C. K. Chu

Hybrid pharmacokinetic models to describe anti-HIV nucleoside brain disposition following parent and prodrug administration in mice.

Brain delivery of active anti-HIV compounds is important for successful treatment of the AIDS patient. As an initial step in predicting human brain drug concentrations, hybrid pharmacokinetic models were developed to characterize the disposition of anti-HIV nucleosides following parent and prodrug administrations in mice. Mouse data were obtained following intravenous administration of 3′-azido-2′,3′-dideoxyuridine (AZddU or AZDU), 3′-azido-3′-deoxythymidine (AZT), and their dihydropyridine prodrugs (AZddU-DHP and AZT-DHP). Exponential equations were fitted to the serum concentration–time data for each species, including the pyridinium ion moieties, and subsequently used in differential mass balance equations describing the brain dynamics of each compound. Model parameters for the mass balance equations were estimated by various techniques, including the utilization of in vitro data. In general, model-predicted brain concentrations agreed with the observed data. Similar data in larger animals will permit scale-up of the current model to predict human brain drug concentrations.

DISCONTINUOUS ORAL ABSORPTION PHARMACOKINETIC MODEL AND BIOAVAILABILITY OF 1-(2-FLUORO-5-METHYL-β-L-ARABINOFURANOSYL)URACIL (L-FMAU) IN RATS

1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)uracil (L-FMAU), the L isomer of FMAU, has shown potent activity against hepatitis B virus and Epstein-Barr virus. L-FMAU showed double peaks in the plasma concentration versus time profiles following oral administration to rats, indicating discontinuous oral absorption. The objective of this study was to characterize the bioavailability and pattern of L-FMAU absorption using a pharmacokinetic model which incorporated two separate absorption processes following oral administration of the nucleoside in an animal model, the rat. Simultaneous fitting of differential equations to L-FMAU plasma concentrations following oral and intravenous administration was performed using PCNONLIN. Total clearance of L-FMAU was moderate, averaging 0.47 +/- 0.16 L h-1 (mean +/- SD). Distributional clearance averaged 0.18 +/- 0.14 L h-1. The volume of the central compartment averaged 0.30 +/- 0.09 L, and the volume of the peripheral compartment averaged 0.15 +/- 0.08 L. The first-order absorption rate constants describing the first and second absorption phases averaged 1.22 +/- 1.56 and 4.14 +/- 5.42 h-1, respectively. Oral bioavailability was calculated by three methods: AUC, urinary excretion data, and a discontinuous oral absorption pharmacokinetic model. Bioavailability averaged 0.59 +/- 0.16, 0.64 +/- 0.23, and 0.63 +/- 0.13, respectively, for the three methods. The discontinuous oral absorption pharmacokinetic model is a promising new method for estimating absorption from two phases and for calculating oral bioavailability.

Physicochemical Properties, Bioconversion and Disposition of Lipophilic Prodrugs of 2′,3′-Dideoxycytidine

Lipophilic prodrugs of 2′,3′-dideoxycytidine (ddC), 4,5′-diacetyI-ddC (DAC), 4,5′-ditrimethylacetyl-ddC (DTMAC), 4,5′-dicyclopentylpropionyl-ddC (DCYPP) and 5′-cholesteryl-ddC (CHOL), were evaluated for their utility in improving brain delivery of the parent nucleoside. The lipophilicity of the prodrugs was greater, compared to ddC., with partition coefficient values increasing from 0.03 for ddC to 0.37,28, 63 and 483 for DAC., DTMAC., DCYPP and CHOL., respectively. Aqueous solubility was decreased proportionally to the increase in lipophilicity. Bioconversion studies were performed in phosphate buffer (pH 7.4), human serum, mouse serum, and mouse brain and liver homogenates. Whereas CHOL was stable in vitro in all media, DAC., DTMAC and DCYPP exhibited stability only in buffer, indicating that the hydrolytic reaction for these compounds was, predominately, enzymatically triggered. DCYPP was rapidly hydrolysed in mouse serum and liver and brain homogenates with degradation half-life values of 0.04, 0.35 and 0.34 h respectively. DAC had a longer half-life in mouse serum than did DTMAC (0.82 h vs. 0.38 h), however, in mouse brain homogenate DTMAC (t1/2=3.9 h) was more stable than DAC (t1/2= 1.6 h). Both of these pro-drugs were rapidly metabolized in the mouse liver homogenate with half-life values of 0.36 h for DAC and 0.23 h for DTMAC. In-vivo studies performed for ddC., DAC and DTMAC in mice showed that the relative brain exposure (re) of ddC was not improved by administering the prodrugs. DTMAC yielded a re value of 0.023 which was similar to that for ddC (re = 0.028), while no ddC was detected in brain after DAC administration. Thus, although all of the prodrugs were more lipophilic than ddC., delivery of ddC to the brain was not enhanced in vivo.

