Methotrexate accumulates to similar levels in animals transplanted with normal versus drug-resistant transgenic marrow.

Gene transfer and expression of methotrexate (MTX)-resistant variants of dihydrofolate reductase (DHFR) in normal hematopoietic cells is a potential strategy to permit administration of larger doses of MTX by alleviating drug toxicity in normal cells and tissues that are drug sensitive. We have previously demonstrated that transplantation of marrow from transgenic mice expressing drug-resistant DHFRs conferred upon normal recipient animals resistance to MTX at levels that are usually toxic for hematopoietic and gastrointestinal (GI) tissues. One explanation for the observed protection from GI toxicity by drug-resistant marrow is that MTX could be cleared more rapidly in animals maintaining a more healthy hematopoietic system. To evaluate this possibility, we carried out MTX pharmacokinetic studies in mice that received transplanted transgenic marrow expressing either of two different DHFR variants, administering increasing doses of MTX up to 4 mg/kg/day. Animals received i.p. injection precisely every 24 h. Every 4 days, three animals from each group were sacrificed, and their plasma and intestines were assayed for MTX. Animals transplanted with transgenic Arg-22 DHFR drug-resistant marrow maintained hematocrit levels that were about 4-fold higher at 3 weeks after transplant than those of untreated animals or animals that received normal marrow cells. Animals that received normal marrow did not survive beyond 25 days and did not accumulate higher levels of MTX than animals that received a transgenic marrow transplant. Untreated animals exhibited a higher rate of survival (36 days) but again did not accumulate higher levels of MTX than the transgenic marrow recipients. When the experiment was repeated using transgenic Tyr-22 DHFR marrow, the levels of MTX in the plasma or GI tissues did not differ significantly between groups. Intestinal concentrations of MTX in both experiments were about 4-5-fold higher than those in the plasma. These results indicate that protection from MTX toxicity conferred by expression of drug-resistant DHFR activity in the marrow is not the result of a higher rate of MTX clearance from the circulation in comparison with control animals but a true resistance of hematopoietic and GI tissues to MTX. The maintenance of antifolate levels in animals protected from MTX toxicity implies that this procedure should not compromise the antitumor efficacy of MTX.

The absorption of retinoic acids from the gastrointestinal tract is dependent upon chemical structure.

PURPOSE: The gastrointestinal permeability of a number of retinoic acids was determined in order to evaluate whether the gastrointestinal membrane was able to distinguish between retinoids in which the polyene chain was present in several different isomeric forms. In addition, the structure of the six-membered ring was varied in order to determine which portion of the molecule was most important for its recognition by the membrane. The role of bile salt micelle composition in the intestinal absorption of retinoids was also evaluated. METHODS: In situ perfused rat intestinal segment preparations (= 78) were used, and the retinoids were each perfused at a concentration of approximately 1 microg/ml in either simple micelles of sodium taurocholate (10 mM) or mixed micelles of sodium taurocholate/egg phosphatidylcholine (10 mM/10 mM). The flow rate of the perfusate was either 0.1 or 0.35 ml/min. RESULTS: For each retinoid, the mixed micelles were associated with a higher degree of retinoid uptake into the jejunal cells than were the simple micelles. In addition, the permeability was higher when the perfusate flow was greater, indicating that the aqueous boundary layer of the intestine contributes to the resistance to the disappearance of the retinoid from the intestinal lumen. Retinoid structure was also found to have a significant effect on the permeability in the mixed micelle systems at both low and high flow rates, but not with simple micelles. The structure of the six-membered ring was not a major determinant of the permeability. However, the permeability of the retinoids with the polyene chain in the 13-cis position was significantly greater than when the chain was all-trans or in the 9-cis position. CONCLUSIONS: The isomeric position of the polyene chain and the presence of phospholipid in the micellar vehicle have a significant influence on the membrane transport of the retinoic acids.

The intestinal uptake of phenol from micellar systems does not conform to the aqueous transfer model.

