The reproductive and developmental toxicity of the antifungal drug Nyotran (liposomal nystatin) in rats and rabbits.

Nyotran is a liposomally encapsulated i.v. formulation of the antifungal polyene nystatin. This drug was evaluated in a series of reproductive toxicity studies, according to the guidelines outlined by the International Conference on Harmonization (ICH). A fertility and early embryonic development study (SEG I) and a prenatal and postnatal development (SEG III) study were conducted in rats, and embryo-fetal development (SEG II) studies were conducted in rats and rabbits. Nyotran was administered iv in all studies. In SEG I and SEG III, rats were administered daily doses of 0.5, 1.5, or 3.0 mg/kg Nyotran. In both studies, parental mortality and toxicity in the 3.0 mg/kg dose group necessitated the lowering of the high dose to 2.0 mg/kg/day. Parental toxicity, in the form of decreased body weights, decreased food consumption, and piloerection were also observed at the 1.5 mg/kg/day dose level in the SEG I and SEG III studies. Despite the parentally toxic doses in the SEG I study, there was no effect of Nyotran on F0 male or female fertility or early embryonic development of F1 offspring. In the SEG III study, lactational body weights of the F1 generation were decreased at all Nyotran dose levels. There was no effect on pre-wean developmental landmarks, but post-wean development was affected by Nyotran administration at all dosage levels. Preputional separation was delayed in the 1.5 and 3.0/2.0 mg/kg/day F1 offspring, auditory startle function was decreased in F1 females at all dose levels, and motor activity was decreased in male F1 offspring at all dose levels. However, there were no treatment-related effects on the subsequent mating of the F1 generation and resulting F2 offspring. In SEG II studies, rats and rabbits were also administered 0.5, 1.5, or 3.0 mg/kg/day of Nyotran during gestation. The high dose in these SEG II studies was not lowered, as the maternal animals were able to tolerate the shorter duration of dosing. Maternal effects in rabbits were observed only in the high-dose group and were limited to decreased food consumption and decreased absolute and relative liver weight. Decreased food consumption in high-dose dams and clinical weight loss in some animals at the mid- and high-dose levels evidenced maternal toxicity in rats. Nyotran did not have any effect on Caesarian section parameters in either rats or rabbits and no effect on the incidence of fetal malformations in rabbits. A statistically significant increase in mild hydrocephaly, observed in 4 rat fetuses, was seen at the highest dose level of 3.0 mg/kg/day. The biological significance and relationship to Nyotran treatment of this finding is not clear. This finding may represent a change in the background incidence or a change in the pattern of responsiveness of this strain of rat fetus to the test chemical. Toxicokinetic data were also collected in the SEG II rabbit and rat studies for comparison to human exposures. In both species, systemic exposure to the nystatin at effective antifungal concentrations was demonstrated. The systemic exposures in rats and rabbits were, however, considerably less than have been reported in humans administered clinical doses of 2 or 4 mg/kg/day Nyotran. Thus, humans tolerate higher dosages and systemic exposures of Nyotran relative to rats and rabbits and there is no margin of safety in either dosage level or systemic exposure to drug. Given this lack of a margin of safety and the effects on postnatal development in F1 rats, caution should be exercised when using this drug in females of childbearing potential.

Acute, multiple-dose, and genetic toxicology of AR177, an anti-HIV oligonucleotide.

AR177 (Zintevir) is a 17-mer oligonucleotide that has been shown to have anti-HIV activity and to be a potent HIV-1 integrase inhibitor in vitro, and is among the first oligonucleotides to enter human clinical trials. Acute and multiple-dose intravenous toxicity studies were performed in mice, and genetic toxicity studies were performed in vitro and in vivo in order to determine the toxicity profile of AR177. The acute toxicity study in mice showed that AR177 had an LD50 of > or = 1.5 g/kg body weight. The multipledose toxicity study in mice showed that AR177 caused male-specific mortality, and changes in serum chemistry, hematology, and histology at doses of 250 and 600 mg/kg. Clinical chemistry findings included changes in liver function, and decreased erythrocyte values at 250 and 600 mg/kg. Histopathologic findings included vacuolization of reticuloendothelial cells in phagocytic cells in lymphoid tissue, liver, lungs, heart and uterus, and extramedullary hematopoeisis in the spleen. Renal toxicity was exhibited as nephropathy and tubular necrosis in the two high-dose groups of males. A no-effect dose was not established. AR177 did not exhibit genetic toxicity in any of three mutagenic assays. In combination with previously reported toxicity studies of AR177 in monkeys, this study showed that the toxicity of AR177 is species specific.

