Pharmacokinetics of zidovudine in HIV-infected patients with end-stage renal disease.

We determined the pharmacokinetics of zidovudine (AZT) and its metabolite (GAZT) in HIV-infected patients with end-stage renal disease (ESRD), between dialysis sessions, and compared these to HIV-infected patients with normal renal function. Clearance of AZT in ESRD patients was not significantly different from controls. The mean serum AZT levels in ESRD patients were six times greater than the levels in normal controls at 4 h. Serum levels of GAZT were higher in ESRD patients at 90 min, and by 4 h were more than an order of magnitude greater than normal controls. If the AZT serum level is a good index of toxicity, we conclude that the currently recommended dose of 200 mg AZT three times a day is probably safe for use in HIV-infected patients with ESRD. The enterohepatic metabolism of AZT, the effect of such a dosing schedule and the effects of circulating levels of GAZT on outcomes in HIV-infected patients with ESRD must be further investigated.

The excretion of 7-methyladenine in the urine of rats exposed to carcinogenic methylating agents.

Earlier studies showed that urine of rats which had been injected with the methylating agent N-[3H-methyl]-N-nitrosourea contained a previously undetected metabolic product, 7-[3H-methyl]adenine. This methylpurine, undoubtedly derived from alkylation of nucleic acids followed by depurination, was not labeled when 14C-methyl-labeled methionine was administered concurrently. To establish whether urinary 7-methyladenine (7-MA) might serve as a marker of exposure to exogenous and carcinogenic methylating agents, the excretion of 7-MA following injection of methylating agents was measured. A GC-MS method, using pentafluorobenzyl derivatives and an internal standard of tri-deutero-7-MA, was developed to assay levels of 7-MA. Increasing the i.p. dose of N-methylnitrosourea (MNU) from 2 to 80 mg/kg/rat resulted in a linear increase in urinary 7-MA, which at the highest dose was 1.6 micrograms during the first day and another 0.4 microgram during day 2. Doses of 5 mg/kg MNU led to elevated urinary levels of 7-MA (144 ng) compared to controls (26 ng). Other methylating agents, such as dimethylnitrosamine, N-methyl-N'-nitro-N-nitrosoguanidine and dimethyl sulfate, also provided urinary 7-MA. To determine the fate of injected 7-MA, the administration of 2 micrograms 7-[3H-methyl]adenine led to an 80% recovery of radioactivity in the urine, almost all of it during the first 24 h. No other labeled metabolites were detected. At least for the rat, urinary 7-MA serves as an indicator of exposure to methylating agents.

Transurethral surgery using intravesical bupivacaine and intravenous sedation.

We report our experience using intravesical 0.5% bupivacaine as a topical anesthetic along with intravenous fentanyl and midazolam sedation to perform a variety of transurethral procedures in 78 patients. We achieved adequate pain control in all patients and observed no anesthetic complications. Use of this combination of intravesical topical anesthesia and intravenous sedation provided safe, adequate anesthesia to our patients undergoing various transurethral procedures in an outpatient clinic setting.

Pharmacokinetics of potential anti-AIDS agents thiofoscarnet and foscarnet in the cat.

The pharmacokinetics of thiophosphonoformate (TPFA) and phosphonoformate (foscarnet, PFA) were studied in normal adult cats, a species susceptible to feline immunodeficiency virus (FIV) infection. Parent drugs and metabolites were quantitated by high-performance liquid chromatography (HPLC). TPFA had a mean terminal plasma half-life of 42 min, a total clearance of 4.58 ml/min/kg, and a renal clearance of 1.24 ml/min/kg (N = 4). TPFA underwent in vivo metabolism to PFA and thiophosphonic acid (TPA); the latter was inactive against HIV reverse transcriptase. The 6-h cumulative urinary excretion was 42.3% of the intravenous administered dose of TPFA, consisting of 23.5% unchanged TPFA, 13.8% PFA, and 5.0% TPA. In comparison, PFA had a mean (N = 5) terminal half-life of 172 min and a total clearance of 1.88 ml/min/kg, approximating its renal clearance. There was no evidence of PFA metabolism. Oral doses of TPFA were administered either in enteric-coated capsules or in solution by gavage. The mean oral bioavailability of encapsulated TPFA and PFA was 22 and 8%, respectively. When given by gavage, TPFA had a higher mean bioavailability (33%), but with a greater variability. Based on the 6-h cumulative urinary excretion of TPFA, the mean oral bioavailability of TPFA was 44%, similar to that based on plasma data. The TPFA appears to be superior to PFA because of its greater oral bioavailability and its ability to deliver an active metabolite, PFA, to the systemic circulation after oral dosing.