PURPOSE: To evaluate the aqueous transfer model as the mechanism for the micelle-mediated uptake of phenol in the rat in situ intestinal perfusion model. METHODS: Phenol in isotonic HEPES buffer was perfused through the jejunal segment at two flow rates and at various concentrations. Phenol was then dispersed in two, distinct mixed micelle systems composed of sodium taurocholate and phosphatidylcholine at 10 mM:2.5 mM (10:2.5 system) and at 10 mM: 10 mM (10:10 system) and its uptake studied in each case. Equilibrium dialysis was done to determine the aqueous fraction of phenol in each system. RESULTS: The P(eff) of phenol in isotonic HEPES buffer at a low flow rate (n = 6) was 1.7 +/- 0.4 x 10(-4) cm/s and at a high flow rate (n = 13) was 1.8 +/- 0.5 x 10(-4) cm/s. The P(eff) for the 10:2.5 system at the high flow rate (n = 3) was 1.5 +/- 0.4 x 10(-4) cm/s and at the low flow rate (n = 3) was 1.4 +/- 0.3 x 10(-4) cm/s. Uptake was membrane rate-limited in both the non-micellar and 10:2.5 systems. P(eff) at a high flow rate (n = 3) in the 10:10 system was 1.3 +/- 0.1 x 10(-4) cm/s. Equilibrium dialysis (n = 4) revealed free fractions of 0.60 +/- 0.05 and 0.50 +/- 0.03 for the 10:2.5 and 10:10 systems. CONCLUSIONS: The uptake of micellized phenol did not follow the aqueous transfer model of uptake.

First-pass disposition of (-)-6-aminocarbovir in rats: II. Inhibition of intestinal first-pass metabolism.

A CBV [(-)-carbovir, (-)-carbocyclic 2',3'-didehydro-2', 3'-dideoxyguanosine] prodrug, 6AC [(-)-6-aminocarbovir, (-)-carbocyclic 2',3'-didehydro-2', 3'-dideoxy-6-deoxy-6-aminoguanosine], was previously evaluated in rats, and it exhibited superiority to the parent drug in increasing systemic and central nervous system exposure to CBV. The gut wall was determined to be the dominant site of the first-pass activation of 6AC after lumenal administration. If subsequent delivery to the brain is desired, then such a first-pass effect might not be viewed favorably. Because the first-pass conversion of 6AC primarily takes place in the intestine by adenosine deaminase (ADA), quenching of the intestinal activation of 6AC by oral administration of ADA inhibitors may result in an increased 6AC bioavailability, and thus an improved brain exposure to CBV. The objectives of the study were to determine whether the ADA inhibitors 2'-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)adenine were capable of achieving a substantial and selective inhibition of gut wall activation of 6AC, and to determine whether the systemic concentrations of 6AC would be thus increased. Thirty-nine male Sprague-Dawley rats were divided into two groups. One group received 6AC by either the portal vein or intralumenally with the coadministration of intralumenal 2'-deoxycoformycin. Similarly, the other group received 6AC with coadministration of erythro-9-(2-hydroxy-3-nonyl)adenine. Substantial suppression of the first-pass conversion of 6AC was achieved with both inhibitors. This inhibition appeared to be relatively selective, allowing the choice of dose of inhibitor that would sufficiently inhibit the first-pass metabolism while leaving the activation capacity in the systemic circulation unaltered. The systemic level of 6AC increased with the escalating dose of inhibitors, thus increasing the driving force for passive uptake into the brain.

Synthesis and antiviral activity of amino acid carbamate derivatives of AZT.

Lipophilic amino acid methyl ester and methyl amide carbamates of 3'-azido-3'-deoxythymidine (AZT) were synthesized and their anti-HIV-1 activity in PBMCs was determined. The methyl amides were more potent (EC50s = 1.8-4.0 microM) than the methyl esters (EC50s = 2.0-20 microM). Carbamate hydrolysis by cell lysates and liberation of AZT was not observed for representative methyl ester or methyl amide AZT carbamates. No evidence of direct inhibition of HIV reverse transcriptase or integrase was observed.

Chemoprevention of pulmonary carcinogenesis by brief exposures to aerosolized budesonide or beclomethasone dipropionate and by the combination of aerosolized budesonide and dietary myo-inositol.

This investigation is part of an effort to develop chemoprevention for carcinogenesis of the lung. It focuses on the efficacy of low doses of synthetic glucocorticoids administered either as single agents or in combination with a second compound, myo-inositol. Glucocorticoids are potent inhibitors of carcinogenesis. The use of low doses is important to avoid potential side-effects. The synthetic glucocorticoid budesonide, administered by aerosol for 20 s three times a week, was studied to determine its effects on benzo[a]pyrene-induced pulmonary adenoma formation in female A/J mice. Two dose levels were employed, 10 and 25 microg/kg body wt. The lower dose produced a 34% reduction in lung tumor formation and the higher dose level a 60% reduction in lung tumors. In additional groups of mice, the effects of 0.3% myo-inositol added to the diet was found to reduce pulmonary tumor formation by 53%. The two agents given in combination resulted in a greater inhibition of lung tumor formation than either by itself. Budesonide at 10 microg/kg body wt plus 0.3% myo-inositol reduced the number of tumors by 60% and budesonide at 25 microg/kg body wt plus 0.3% myo-inositol reduced lung tumor formation by 79%. To determine whether a glucocorticoid other than budesonide would have inhibitory effects in this experimental model, beclomethasone dipropionate administered by aerosol for 20 s three times a week was studied as a single agent and showed almost identical inhibitory properties to budesonide. The doses of the glucocorticoids calculated on a daily basis are within the range of those used widely for control of chronic allergic respiratory diseases in the human. The capacity of low doses of inhaled glucocorticoids to prevent pulmonary neoplasia and the enhancement of this preventive effect by myo-inositol, an essentially non-toxic compound, are findings that should encourage further work to evaluate the applicability of these agents to the prevention of neoplasia of the lung in the human.