[In vitro activity of a liposomal nystatin formulation (Nyotran) against Cryptococcus neoformans]. / Actividad in vitro de una formulación liposómica de nistatina (Nyotran) frente a Cryptococcus neoformans.

The in vitro antifungal activity of a new liposomal nystatin formulation (NISTL, Nyotran, Aronex Ltd., EE.UU.) was evaluated by a microdilution method with RPMI based on the M27A document of the National Committee for Clinical Laboratory Standards (NCCLS) against 22 isolates of Cryptococcus neoformans. This antifungal activity was compared with those of other seven antifungal agents, such as nystatin (NIST), amphotericin B deoxycholate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B colloidal dispersion, fluconazole, and itraconazole. NISTL was more active in vitrothan NIST, showing MIC values 2-3 fold smaller in 90% of the isolates. The results obtained suggest that this new formulation would be very helpful for the treatment of cryptococcosis.

In-vitro antifungal activity of liposomal nystatin in comparison with nystatin, amphotericin B cholesteryl sulphate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B desoxycholate, fluconazole and itraconazole.

The in-vitro susceptibilities of 120 clinical isolates of yeasts to liposomal nystatin were compared with those to amphotericin B lipid complex (ABLC), liposomal amphotericin B (LAB), amphotericin B cholesteryl sulphate (ABCD), amphotericin B desoxycholate, nystatin, fluconazole and itraconazole. Yeast isolates examined included strains of Candida albicans, Candida parapsilosis, Candida glabrata, Candida krusei, Candida guilliermondii, Candida tropicalis, Candida kefyr, Candida viswanathii, Candida famata, Candida rugosa, Rhodotorula rubra, Trichosporon spp., Cryptococcus laurentii and Cryptococcus neoformans. The mean MICs for all strains examined were: liposomal nystatin 0.96 mg/L; nystatin 0.54 mg/L; ABLC 0.65 mg/L; LAB 1.07 mg/L; ABCD 0.75 mg/L; amphotericin B 0.43 mg/L; fluconazole 5.53 mg/L; and itraconazole 0.33 mg/L. No significant differences were seen between the activity of liposomal nystatin and the polyene drugs or itraconazole, but liposomal nystatin was more active than fluconazole. MICs were lower than the reported blood concentrations following therapeutic doses of this drug, indicating the potential for a therapeutic use of liposomal nystatin in humans. These results indicate good activity in vitro against medically important yeasts, which compares favourably with the activities of other currently available antifungal drugs. Liposomal nystatin may have a role in the treatment of disseminated and systemic mycoses.

Species differences in the proportion of plasma lipoprotein lipid carried by high-density lipoproteins influence the distribution of free and liposomal nystatin in human, dog, and rat plasma.

The objective of this study was an interspecies comparison of free nystatin (NYS) and liposomal NYS (Nyotran) distribution in plasma. NYS and liposomal NYS at concentrations of 5, 10, and 20 microg of NYS/ml were incubated in human, dog, and rat plasma for 5, 60, and 180 min at 37 degrees C. Following these incubations, plasma samples were separated into their high-density lipoprotein (HDL), triglyceride-rich lipoprotein, low-density lipoprotein, and lipoprotein-deficient plasma (LPDP) fractions by density-gradient ultracentrifugation, and each fraction was assayed for NYS by high-pressure liquid chromatography. Total plasma and lipoprotein cholesterol, triglyceride, and protein concentrations in each human, dog, or rat plasma sample were determined by enzymatic assays. When NYS and liposomal NYS were incubated in human, dog, or rat plasma, the majority of the NYS was recovered in the LPDP fraction. For the 5- and 60-min incubation times for all plasmas measured, a significantly greater percentage of NYS was recovered in the lipoprotein fraction (primarily HDL) following the incubation of liposomal NYS than following the incubation of NYS. There was a significant correlation between the lipoprotein lipid and protein profiles in human, dog, and rat plasmas and the distribution of NYS and liposomal NYS in plasma. In particular, differences in the proportion of plasma lipoprotein cholesterol, triglyceride, and apolar lipids (cholesteryl ester and triglycerides) carried by HDL influenced the distribution of NYS and liposomal NYS within plasmas of different species. These findings suggest that the distribution of NYS among plasma lipoproteins of different species is defined by the proportion of lipid carried by HDL, and this is possibly an important consideration when evaluating the pharmacokinetics, toxicities, and activities of these compounds following administration to different animal species.