Inhibition of DNA excision repair by methotrexate in Chinese hamster ovary cells following exposure to ultraviolet irradiation or ethylmethanesulfonate.

Previous results have suggested that methotrexate (MTX) could interfere with the repair of spontaneous DNA damage. To determine its effects on induced DNA damage, MTX was compared to hydroxyurea and arabinofuranosylcytosine (H/A), a drug combination known to block the DNA polymerase step of excision repair, for its ability to cause the accumulation of single-strand breaks (SSB) following exposure to either UV light or the alkylating agent ethylmethanesulfonate in Chinese hamster ovary cells. SSB were measured by alkaline elution 1, 2, and 6 h after exposure to either 1.8 mg/ml of ethylmethanesulfonate or 10 J/m2 of UV in cells pretreated with MTX or H/A. Following exposure to ethylmethanesulfonate, significant accumulation of SSB occurred in cells pretreated with either H/A or MTX. Coadministration of hypoxanthine and thymidine in MTX-treated cells prevented SSB accumulation, indicating that nucleotide depletion by MTX had inhibited repair synthesis. After UV irradiation, SSB accumulation was much less in MTX- than in H/A-treated cells. MTX was found to have no effect on the incision of UV damage. These results indicate that nucleotide depletion by MTX can affect the repair of DNA damage by exogenous agents, and that the extent of inhibition is dependent on the type of damage induced.

Urinary O6-methylguanine excretion in the rat after O6-methylguanine but not N-methylnitrosourea administration.

Urinary makers that permit monitoring the exposure of rats to methylating carcinogens could be of considerable value. Because of the association of the formation and retention of O6-methylguanine (m6Gua) with carcinogenesis, the urinary recovery of this compound would be of particular interest. Urine of rats receiving 200 muCi 3H-methyl nitrosourea (MNU) was therefore subjected to HPLC analysis to detect labeled m6Gua. Following the intraperitoneal injection of 3H-MNU (16 or 80 mg/kg), 3H-m6Gua could not be detected in the urine, although its presence was demonstrated in liver DNA under such conditions. Rats were also administered 3H-MNU followed by pretreatment with unlabeled m6Gua to block the repair enzyme, m6G-DNA-transmethylase, which might have obscured an excision mechanism. This procedure permitted the recovery of about half of the administered dose of m6Gua in the urine, but none of this compound contained tritium. We conclude that m6Gua, though formed in the DNA from MNU, is not excised and excreted as such in the urine. Thus m6Gua could not be used to serve as a suitable urinary marker for exposure to methylating carcinogens.

Pharmacokinetics of diastereoisomers of (6R,S)-folinic acid (leucovorin) in humans during constant high-dose intravenous infusion.

Eleven patients treated with a 5.5-day continuous i.v. infusion of 500 mg/m2/day of (6R,S)-folinic acid in combination with daily bolus 5-fluorouracil had a median steady-state plasma concentration of 3.25 microM (6S)-folinic acid (the bioactive diastereoisomer). The bioactive metabolite (6S)-5-methyltetrahydrofolic acid, analyzed in six patients, reached a median steady-state plasma concentration of 5.7 microM. The lowest plasma concentrations at steady-state were 1.86 microM (6S)-folinic acid and 3.12 microM (6S)-5-methyltetrahydrofolic acid. These concentrations are above the minimum concentrations shown by other investigators to produce synergism between (6R,S)-folinic acid and 5-fluorouracil in vitro. The median steady-state plasma concentration of (6R)-folinic acid was 38.2 microM, more than 10 times the concentration of (6S)-folinic acid. Along with other plasma pharmacokinetic parameters, terminal half-lives were estimated for (6S)-folinic acid (median, 45.4 min), (6R)-folinic acid (median, 388 min), and (6S)-5-methyltetrahydrofolic acid (median, 446 min). Investigation of the renal pharmacokinetics confirmed the marked difference in the renal clearance of the two diastereoisomers of folinic acid which had been observed after low doses of (6R,S)-folinic acid (J. A. Straw, D. Szapary, and W. T. Wynn, Cancer Res., 44: 3114-3119, 1984). However, the low renal clearance of (6R)-folinic acid (median, 8.2 ml/min/m2) was attributable to the extensive binding of (6R)-folinic acid to plasma proteins (median, 8.7% free), not to reabsorption in the kidney.