Antiviral nucleoside drug delivery via amino acid phosphoramidates.

Stable and water soluble amino acid phosphomonoester amidates of AZT were synthesized and shown to have potent anti-HIV-1 activity. Intracellular and cell extract metabolism studies revealed that these compounds are likely to be enzymatically converted to the corresponding monophosphates. In addition, we have shown that the half life and tissue distribution of a phosphoramidate of AZT is 5 and 10-fold greater, respectively, than AZT.

Quantitation of urinary metabolites of a tobacco-specific lung carcinogen after smoking cessation.

We quantified urinary levels of two metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in people who had stopped smoking: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its O-glucuronide, 4-[(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). Twenty-seven people completed the study. Thirteen used the nicotine patch starting at the quit date, whereas the others used no patch. Two 24-h urine samples were collected on 2 consecutive days before smoking cessation; blood was also obtained. Beginning at their quit date, subjects provided 24-h urine samples on days 7, 21, 42, 70, 98, and 126, and some subjects also provided samples at later times. The urine was analyzed for NNAL, NNAL-Gluc, nicotine plus nicotine-N-glucuronide, and cotinine plus cotinine-N-glucuronide. Some blood samples were also analyzed for NNAL. The decline of urinary NNAL and NNAL-Gluc after smoking cessation was much slower than expected. This was clearly demonstrated by comparison with cotinine and nicotine levels in urine. One week after smoking cessation, 34.5% of baseline NNAL plus NNAL-Gluc was detected in urine, whereas the corresponding values for cotinine and nicotine were 1.1 and 0.5%, respectively. Even 6 weeks after cessation, 7.6% of the original levels of NNAL plus NNAL-Gluc remained. In some subjects, NNAL plus NNAL-Gluc were detected 281 days after cessation. The distribution half-life for NNAL and NNAL-Gluc was 3-4 days, whereas the elimination half-life was 40-45 days. Total body clearance of NNAL was estimated to be 61.4 +/- 35.4 ml/min, and volume of distribution in the beta-phase was estimated to be 3800 +/- 2100 liters, indicating substantial distribution into the tissues. Parallel studies in rats treated chronically or acutely with NNK in the drinking water support the conclusion that NNAL has a large volume of distribution. There was no effect of the nicotine patch on levels of NNAL plus NNAL-Gluc, indicating that NNK is not formed endogenously from nicotine. The results of this study demonstrate that NNAL and NNAL-Gluc are slowly cleared from the body after smoking cessation, indicating the presence of a high-affinity compartment where NNK, NNAL, and/or NNAL-Gluc are retained or sequestered and slowly released.

First-pass disposition of (-)-6-aminocarbovir in rats. I. Prodrug activation may be limited by access to enzyme.

Several in vitro and in situ approaches were used to determine the dominant presystemic activation site for (-)-6-aminocarbovir, (-)-carbocyclic 2',3'-didehydro-2', 3'-dideoxy-6-deoxy-6-aminoguanosine (6AC) conversion in rats. The in vitro disappearance half-lives (mean +/- S.D.) in the cytosolic fractions obtained from homogenates of the intestine, liver, and intestinal contents were 0.4 +/- 0.1 (n = 3), 12.2 +/- 1.1 (n = 3), and 15.5 (n = 1) min, respectively. An in situ vascularly perfused intestine-liver (IPIL) study was then carried out (n = 6) to determine the relative contribution of each presystemic organ to the overall first-pass extraction of 6AC. The 6AC extraction ratios in the intestine and liver in the IPIL were found to be 0.08 +/- 0.02 and 0.11 +/- 0.03, respectively. The intestinal extraction ratio was in dramatic contrast to the in vitro results. It was postulated that vascularly delivered 6AC had limited access to the metabolic site in the intestine. A theoretical analysis suggested that the extent of intestinal wall extraction of 6AC would be underestimated by the IPIL and should be determined after oral dosing. To compare intestinal extraction ratio in the IPIL with that after an oral administration, in situ intestinal lumen perfusions (n = 4) and intraportal infusions (n = 3) of 6AC were conducted in two groups of rats. The lumenally administered 6AC was extracted to a much greater extent by the intestine as compared with the IPIL, which presents 6AC to the intestine by the vascular route. The extraction ratio was found to be 0.54 +/- 0.06, which was significantly larger than that obtained in the IPIL.