Differences in the lipoprotein distribution of free and liposome-associated all-trans-retinoic acid in human, dog, and rat plasma are due to variations in lipoprotein lipid and protein content.

The objective of the proposed study was to determine the distribution in plasma lipoprotein of free all-trans retinoic acid (ATRA) and liposomal ATRA (Atragen; composed of dimyristoyl phosphatidylcholine and soybean oil) following incubation in human, rat, and dog plasma. When ATRA and Atragen at concentrations of 1, 5, 10, and 25 micrograms/ml were incubated in human and rat plasma for 5, 60, and 180 min, the majority of the tretinoin was recovered in the lipoprotein-deficient plasma fraction. However, when ATRA and Afragen were incubated in dog plasma, the majority of the tretinoin (> 40%) was recovered in the high-density lipoprotein (HDL) fraction. No differences in the plasma distribution between ATRA and Atragen were found. These data suggest that a significant percentage of tretinoin associates with plasma lipoproteins (primarily the HDL fraction) upon incubation in human, dog, and rat plasma. Differences between the lipoprotein lipid and protein profiles in human plasma and in dog and rat plasma influenced the plasma distribution of ATRA and Atragen. Differences in lipoprotein distribution between ATRA and Atragen were not observed, suggesting that the drug's distribution in plasma in not influenced by its incorporation into these liposomes.

Activity of liposomal nystatin against disseminated Aspergillus fumigatus infection in neutropenic mice.

The purpose of this study was to examine the activity of liposomal nystatin against a disseminated Aspergillus fumigatus infection in neutropenic mice. Mice were made neutropenic with 5-fluorouracil and were administered the antifungal drug intravenously for 5 consecutive days beginning 24 h following infection. Liposomal nystatin, at doses as low as 2 mg/kg of body weight/day, protected neutropenic mice against Aspergillus-induced death in a statistically significant manner at the 50-day time point compared to either the no-treatment, the saline, or the empty-liposome group. This protection was approximately the same as that for free nystatin, a positive control. Histopathological results showed that liposomal nystatin cleared the lungs, spleen, pancreas, kidney, and liver of Aspergillus and that there was no organ damage at the day 5 time point, which was after only three doses of liposomal nystatin. Based on these results in mice, it is probable that liposomal nystatin will be effective against Aspergillus infection in humans.

Pharmacokinetics and distribution of a 33P-labeled anti-human immunodeficiency virus oligonucleotide (AR177) after single- and multiple-dose intravenous administration to rats.

AR177 is a 17-mer oligonucleotide that has anti-human immunodeficiency virus activity in vitro. The disposition of internally labeled 33P-AR177 was studied after the tail vein injection of single and multiple doses (0.7 mg/kg) to rats. After a single dose, the terminal half-life of AR177 in the blood and plasma was 367 and 271 hr, respectively, significantly longer than values reported for other oligonucleotides. Analysis of the AR177 tissue distribution showed that the majority of the dose was distributed to the liver (40%), bone marrow (17%) and renal cortex (15%) at 8 hr after single dosing. Analysis of the AR177 concentrations in tissues showed that the highest concentrations were achieved in the renal cortex (15.0 microg-eq/g), liver (7.4 microg-eq/g), bone marrow (3.9 microg-eq/g), mesenteric lymph node (3.0 microg-eq/g) and spleen (2.4 microg-eq/g) at 8 hr after single dosing. The half-life in these tissues was 9.6, 7.7, 36.8, 10.0 and 30.8 days, respectively. Forty-eight hours after the last of seven i.v. doses given every other day, the concentrations in tissues were as follows: renal cortex, 39.9 microg-eq/g; liver, 33.9 microg-eq/g; bone marrow, 12.7 microg-eq/g; spleen, 9.3 microg-eq/g; mesenteric lymph node, 5.1 microg-eq/g. Twenty-one days after administration of the last dose, tissue concentrations were still high, as follows: renal cortex, 18.6 microg-eq/g; liver, 6.2 microg-eq/g; bone marrow, 12.5 microg-eq/g; mesenteric lymph node, 3.9 microg-eq/g; spleen, 8.1 microg-eq/g. There was low urinary and fecal excretion (urinary excretion of 12.8% and fecal excretion of 6.0% of the total dose over 21 days) after a single dose. Gel filtration and anion-exchange high-performance liquid chromatography and electrophoretic analysis of the radioactivity in tissues indicated that >90% of the radioactivity represented intact AR177 for at least 7 days after drug dosing. These results demonstrate that AR177 has an extended plasma, blood and tissue half-life, is widely distributed and achieves high concentrations in lymphoid and nonlymphoid tissues in rats.