Chromatographic detection of 7-methyladenine in urine of rats administered N-methylnitrosourea: a potential marker for monitoring exposure to methylating carcinogens.

Relatively simple and rapid analytical procedures involving two sequential HPLC separations were developed for the isolation of methylated purines in the urine of rats administered radiolabeled methylating carcinogens. Following a dose of [3H]N-methyl-N-nitrosourea (MNU), 7-methyl-adenine (m7Gua) was detected by chromatography as a urinary methylpurine in addition to the expected 7-methylguanine (m7Gua) and 3-methyladenine (m3Ade). When methyl-labeled methionine was given to rats concurrently with MNU, urinary m7Gua was labeled, but no radioactivity was recovered in either of the two methyladenine fractions. The profile of urinary methylated purines following a dose of dimethyl sulfate to the rat was similar. Small amounts of 1-methyladenine (m1Ade) and 3-methylguanine (m3Gua) were also detected in the urine. The excretion of m7Ade derived from the methyl group of the carcinogen rather than from the normal precursor for methylation, implies that this adduct, like m3Ade, may serve as an indicator in urine for exposure to methylating carcinogens.

Enhanced cytotoxicity with methotrexate in conjunction with hypoxanthine in L1210 cells in culture.

By inhibiting dihydrofolate reductase, methotrexate (MTX) depletes cellular stores of reduced folates, resulting in the inhibition of DNA and RNA synthesis. Inhibition of RNA synthesis arrests cells in the G1 phase of the cell cycle, preventing these cells from entering S phase and rendering them insensitive to MTX. Because MTX cytotoxicity can be enhanced by concurrent administration of hypoxanthine (HX), we examined the hypothesis that this modulation can allow normal rates of RNA synthesis and cell cycle progression from G1 to S phase. For L1210 cells exposed to MTX for 12 h or 24 h, the addition of HX enhanced the cytotoxicity of MTX; however, no enhancement was observed with a 6-h exposure. Inhibition of RNA synthesis by MTX was prevented by concurrent administration of HX. The effect of HX on cell cycle progression was first examined using flow cytometry, which indicated that MTX treatment alone or with concurrent HX caused a buildup of cells with a G1 content of DNA. Because this technique may fail to distinguish between cells in late G1 phase, the G1/S border, or early S, the method of premature chromosome condensation was used to determine cell cycle position based on chromatin morphology. A shift to a higher degree of chromatin decondensation was observed when HX was coadministered with MTX during a 12-h exposure, suggesting progression from G1 towards S. This correlated with the enhancement of MTX cytotoxicity by HX after 12 h exposure. The results of these studies suggest that HX potentiates MTX cytotoxicity by maintaining RNA synthesis, allowing cells that might ordinarily be arrested in G1 to progress into the cytotoxic S phase.

Effects of high potassium or low sodium diet on vascular Na+,K+-ATPase activity and blood pressure in young spontaneously hypertensive rats.

These studies were designed to investigate whether the antihypertensive effects of high potassium or low sodium diets are related to changes in vascular Na+,K+-adenosine triphosphatase (ATPase) activity. Vascular Na+,K+-ATPase was measured as ouabain-sensitive rubidium uptake in aorta incubated in buffer or plasma from spontaneously hypertensive rats (SHR) fed either a high potassium, a low sodium, or a normal diet for 2 weeks. The high potassium diet significantly increased Na+,K+-ATPase activity, whereas the low sodium diet significantly decreased activity. There was no evidence of a ouabainlike factor in plasma. The increased pump activity on the high potassium diet appeared to be due to an increase in maximum activity (Vmax) of the enzyme, rather than to an increased affinity for potassium. Potentially, an increase in Na+,K+-ATPase activity could contribute to the antihypertensive effect of potassium by hyperpolarizing the cell membrane. The decrease in vascular Na+,K+-ATPase activity on a low sodium diet probably is unrelated to its depressor effect, but it may be a homeostatic mechanism for maintaining sodium balance in the animal.

Effects of a high K+/low Na+ diet on blood pressure in young spontaneously hypertensive rats.

Blood pressure was measured after treatment with a high K+, a low Na+ and a combined high K+/low Na+ diet in young spontaneously hypertensive rats (SHR). A high K+ diet reduced blood pressure by approximately 10 mmHg during the development of hypertension. This decrease was accompanied by a significant increase in water intake and urine volume and a significant decrease in plasma renin activity (PRA). A low Na+ diet also decreased blood pressure significantly, but, in contrast to the high K+ diet, water intake and urine volume significantly decreased and PRA increased. When both diets were given together, the antihypertensive effects of both were eliminated. Thus while an increase in dietary K+ and a decrease in dietary Na+ are both effective antihypertensive regimens in SHR, the mechanism of action of each appears to be different and may be antagonistic in these animals.