Use of the deconvolution principle in the estimation of absorption and pre-systemic intestinal elimination of drugs.

The deconvolution principle was used to evaluate the extent of absorption and first-pass elimination of selected drugs. In the first example, deconvolution of the portal blood profiles of etretinate (ET, a synthetic retinoid) indicated that there was significant gut-wall conversion of ET to acitretin (ETA, the primary metabolite of ET) during a 60-min intestinal perfusion of ET. In the second example, deconvolution was used to confirm that the extent of carbovir disappearing from the gastrointestinal lumen was matched by the extent of carbovir appearance in the portal blood. Thus, deconvolution has several important applications in the study of absorption and intestinal first-pass metabolism.

Chemoprevention of pulmonary carcinogenesis by aerosolized budesonide in female A/J mice.

This investigation is part of a continuing effort to develop effective chemoprevention for carcinogenesis of the lung. The present study explores the use of aerosol administrations for this purpose. The agent selected for initial study was the synthetic glucocorticoid budesonide. This selection was based on previous work in which budesonide added to the diet was found to inhibit pulmonary adenoma formation in female A/J mice. However, high dose levels were required, i.e., of the order of 300 microg/kg, of body weight [L. W. Wattenberg and R. D. Estensen, Carcinogenesis (Lond.), 18: 2015-2017, 1997]. For aerosol administration of budesonide, a nose-only technique has been developed that entails nebulization of the compound dissolved in ethanol and subsequent stripping off of the solvent (less than 3 microl ethanol/liter of air remaining at the site of inhalation). The budesonide particles produced by the apparatus had a mass median aerodynamic diameter of less than 1 microm. An experiment has been carried out in which the inhibitory effects of aerosolized budesonide, given for 1 min six times a week, were studied. Concentrations of budesonide of 26, 81, and 148 microg/liter of air (calculated doses of 23, 72, and 126 microg/kg of body weight) were used. The aerosols were started 1 week after three oral administrations of benzo(a)pyrene (2 mg/20 g of body weight) to female A/J mice. All three doses of budesonide resulted in more than 80% inhibition of pulmonary tumor formation compared to the aerosol control and 90% or greater compared to mice not exposed to aerosol. The difference in inhibition is due to the aerosol procedure itself, which produces a reduction in tumor formation. A decrease in splenic weight (evidence of a systemic effect) occurred at all doses of budesonide. To the best of our knowledge, this is the first published effort at the use of aerosol administration to prevent neoplasia of the respiratory tract. The results of the present study show that administration of a potential chemopreventive agent by aerosol at a low dose can inhibit the occurrence of pulmonary carcinogenesis in female A/J mice.

Solubilization of retinoids by bile salt/phospholipid aggregates.

PURPOSE: The capacity and specificity of bile salt (BS)/ phosphatidylcholine (PC) mixed lipid aggregated systems in solubilizing four structurally related retinoids, etretinate, motretinid, fenretinide and N-ethyl retinamide, were determined. METHODS: Excess solid drug was dispersed into sodium taurocholate (NaTC)/egg PC systems at lipid ratios of 10:0, 10:2 and 10 mM:10 mM in isotonic HEPES buffer, pH 6.5. A sensitive HPLC method was used to quantify the amount solubilized. The melting point and associated enthalpy change as well as the aqueous solubilities were also measured. RESULTS: The retinoids had aqueous solubilities of less than 25 nM. The predicted aqueous solubility was less than 0.01 nM. The amount of retinoid in 10 mM NaTC was increased from three to four orders of magnitude relative to the aqueous solubility. Further increases in the amount solubilized were observed in the 10:10 mixed micelle dispersion. Fenretinide and N-ethyl retinamide were particularly well solubilized by BS and BS/PC aggregated systems which may be related to the presence of a cyclohexenyl ring. CONCLUSIONS: The discrepancy between the observed and predicted aqueous solubility may be due to self-association of the retinoids. Micellar/aqueous distribution ratios appear to be dominated by the hydrophobic effect, although specific interactions also are important. In considering intestinal absorption, the large increase in solubilization with BS/PC micelles would be capable of dramatically increasing the bioavailability in spite of the smaller effective diffusivity of the solubilized retinoid.