Single-dose hemodynamic toxicity and pharmacokinetics of a partial phosphorothioate anti-HIV oligonucleotide (AR177) after intravenous infusion to cynomolgus monkeys.

5' GTGGTGGGTGGGTGGGT-3' (AR177) is a 17-mer oligonucleotide with anti-human immunodeficiency virus (HIV) activity that is composed of a phosphodiester backbone and single phosphorothioate linkages at the 3' and 5' ends. A hemodynamic toxicity study was conducted in which cynomolgus monkeys were infused i.v. over a 10-minute period with single doses of 5, 20 or 50 mg AR177/kg or saline. Blood pressure, ECG, clinical chemistry, hematology, complement factors, coagulation parameters and the AR177 plasma concentration were determined. AR177 did not cause any mortality in this study, nor did it cause changes in blood pressure, ECG, clinical chemistry or hematology parameters at any dose. There was a minimal, dose-dependent increase in the levels of complement split product Bb and total hemolytic complement. There was a significant dose-dependent and reversible inhibition of coagulation with the 20- and 50-mg/kg doses that lasted up to several hours after infusion. The time course of the inhibition of coagulation closely matched the plasma levels of AR177. There was a no-effect plasma AR177 concentration vs. activated partial thromboplastin time of approximately 60 to 100 micrograms AR177/ml, above which there was prolongation of activated partial thromboplastin time. These data demonstrate that AR177 does not cause significant hemodynamic toxicity at the doses studied and that this drug could be administered as a rapid infusion without any acute, life-threatening effects at doses that produce plasma concentrations that have shown anti-HIV activity in vitro.

Repeat-dose toxicity and pharmacokinetics of a partial phosphorothioate anti-HIV oligonucleotide (AR177) after bolus intravenous administration to cynomolgus monkeys.

5'GTGGTGGGTGGGTGGGT-3' (AR177) is a partial phosphorothioate, 17-mer oligonucleotide that has been shown to have anti-human immunodeficiency virus (HIV) activity in vitro and to be a potent inhibitor of HIV-1 integrase. A repeat-dose toxicity and pharmacokinetic study was conducted in which cynomolgus monkeys were given bolus i.v. injections of 2.5, 10 or 40 mg AR177/kg/day every other day for a total of 12 doses. Control monkeys received saline. ECG, clinical chemistry, hematology, coagulation parameters, histopathology and the AR177 plasma concentration were evaluated. AR177 did not cause any mortality in this study, nor did it cause changes in ECG, clinical chemistry, hematology values or histology. However, there was a dose-dependent inhibition of coagulation measured by a prolongation of activated partial thromboplastin time; this inhibition was reversible with drug washout. Analysis of plasma samples by HPLC demonstrated that there was no difference between the AR177 plasma concentrations that were achieved after the 1st and 12th (last) doses of 2.5, 10 or 40 mg/kg. There was a direct relationship between the AR177 plasma concentration and activated partial thromboplastin time. These results indicate that repeated bolus i.v. administration of AR177 to cynomolgus monkeys at doses as high as 40 mg/kg was well tolerated and was not associated with the serious cardiovascular responses previously observed with other oligonucleotides administered i.v.

Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide.

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.

Pharmacokinetics of a 14C-labeled phosphorothioate oligonucleotide, ISIS 2105, after intradermal administration to rats.

After intradermal administration of 3.7 mg/kg of 14C-labeled 5'-TTGCTTCCATCTTCCTCGTC-3' (14C-labeled ISIS 2105) to rats, a phosphorothioate oligodeoxynucleotide, absorption was rapid. Approximately 65% of the administered dose was absorbed within 1 hr after the dose and peak blood levels were achieved within 30 min. After the initial rapid phase of absorption, a slower absorption phase ensued that resulted in more than 95% of the dose being cleared from the injection site. Slow metabolism of 14C-labeled ISIS 2105 occurred at the injection site. The rate and characteristics of metabolism in the skin were similar to those observed in other tissues. Once absorbed, the pharmacokinetics, distribution and metabolism of 14C-labeled ISIS 2105 after intradermal administration were comparable to those after an i.v. dose. The distribution and terminal half-lives were 0.5 and 53 hr, respectively. Levels of 14C-labeled ISIS 2105 in the blood were found in the plasma and the drug distributed broadly to all peripheral tissues; the liver, renal cortex and bone marrow accumulated the highest levels of drug. The 14C-labeled ISIS 2105 was eliminated principally by metabolism. Approximately 50% of the dose was found in expired air and 15% and 5% were found in urine and feces, respectively. No intact oligonucleotide was found in urine or feces at any time.