Pharmacokinetics of 1-(2-deoxy-2-fluoro-β-L-arabino-furanosyl)-5-methyluracil (L-FMAU) in rats

AbstractPurpose. The objective of this study was to characterize the pharmacokinetics of 1 -(2-deoxy-2-fluoro-β-L-arabinofuranosyl)-5-methyluracil (L-FMAU), a nucleoside analogue with potent activity against the hepatitis B virus and the Epstein-Barr virus, in rats. Methods. Three doses of L-FMAU were administered intravenously (10, 25, and 50 mg/kg) to rats, and L-FMAU concentrations in plasma and urine were measured by HPLC. Pharmacokinetic parameters were generated by using area-moment analysis. Results. There were no significant differences in the pharmacokinetic parameters between the three doses (α < 0.05). Thus, the disposition of L-FMAU was linear over the dosage of 10 to 50 mg/kg. Plasma concentrations of L-FMAU declined rapidly with a terminal phase half-life of 1.33 ± 0.45 h (mean ± SD). Total clearance of L-FMAU was moderate, averaging 1.15 ± 0.28 L/h/kg. The fraction of compound excreted unchanged in urine was 0.59 ± 0.13. No glucuronide metabolite was found in the urine. The steady-state volume of distribution was 1.12 ± 0.26 L/kg indicating intracellular distribution of the compound. The fraction of L-FMAU bound to plasma proteins was approximately 15% and was independent of nucleoside concentration. Conclusions. The pharmacokinetics of L-FMAU in rats were independent of dose over the dosage range of 10 to 50 mg/kg.

High-performance liquid chromatographic determination of (−)-β-d-2-aminopurine dioxolane and (−)-β-d-2-amino-6-chloropurine dioxolane, and their metabolite (−)-β-d-dioxolane guanine in monkey serum, urine and cerebrospinal fluid

Abstract (−)-β- d -2-Aminopurine dioxolane (APD), (−)-β- d -2-amino-6-chloropurine dioxolane (ACPD) and dioxolane guanine (DXG) are nucleoside analogues possessing potent activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) in vitro. APD and ACPD are metabolized in vivo to yield DXG. Reversed-phase HPLC analytical methodologies were developed for the simultaneous determination of APD and DXG, and for ACPD and DXG in monkey serum, urine and cerebrospinal fluid (CSF). 2-Fluoro-2′,3′-dideoxyinosine (FDDI) served as the internal standard. The extraction recoveries of the nucleoside analogues from serum samples were similar, averaging approximately 90%. The limit of quantitation of the analytical method for serum samples was 0.1 μg/ml for DXG, and 0.25 μg/ml for APD and ACPD. The intra- and inter-day relative standard deviations for each compound at low, medium and high nucleoside concentrations were less than 9.0%. The accuracy of the assay methods was greater than 90% for prodrugs and parent compound. Similar results were observed with urine and CSF samples. Thus, these methods provide sensitive, accurate and reproducible determination of the prodrugs and parent nucleoside in biological samples.