Na+, K+ and water balance in young spontaneously hypertensive rats: relationship to blood pressure after high K+ treatment.

These studies were designed to investigate the effects of high dietary K+ on electrolyte and water balance in young spontaneously hypertensive rats (SHR) and to relate these effects to changes in blood pressure. The high K+ diet reduced blood pressure by approximately 10 mmHg during the development of hypertension. Blood pressure, however, plateaued at the same maximum level as control by age 13 weeks. Rats fed the high K+ diet showed a significant increase in water intake and urine volume throughout the treatment period but no change in plasma volume or extracellular fluid volume occurred. A slight natriuresis was also observed in rats on the high K+ diet, but this was not of sufficient magnitude to decrease total body Na+. These results confirm previous findings that K+ causes a diuresis and a natriuresis, but demonstrate that the diuretic action of K+ cannot explain its antihypertensive properties in young SHR.

Pharmacokinetics of leucovorin (D,L-5-formyltetrahydrofolate) after intravenous injection and constant intravenous infusion.

The pharmacokinetics of the active and inactive diastereoisomers of leucovorin and its active metabolite, 5-methyltetrahydrofolate (5-CH3-THF) were studied after iv injection of leucovorin in normal human subjects at a dose of 28 mg/m2, and in patients given 500 mg/m2 daily by constant iv infusion for a 5.5 day period. In both studies the plasma half-life (t1/2) of the active isomer, L-formyltetrahydrofolate (CHO-THF), was only 32 to 35 minutes, whereas the inactive isomer, D-CHO-THF had a plasma t 1/2 of 352 to 485 minutes. During constant infusion, the plasma levels reached plateaus of 2.33 and 37.5 microM for L-CHO-THF and D-CHO-THF, respectively. The inactive isomer was cleared from plasma only by urinary excretion of the unchanged drug. The active isomer was also excreted unchanged in the urine but in addition was extensively metabolized to the active metabolite L-5-CH3-THF. The active metabolite achieved a plasma level of 4.85 microM during constant infusion and appeared to have a longer t 1/2 after constant infusion than was observed after iv injection. Furthermore a larger apparent volume of distribution (Vd) of 5-CH3-THF was obtained in the constant infusion study. These findings suggest that constant iv infusion of large doses of leucovorin can considerably expand the intracellular pools of active folate. The consequence of the extensive accumulation of the inactive isomer, D-CHO-THF, is not known. However, the small Vd of D-CHO-THF suggests that it does not extensively accumulate in tissues.

Increased aortic DNA synthesis precedes renal hypertension in rats. An obligatory step?

The rate of DNA synthesis was determined in rats with developing and established two-kidney, one clip renal hypertension. Rate of DNA synthesis was measured as [3H]thymidine incorporation into DNA per hour. After stenosis of the renal artery, blood pressure increased over a 2-week period. Five days after clipping, there was an increase in the rate of aortic DNA synthesis before an increase in blood pressure was detected, whereas there was no DNA effect in sham-operated animals. This difference in [3H]thymidine incorporation into aortic DNA could not be accounted for by alterations in thymidine pool sizes. The increase in DNA synthesis was still present 3 weeks after renal artery stenosis, although by that time blood pressure had plateaued. The role of DNA synthesis in the development of renal hypertension was investigated by determining whether inhibition of DNA synthesis with cytosine arabinoside could prevent the increase in blood pressure. Treatment of clipped rats with cytosine arabinoside for 5 days delayed the increase in blood pressure for more than 4 days, as compared with the effect of saline treatment in clipped rats. Although the possibility remains that some effect of cytosine arabinoside other than its effect on DNA synthesis could have influenced blood pressure, there were no differences in body weight, food intake, water intake, or urine output between cytosine arabinoside-treated and saline-treated rats with renal artery clips, and cytosine arabinoside treatment had no effect on blood pressure or body weight in normal rats. These results suggest that an increase in DNA synthesis may be an obligatory step in the genesis of renal hypertension.

Failure of quality control to detect errors in the preparation of technetium-99m disofenin (DISIDA).