Disposition of the 14C-labeled phosphorothioate oligonucleotide ISIS 2105 after intravenous administration to rats.

5'-TTGCTTCCATCTTCCTCGTC-3' (ISIS 2105) is a phosphorothioate oligodeoxynucleotide currently being evaluated as an intralesional antiviral drug for the treatment of genital warts that are caused by the human papillomavirus. ISIS 2105, labeled with 14C (at the carbon-2 position of thymine) was administered as a single i.v. injection (3.6 mg/kg) to female Sprague-Dawley rats to assess the disposition of the drug. After i.v. administration of [14C]2105, blood radioactivity disappeared in a multiexponential manner with the half-lives of the phases equal to 0.4, 1.9, 7.1 and 5.1 hr. The initial volume of distribution was 22 ml and the postdistribution volume of distribution was 1076 ml, which indicated an extensive distribution of radioactivity. The apparent blood clearance was 14.7 ml/hr. The radioactivity in the expired air accounted for 51% of the administered dose over the 10-day period. Urinary and fecal radioactivity accounted for 15% and 5% of the administered dose, respectively. The major sites of radioactivity uptake were the liver (up to 22.6% of the dose), kidneys (renal cortex, up to 14% of the dose), bone marrow (up to 14% of the dose), skin (up to 13% of the dose) and skeletal muscle (up to 9% of the dose). Other tissues contained approximately 1% or less of the dose. The overall recovery of radioactivity 10 days postdosing was 95.1 +/- 7.5% (mean +/- S.D.) of the administered single dose. The radioactivity in the blood was almost completely in the plasma during the course of the study. In the plasma, the radioactivity was extensively bound to proteins, as assessed by size-exclusion high-performance liquid chromatography (HPLC), in samples up to 8 hr postdosing. Retention data on size-exclusion HPLC and in vitro incubations using purified proteins suggested that the plasma proteins that bound [14C]2105 were albumin and alpha 2-macroglobulin. The complex formed between the plasma proteins and [14C]2105-derived radioactivity was dissociated on anion-exchange HPLC to indicate that the great majority of plasma radioactivity coeluted with intact [14C]2105 in samples that contained sufficient radioactivity for analysis. There was a time-dependent decrease in the proportion of hepatic and renal radioactivity that coeluted with the intact [14C]2105 during the course of the study. The urine did not contain radioactivity that eluted with intact [14C]2105 on anion-exchange HPLC.

The disposition of a human relaxin (hRlx-2) in pregnant and nonpregnant rats.

The pharmacokinetics and tissue distribution of a human relaxin were investigated after intravenous (iv) bolus administration to pregnant or nonpregnant rats. Human gene-2 relaxin (hRlx-2) serum concentrations after iv bolus administration were described as the sum of three exponentials. The pharmacokinetics were comparable in pregnant and nonpregnant rats. The serum clearance (CL) was 7.4-10.2 ml/min/kg at doses of 46-93 micrograms/kg and was linear in this range. The half-lives were 1.1-2.0, 15.1-16.4, and 53.7-67.9 min, respectively. The volume of the central compartment (Vc) was 48-79 ml/kg and the volume of distribution at steady state (Vss) was 271-336 ml/kg. Increasing the dose to 463 micrograms/kg increased the dose-corrected area under the serum concentration-time curve and significantly decreased CL and Vss. The distribution of radioactivity in the tissues of pregnant rats was followed after iv bolus dosing with hRlx-2 internally labeled with 35S-cysteine. Comparison of the extent of organ uptake of radiolabel after 35S-hRlx-2 or 35S-cysteine administration suggested that the kidneys were the principal site of uptake; the liver was of secondary importance. In perfusion experiments utilizing livers isolated from pregnant or nonpregnant rats, 36-52% of the dose of hRlx-2 was cleared from the perfusate in 2 hr. These studies showed that the pharmacokinetics of hRlx-2 in rats appeared to be unaffected by pregnancy and suggested that the kidneys and liver both play a role in the elimination of hRlx-2.

Transplacental passage of a human relaxin administered to rhesus monkeys.

