charge ratios of 319, 245, and 261, as detected by mass spectrometry), but in different proportions. In rats and monkeys, the metabolite with a mass-to-charge ratio of 245 accounted for 65% of the total metabolites and was preliminarily identified as BIU. The other two metabolites were present in roughly equal amounts. In contrast, the metabolite with a mass-to-charge ratio of 319 was the major metabolite in human microsomes, accounting for 54% of the total metabolites measured, followed by the presumed metabolite BIU (37%). The rates of production of metabolites with mass-to-charge ratios of 319 and 261 were similar in all three species, but at 60 minutes, the amount of BIU produced by rat and monkey microsomes was five times that of the human sample. Comparison with results from standard cytochrome P450-related reactions (e.g., testosterone 6β-hydroxylation, catalyzed by cytochrome P450 3A4 or -5) and inhibition assays using nifedipine and traromycin indicated that EMV is metabolized by cytochrome P450 enzymes 3A4 and 3A5 in humans. However, at low pharmacological concentrations (10 μM) of EMV, 40% of the total BIU formed in human microsomes could not be eliminated by traromycin, a cytochrome P450 3A4 and P3A5-specific inhibitor. Combined with correlation analysis of cytochrome P450 1A2 activity (7-ethoxyhalothrin O-dealkylase) in individual samples, this result suggests that BIU formation in humans is partially catalyzed by cytochrome P450 1A2. We then observed that furazolidone (a cytochrome P450 1A2-specific inhibitor) inhibited the formation of BIU in human microsomes treated with 10 μM EMV by approximately 50%, without affecting the formation of the other two major metabolites.

rattLDLotoralt2500 mg/kgt Antibacterial Drugs and Chemotherapy, 44(123), 2000 [PMID:10602732]

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Safety pharmacology studies. [2]
In the various safety pharmacology studies conducted, no significant or consistent pharmacological effects were found in imivirine (EMV). In mice, oral administration of 300 mg/kg of EMV significantly prolonged the duration of hexobarbital anesthesia, but this was not observed at a dose of 100 mg/kg. Similarly, oral administration of 300 mg/kg of EMV in mice accelerated intestinal transit, but this was not observed at a dose of 100 mg/kg. In anesthetized dogs, administration of 300 mg/kg of EMV did not affect respiratory rate, blood pressure, heart rate, or electrocardiogram.

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Toxicology studies. [2] In single-dose experiments, the approximate lethal dose of oral EMV in rats was ≥3 g/kg for males and ≥2.5 g/kg for females. BIU is the presumed metabolite of EMV, and a single oral dose of 3 g/kg of BIU did not cause death in rats, indicating that its toxicity is no higher than that of EMV. In a one-month rat experiment, 50 mg/kg/day was determined as the no-effect dose, at which the mean peak plasma concentration of EMV was 3,090 ng/ml and the mean AUC∞ was 5,064 ng·h/ml (determined by pharmacokinetic experiments). At the next dose level (150 mg/kg/day), animals showed weight loss, increased blood urea nitrogen (BUN), vacuolation in renal tubules, increased serum alanine aminotransferase activity, and hepatocyte hypertrophy. Histopathological examination showed that the liver was normal. A one-month study in monkeys determined a no-effect dose of 40 mg/kg/day, at which the mean peak plasma EMV ranged from 5 to 67 ng/ml, and the AUC ranged from 30 to 412 ng·h/ml. At the next higher dose level (200 mg/kg/day), monkeys also exhibited similar vomiting, mild diarrhea, and hepatic and renal impairment as in rats. At this dose, the mean peak plasma EMV ranged from 30 to 170 ng/ml, and the AUC0-24 ranged from 324 to 1,912 ng·h/ml. As shown in Table 2, the no-effect doses in the 3-month and chronic toxicity studies were the same as those in the 1-month studies in rats and monkeys. Similarly, as mentioned above, the toxicity indications at higher doses were primarily limited to effects on the kidneys. In all toxicology studies, renal histology and relevant laboratory parameters were normal 4 weeks after administration. In a one-year monkey study, vomiting and diarrhea occurred in the high-dose (180 mg/kg/day) group, but were mainly limited to the first five weeks of administration. Elevated blood urea nitrogen (BUN) and creatinine levels were also observed, in addition to a slight increase in alanine aminotransferase activity and a slight decrease in red blood cell count. In the one-year study, one of the six male monkeys in the high-dose group underwent a necropsy at week 5 when the animal was near death. This animal suffered from persistent diarrhea and was unresponsive to treatment. Histopathological examination determined that the diarrhea was caused by moderate to severe enteritis. Several animals in the high-dose group and one monkey in the medium-dose group experienced toxicities sufficient to lead to brief (one-week) discontinuation of treatment during the second to fourth months of administration. However, no further toxicities were observed in any dose group after the aforementioned toxicities subsided. In chronic rat studies, sperm count and motility were normal in rats treated with EMV for three months. In chronic monkey studies, nerve conduction velocity was measured at 6 months and 1 year, showing no effect of EMV treatment.
The toxicokinetic analysis results were consistent with the expected induction of hepatic drug-metabolizing enzymes in rats and monkeys, as drug exposure levels at week 1 were higher than at subsequent time points. However, drug exposure was dose-dependent in all experiments. In the 6-month rat study, EMV exposures at weeks 13 and 26 were comparable, indicating that the autoinduction of drug-metabolizing enzymes had reached a plateau. In the high-dose group (160 mg EMV/kg/day), the mean AUC0-24 at week 13 was 2.6 μg·h/ml in male rats and 16.6 μg·h/ml in female rats. No sex differences were observed in the 3-month and 1-year monkey studies. In these studies, after administration of an EMV dose of 180 mg/kg/day on day 2, the AUC0-24 was 1.1 μg·h/ml in male monkeys and 0.9 μg·h/ml in female monkeys. The corresponding value at week 13 was 0.2 μg·h/ml in both male and female monkeys, again indicating that EMV is enzyme-induced and first-pass metabolized.
In developmental toxicology experiments in rats and rabbits, no adverse effects on fetal development were found. However, at high doses (160 mg/kg/day), maternal toxicity was sufficient to cause abortion and death in rabbits. In rat fertility experiments, all doses of EMV (10, 40 and 160 mg/kg/day) did not cause abnormal fertility. In rat prenatal and postnatal experiments, no effect was observed at doses of 10 and 40 mg/kg/day. When the dose reached 160 mg/kg/day, the feed intake and body weight of the mother rats were significantly reduced, and the body weight of the offspring was significantly lower than that of the control group throughout the lactation period (Dunnett test, P < 0.01 [8]). However, regardless of the dosage, all other measures of the offspring, such as activity level, learning ability, memory, and reproductive function, were not affected by EMV treatment. No signs of genotoxicity were found in any of the aforementioned genotoxicology studies.