Diffusivity of bile salt/phospholipid aggregates in mucin.

PURPOSE: The objective of this study is to evaluate the effect of the mucous layer on the transport of the drug-solubilizing bile salt/phosphatidylcholine (BS/PC) aggregates. METHODS: The self-diffusion coefficient of BS/PC aggregates in bovine submaxillary mucin (BSM) was measured by Fourier-transform pulsed-field gradient spin-echo (FT-PGSE) 1H NMR spectroscopy. RESULTS: In spite of the complexity of the mixture, the FT-PGSE technique allowed the unambiguous determination of the diffusivity of PC and 1H2HO (HDO, natural abundance in D2O). With a series of BS/PC total lipid concentrations ranging from 1 to 7 g/dl, a progressive decrease in the effective diffusivity of HDO was observed with an increase in the both the BSM and total lipid concentration. The effective diffusivity of PC decreased with increasing lipid concentrations in the presence of mucin, while in the controls it increased. After correcting the effective diffusivity of PC for the obstruction effect of mucin, the size of the BS/PC mixed micelle was assessed. It appears that PC associates with BSM resulting in a decrease in the available PC for micellization. This reduces the average size of the mixed micelle within the mucous layer. CONCLUSIONS: The aggregation state of BS/PC micelle is altered by the presence of mucin which would have a direct impact on the transport of dietary lipid and solubilized drug through the aqueous boundary layer of the intestinal tract.

Retinoid absorption from simple and mixed micelles in the rat intestine.

The absorption of three retinoid analogs etretinate (ET), acitretin (ETA), and motretinid (MOE) from two distinct micellar systems was studied in the rat intestine. Each of the three drugs was loaded into simple micelles consisting of 10 mM sodium taurocholate (NaTC) and mixed micelles consisting of 10 mM egg phosphatidylcholine (PC) and 10 mM NaTC. Following perfusion through the jejunum segments, both the fraction of drug disappearing from the segment and the permeability of the drug from the lumen into the gut wall (Peff) was greater with the mixed micelles as compared to the simple micelles. Perfusion flow rate had an influence on the Peff for ET and ETA. Similar trends as for the jejunum were seen in the ileum perfusions. The simultaneous uptake of PC and NaTC during the retinoid perfusions was monitored. There appeared to be a correlation between the Peff values for PC and that of the retinoids. The viability of the in-situ perfusion system was confirmed histologically. There is evidence to indicate that the permeability of the intestine is sensitive to subtle differences in the chemical structure of the retinoids.

Comparative brain exposure to (-)-carbovir after (-)-carbovir or (-)-6-aminocarbovir intravenous infusion in rats.

PURPOSE: Evaluate the ability of (-)-6-aminocarbovir ((-)-6AC) to improve the CNS exposure to (-)-carbovir ((-)-CBV). METHODS: Activation of (-)-6AC in vitro was assessed by incubations of rat brain tissue homogenates. The in vivo brain exposure to (-)-CBV was then examined in rats after iv infusions of either (-)-CBV (n = 4) or (-)-6AC (n = 5). The drugs were infused to steady-state via the jugular vein. At the end of the infusion, a bolus of [3H]inulin was injected via the femoral vein in order to obtain an estimate of the brain vascular space. RESULTS: (-)-6AC was converted to (-)-CBV by incubations of rat brain tissue homogenates. After iv infusion of (-)-CBV, the brain/blood concentration ratio of (-)-CBV was 0.032 +/- 0.009. The brain/blood concentration ratio of (-)-CBV after iv infusion of (-)-6AC was 0.080 +/- 0.020. CONCLUSIONS: (-)-6AC improved the brain delivery of (-)-CBV, although the absolute exposure of the brain tissue to (-)-CBV was still quite low.

Pharmacokinetics and hepatic first-pass effect of (-)-carbovir in the anesthetized rat.

Studies were designed to allow an in vivo estimation of the hepatic extraction ratio and to test the hypothesis that the pharmacokinetic parameters of (-)-CBV are significantly different during anesthesia. (-)-CBV was administered as an i.v. bolus followed by i.v. infusion into either the portal (n = 3) or the jugular (n = 3) veins of anesthetized male Sprague-Dawley rats. These studies indicate that (-)-CBV had a very low hepatic extraction ratio, in agreement with previous (-)-CBV in situ liver perfusion studies. Additionally, anesthesia was shown to alter the pharmacokinetics of (-)-CBV by reducing the total body clearance of this drug.