Brain Targeting of anti-HIV Nucleosides: Ether Prodrugs of 3′-Azido-2′,3′-Dideoxyuridine (AZdU) and 3′-Azido-3′-Deoxythymidine (AZT)

In an effort to increase the brain delivery of anti-HIV nucleosides, 5-0-benzyl and glucose derivatives of 3′-azido-2′,3′-dideoxyuridine (AZdU or CS-87) and 3′-azido-3′-deoxythymidine (AZT) were synthesized. In vitro stability and pharmacokinetic studies in mice were conducted with benzyl AZdU (BzlAZdU), benzyl AZT (BzlAZT), and glucose AZdU (GAZdU) prodrugs. In vitro studies indicated that the prodrugs were stable in phosphate buffer (pH 7.4), human serum and mouse serum. In mouse brain homogenate, the degradation half-lives for BzlAZdU, BzlAZT, and GAZdU were 1.66, 2.06, and 0.98 h, respectively, and in liver homogenate the degradation half-lives were 0.49, 0.29, and 1.97h, respectively. Following intravenous administration of BzlAZdU, BzlAZT, or GAZdU to mice, prodrug and parent drug concentrations were measured in serum and brain by HPLC, and pharmacokinetic parameters determined. The brain:serum area under the concentration time-curve (AUC) ratio, a parameter indicative of prodrug uptake into brain, was 0.55 for BzlAZdU and 0.56 for BzlAZT, compared to 0.05–0.08 when the parent drugs AZdU and AZT were administered intravenously. GAZdU had poor brain penetration, achieving brain concentrations of only 5% of the serum concentrations. Parent drug concentrations in brain were, for the most part, not detected after administration of any of the prodrugs. Consistent with in vitro data, it is apparent that the prodrugs were converted to metabolites other than the parent drug species.

Pharmacokinetics of 3′-azido-2′,3′-dideoxy-5-methylcytidine in Rats

3′-Azido-2′,3′-dideoxy-5-methylcytidine (AzddMeC) has been shown to have potent activity against human immunodeficiency virus (HIV) in vitro. The purpose of this study was to characterize the pharmacokinetics of AzddMeC in rats. AzddMeC was administered intravenously at doses of 10, 50 and 100 mg kg−1. Plasma and urine AzddMeC concentrations were determined by HPLC. Pharmacokinetic parameters were generated by area/moment analysis. Plasma AzddMeC concentrations after 10mg kg−1 were too low to accurately calculate pharmacokinetic parameters. Following 50 and 100mg kg−1 AzddMeC, plasma drug concentrations declined rapidly with a terminal half-life of approximately 2.5 h. No statistically significant differences were noted in pharmacokinetic parameters between the two higher doses. Total clearance was 1.57 ± 0.33 (mean ± SD) and 1.76 ± 0.32I h−1 kg−1 after 50 and 100 mg kg−1 AzddMeC, respectively. Renal excretion accounted for approximately half of total clearance with 55 ± 11% of the dose recovered as unchanged drug in urine. AzddMeC was not metabolized by deamination to AZT in the rat. No glucuronide metabolite was found in urine. Steady-state volume of distribution of AzddMeC averaged 1.73 ± 0.78 and 1.46 ± 0.441 kg−1 following 50 and 100 mg kg−1, respectively. Thus, the disposition of AzddMeC in rats is independent of dose over the range of 50–100 mg kg−1. The pharmacokinetics of AzddMeC in rats are similar to those of 2′,3′-dideoxycytidine, while the clearance of AzddMeC is 40% less than that of 3′-azido-3′-deoxythymidine.

Brain Targeting of anti-HIV Nucleosides: in vitro and in vivo Evaluation of 6-chloro-2′,3′-dideoxypurine, a Lipophilic Prodrug of 2′,3′-dideoxyinosine