A synthetic version of the human relaxin encoded by the human gene 2 (hR1x-2) was administered to pregnant rhesus monkeys (Macaca mulatta) on gestational days 141-158. Monkeys (three per group) received doses of 100 micrograms/kg or 2000 micrograms/kg as a continuous i.v. infusion over 2 h into a radial vein. One monkey in the low-dose group received, along with the unlabelled hR1x-2, 25.5 microCi/kg of the test material internally labelled with [35S]cysteine. Immunoreactive hR1x-2, as measured by enzyme-linked immunosorbent assay, appeared in all fetuses within 30 min (the first sampling time) of beginning the infusions. Peak fetal plasma levels of hR1x-2 were only 0.8-1.5% of the maternal values. Only 8-15% of the fetal serum radioactivity was hR1x-2. Radioactivity from maternal urine pooled over the 4-h experiment did not elute at the volume corresponding to hR1x-2, but near the column volume.

Role of the red blood cell in drug metabolism.

Contrary to the belief that the RBC is not metabolically active towards pharmacologically active endogenous and exogenous substances, it is evident that the RBC contains moderate cytochrome P-450-like activity, in addition to the ability to catalyse various other transformations of a range of drugs. The list of drugs for which there is evidence of metabolism by RBC (Table 1) contains examples from several drug classes. However some major classes of drugs which are principally cleared in vivo by metabolism are missing (for example, benzodiazepines). Moreover, there is as yet no evidence for the RBC having the capacity for the more important drug conjugation reactions (glucuronidation, sulphation) although there is evidence of other conjugation reactions (methylation, acetylation, glutathione conjugation). It is conceivable that the RBC could be used as a convenient tissue to add to other metabolism screening procedures used in drug development. Already use has been made of the RBC in identifying fast and slow acetylators. Others have used RBC to identify a possible sex-based difference in drug metabolism. Hopefully, this review has stimulated interest in the ability of the RBC to metabolize drugs and this interest will result in further discoveries.

Metabolism and toxicity of dinitrobenzene isomers in erythrocytes from Fischer-344 rats, rhesus monkeys and humans.

The metabolism of dinitrobenzene (DNB) isomers in Fischer-344 rat, rhesus monkey and human erythrocytes was investigated. Erythrocytes from all species metabolized o-DNB and p-DNB to S-(nitrophenyl)glutathione conjugates although there were species differences in the rate and extent of conjugate formation. No metabolites of m-DNB were detected in the erythrocytes of any species. The rank order of the ability of the DNB isomers to produce methemoglobin in vitro varied from species to species, but p-DNB was always the most effective isomer. The data suggest that although the erythrocyte can conjugate DNB isomers with glutathione, this pathway offers no substantial protection from methemoglobinemia induced by dinitrobenzenes.

Extrahepatic metabolism and distribution of aspirin in vascular beds of sheep.

The dispositions of aspirin, its metabolite, salicylic acid, and its subsequent metabolite, salicyluric acid, were studied in eight anesthetized sheep infused with aspirin (61 and 485 microgram X min-1 X kg-1) for 75 min. Plasma samples were withdrawn from the portal vein, hepatic vein, pulmonary artery, left ventricle, left femoral vein, and left femoral artery. Significant extraction of aspirin occurred across the liver and hind leg, with mean availabilities of 0.75, 0.98, and 0.82 observed across the liver, lung, and hind leg, respectively. The extraction of aspirin was not affected by coadministration of sodium salicylate (30-1200 mg equiv salicylic acid). This extraction reflected hydrolysis of aspirin to salicylic acid; the metabolism of aspirin in the hind leg, lung, and liver being confirmed with tissue homogenate studies. The metabolism of aspirin in the extrahepatic tissues is significant in relation to the proposed selective presystemic acetylation of platelet cyclooxygenase by aspirin and the use of low-dose aspirin for thrombotic indications.

Distribution and metabolism of nitroglycerin and its metabolites in vascular beds of sheep.

The disposition of nitroglycerin (NTG) and its metabolites has been examined in anesthetised sheep after infusion of 0.4, 5.7 and 22.1 micrograms of NTG/min/kg through the right femoral vein. Several blood samples were collected from the left ventricle, pulmonary artery, left femoral artery, left femoral vein, portal vein and hepatic vein. Significant extraction of NTG across all vascular beds was demonstrated. The extraction was shown to be dose-dependent reflecting hemodynamic and metabolic events. Evidence for metabolism of NTG was provided by the formation of the dinitro and mononitrate metabolites. A preliminary study also showed that administration of the dinitroglycerin significantly impaired NTG metabolism across the hind leg without affecting markedly the hemodynamic response associated with the infusion of NTG.