[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.

Emiverine is a pyrimidinone compound with the structure uracil, substituted at positions 1, 5, and 6 with ethoxymethyl, isopropyl, and benzyl groups, respectively. Emiverine is a non-nucleoside HIV-1 reverse transcriptase inhibitor (NNRTI) that was used as an experimental drug for HIV treatment but without success. It is both an HIV-1 reverse transcriptase inhibitor and an antiviral drug. Its structure is similar to uracil. Emiverine was used in clinical trials for the treatment of HIV infection. Emiverine is a non-nucleoside reverse transcriptase inhibitor. While its structure is similar to nucleoside analogs, it has been shown to bind directly to reverse transcriptase and act as a non-nucleoside reverse transcriptase inhibitor (NNRTI). However, because it leads to increasingly faster degradation of other drugs metabolized by cytochrome P450 enzymes, development of this drug has been discontinued. Emivirine (EMV), formerly known as MKC-442, is 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil, a novel non-nucleoside reverse transcriptase inhibitor that exhibits potent and selective anti-HIV-1 activity in vivo. EMV has virtually no toxicity to human mitochondria or human bone marrow progenitor cells. Pharmacokinetics were linear in both rats and monkeys, with an oral absorption rate of 68% in rats. Whole-body autoradiography showed that 30 minutes after oral administration of 10 mg/kg body weight of [(14)C]EMV, the drug was widely distributed in tissues in rats. EMV concentrations in plasma and brain tissue were equal after oral administration of 250 mg/kg dose in rats. In vitro liver microsomal assays showed that the metabolic rate of EMV by human liver microsomes was approximately one-third that of rat and monkey liver microsomes. In 1-month, 3-month, and chronic toxicology studies (6 months in rats and 1 year in cynomolgus monkeys), toxicity was limited to reversible effects on the kidneys, manifested as vacuolization of renal tubular epithelial cells and a slight increase in blood urea nitrogen. Increased liver weight in rats and monkeys in the high-dose group was attributed to the induction of drug-metabolizing enzymes. Genetic toxicity tests of EMV were negative. Apart from decreased maternal and fetal weight in the high-dose (160 mg/kg/day) group, EMV did not produce other adverse effects in a series of reproductive toxicology studies in rats and rabbits. These results support the development of EMV as a clinical therapy for the treatment of HIV-1 infection in adults and children. [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.)