Uptake of Tc-99m disofenin is so strongly hepatobiliary that striking appearance of radioactivity in the reticuloendothelial system (RES) is almost certainly caused by improper preparation of the disofenin kit. Four patients who received injections of Tc-99m disofenin demonstrated a pronounced RES biodistribution in addition to the expected hepatobiliary pattern. Since routine quality control testing had not forewarned us of this gross lack of radiochemical purity, Tc-99m disofenin kits were subjected to a variety of insults to elucidate the mechanisms of quality control failure. Assessment of radiochemical purity was conducted using instant thin-layer chromatography (ITLC) and two solvent systems. We concluded that both Tc-99m pertechnetate and Tc-99m sulfur colloid had been injected into the cold disofenin kit. Visual inspections were not reliable for verifying the final radiopharmaceutical product because of dilution of the sulfur colloid by pertechnetate. ITLC testing failed because the assay method being used at that time did not accurately assess the hydrolyzed fraction (colloidal component). Recommendations are made to preclude recurrence of the errors that occurred.

Inhibition of sodium transport in frog skin and iodination of tyrosine in vitro by a peptide of renal origin.

Eluates from a Sephadex G-25 column through which dog renal homogenate was passed contained a factor that inhibited lactoperoxidase (LPO)-catalyzed iodination of tyrosine as well as the short circuit current (active absorptive sodium flux) in isolated skin of the bullfrog (Rana catesbeiana). The active fraction also had a sulfhydryl-containing moiety, the concentration of which was correlated directly with the extent of inhibition of the LPO enzyme. The fractions active in inhibition of the LPO enzyme failed to affect the Na/K-AT-Pase enzyme. Whether the same moiety of the active fraction inhibits both the LPO enzyme and SCC in frog skin is unknown and awaits additional study.

Computer modeling of the pharmacokinetics of Fluorouracil and thymine and their kinetic interaction in normal dogs.

The kinetic behavior of thymine and 5-fluorouracil has been shown to be non-linear and mediated largely by saturable metabolic processes. In vivo estimates of the Michaelis-Menten parameters Vmax and Km were obtained from constant infusion data in normal dogs using a system of balance equations that equate drug input with total output at steady-state. These estimates were then successfully used to simulate both steady-state and post-infusion plasma concentration-time curves for both compounds over a range of saturating and non-saturating conditions. It has been shown previously that estimates of Vmax and Km obtained from dynamic data can be incorrect if an inappropriate compartmental model is used in the analysis. Determining the Michaelis-Menten parameters at steady-state eliminates this difficulty. Moreover, the use of steady-state derived values to simulate post-infusion data confirms the validity of this technique. The kinetic interaction between thymine and 5-fluorouracil was investigated as a case of competitive metabolic inhibition in vivo by calculating Ki values from data obtained during simultaneous constant infusions of the two compounds. These values were then used in conjunction with a series of differential equations incorporating reciprocal metabolic effects to simulate the effect of thymine on FU plasma concentration.

Pharmacokinetics of the diastereoisomers of leucovorin after intravenous and oral administration to normal subjects.

After i.v. administration of d,l-, 1-5-formyltetrahydrofolate (d,l-CHO-THF) CHO-THF was rapidly cleared from the plasma by conversion to 5-methyltetrahydrofolate (5-CH3-THF) and urinary excretion, whereas d-CHO-THF, which was not metabolized and was slowly excreted in the urine, persisted in plasma at concentrations greatly exceeding those of l-CHO-THF and 5-CH3-THF. The plasma half-life (beta) of the unnatural (d) isomer was 451 +/- 24 (S.E.) min compared to 31.6 +/- 1.1 min for the natural (l) isomer, and 227 +/- 20 min for its active metabolite, 5-CH3-THF. The half-lives and volumes of distribution of each of the three compounds were independent of dose over a range of 25 to 100 mg, indicating that mechanisms for distribution, metabolism, and excretion are not saturable over the dose range tested. The urinary clearance of l-CHO-THF or 5-CH3-THF differed only slightly from creatinine clearance, whereas urinary clearance of d-CHO-THF was only one-fifth creatinine clearance, indicating that d-CHO-THF was extensively reabsorbed. Absorption of d,l-CHO-THF after p.o. administration was stereoselective in that absorption of the l-isomer was approximately 5 times that of the d isomer. Thus, p.o. administration resulted in a more favorable ratio of active to inactive folates in plasma. At a dose of 25 mg, absorption approached 100% for l-CHO-THF compared to 20% for d-CHO-THF. However, absorption was saturable, and lower percentages of both compounds were absorbed at doses of 50 and 100 mg.