Lipophilic 6-halo-2′,3′-dideoxypurine nucleosides may be useful prodrugs for the targeting of 2′,3′-dideoxyinosine (ddl) to the central nervous system. The purpose of this study was to evaluate the potential effectiveness of 6-chloro-2′,3′-dideoxypurine (6-CI-ddP) for the targeting of ddl to the brain. In vitro studies indicated that the adenosine deaminase-mediated biotransformation of 6-CI-ddP to ddl was more rapid in mouse brain homogenate than in mouse serum. The brain distribution of 6-CI-ddP and ddl was assessed in vivo in mice following intravenous and oral administration of the prodrug or parent drug. Brain concentrations of ddl were similar after intravenous administration of 6-CI-ddP or ddl. However, after oral administration of the 6-CI-ddP prodrug, significantly greater concentrations of ddl were seen in the brain compared to those found after oral administration of ddl. The brain:serum AUG ratio (expressed as a percentage) of ddl after intravenous administration of 50 mg kg−1 of the active nucleoside was 3%. Following oral administration of 250 mg kg−1 ddl, low concentrations of ddl were detected in the brain. Brain:serum AUC ratios following intravenous and oral administration of the prodrug 6-CI-ddP were 19–25%. Thus, brain:serum AUC ratios were 6- to 8-fold higher after prodrug administration than those obtained after administration of the parent nucleoside. Oral administration of 6-CI-ddP yielded concentrations of ddl in the brain similar to those obtained following intravenous administration. The results of this study provide further evidence that 6-CI-ddP may be a useful prodrug for delivering ddl to the central nervous system, particularly after oral administration.

Dose-dependent pharmacokinetics of 3'-azido-2',3'-dideoxyuridine in rats

The pharmacokinetics of 3′-azido-2′,3′-dideoxyuridine (AzddU; CS-87), an inhibitor of human immunodeficiency virus (HIV) replication in vitro, were characterized in rats. AzddU was administered intravenously at doses of 10, 50, 100 and 250mgkg−1. Plasma and urine AzddU concentrations were measured by HPLC. Plasma AzddU concentrations declined in a biexponential fashion with a terminal half-life of approximately 1.5h. The disposition of AzddU was independent of dose over the dosage range of 10–100mgkg−1; however, the pharmacokinetics of the nucleoside exhibited non-linearities after 250mgkg−1. Over the dose range of 10–100mgkg−1 AzddU, total clearance and renal clearance averaged 2.13lh−1kg−1 and 1.46lh−1kg−1, respectively. Total clearance was significantly lower after 250mgkg−1 (CIT = 1.32lh−1kg−1) owing to a decreased renal clearance (CIR = 0.69lh−1kg−1) of AzddU. Renal clearance exceeded glomerular filtration rate, indicating that active renal tubular secretion was involved in the renal excretion of the compound. The maximum transport capacity (Tmax) and the Michaelis–Menton constant (Km) for the tubular secretion mechanism were 142.2mg h−1 and 60.4mg l−1, respectively. The high values for Tmax and Km explain the high renal clearance of AzddU and the linearity of renal excretion over a wide range of drug concentrations. However, at very high AzddU concentrations active tubular secretion is saturable. Nonrenal clearance was independent of dose with a mean value of 0.66lh−1kg−1. Steady-state volume of distribution was similar at all doses averaging 1.05lkg−1. Thus, the disposition of AzddU is linear over the dose range of 10–100mgkg−1, but becomes dose dependent with decreases in renal and total clearances after 250mgkg−1 AzddU.

Brain Targeting of Anti-HIV Nucleosides

Patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex frequently develop neurological complications due to the human immunodeficiency virus (HIV) infection in the brain. In2 Although the mechanism of HIV-induced CNS dysfunction is unknown, it is believed that HIV is carried into the brain by infected macrophages/monocytes.3 Thus, it is essential that anti-HIV agents cross the blood-brain barrier (BBB) to suppress the viral replication in the brain. 3’-Azido-3’deoxythymidine (AZT) has been demonstrated to penetrate into cerebrospinal fluid and partially reverse the neurological complication^.^^^ It has not been demonstrated, however, that AZT actually crosses the BBB or maintains the sufficient concentration in CNS by which it would be able to effectively suppress the viral replication in the brain6 Thus, it was of interest to develop antiviral prodrugs which could more readily penetrate the BBB than do the parent nucleosides. We have chosen two anti-HIV nucleosides, AZT and AzddU (AZDU, or CS-87). The latter is a compound of which anti-HIV activity was discovered in our laborat~ries,’.~ and it is currently undergoing phase I clinical trials. Among various methods of brain targeting of drugs, Bodor and co-workers’ strategy9 seems to be attractive. The approach uses a dihydropyridine-pyridinium salt redox