Liquid chromatographic-tandem mass spectrometric assay for the simultaneous determination of didanosine and stavudine in human plasma, bronchoalveolar lavage fluid, alveolar cells, peripheral blood mononuclear cells, seminal plasma, cerebrospinal fluid and tonsil tissue.

We have developed a sensitive, high-pressure liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the simultaneous determination of didanosine (ddI) and stavudine (d4T) in human plasma, bronchoalveolar lavage fluid (BALF), alveolar cells (AC), peripheral blood mononuclear cells (PBMC), seminal plasma, cerebrospinal fluid (CSF), and tonsil tissue. Plasma, AC, PBMC and CSF were run with an isocratic HPLC method, while BALF supernatant, semen, and tonsil tissue utilized a gradient elution. Samples were prepared by solid phase extraction. Detection was by electrospray positive ionization with multiple reaction monitoring mode. The lower limits of quantitation for both ddI and d4T were 2.0 ng/ml in plasma; 0.5 ng/ml in CSF; 0.4 ng/ml in AC, PBMC, and BALF; 1.0 ng/ml in seminal plasma; and 0.01 ng/mg in tonsil tissue.

Distributed model analysis of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine distribution in brain tissue and cerebrospinal fluid.

The restricted distribution of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (DDI) in brain tissue and cerebrospinal fluid (CSF) has been analyzed using the distributed model. The distribution volume of AZT and DDI in brain tissue (V(br)) was found to be 1.07 +/- 0.09 and 0.727 +/- 0.030 ml/g brain, respectively, in an in vitro brain slice uptake study. The pharmacokinetic parameters were obtained by fitting the concentration-time profiles of AZT and DDI in brain tissue and CSF after i.v. or i.c.v. administration taking the value of V(br), the CSF bulk flow rate (2.9 microl/min), and the surface area of the cerebroventricular ependyma (2.0 cm2), using a nonlinear least squares program combined with a fast inverse Laplace transform. The efflux transport clearance (PS(BBB,eff)) across the blood-brain barrier (BBB) and the symmetrical permeability clearance (PS(BBB)) across the BBB for AZT were calculated as 179 and 10.3 microl/min/g brain, respectively. The efflux transport clearance (PS(CSF,eff)) across the blood-cerebrospinal fluid barrier (BCSFB) and the symmetrical permeability clearance (PS(CSF)) across the BCSFB for AZT were calculated as 227 and 28.3 microl/min/ml CSF, respectively. For the distribution of DDI, the PS(BBB,eff) and PS(BBB) were 79.2 and 2.03 microl/min/g brain, respectively, while the PS(CSF,eff) and PS(CSF) for DDI were 196 and 5.88 microl/min/ml CSF, respectively. Based on simulation studies using the fitted parameters, a significant degree of efflux transport across the BBB and BCSFB has been suggested to be responsible for the restricted distribution of AZT and DDI in brain tissue and CSF, respectively.

Dose dependence in the plasma pharmacokinetics and uptake kinetics of 2',3'-dideoxyinosine into brain and cerebrospinal fluid of rats.

Dose dependence in the plasma pharmacokinetics of 2',3'-dideoxyinosine (ddI) was examined during and after 2-hr iv infusions in rats at infusion rates of 12.4, 32.7, and 125 mg/kg/hr. After termination of the infusions, the disappearance of ddI from plasma was distinctly biphasic, suggesting that the majority of ddI is eliminated before distribution equilibrium is achieved. The mean alpha t1/2 following the infusions was 2.7 min and was independent of dose. The mean terminal half-life (beta t1/2) was approximately 24 min and also independent of dose. Nonlinear pharmacokinetic behavior in plasma after infusions was manifested in a decreased clearance with increasing dose, as determined from steady state plasma concentrations of ddI during infusions. In parallel with the decreased clearance, the apparent volume of distribution of the central compartment, Vcapp, decreased with increasing dose. Nonlinearity in clearance with increasing dose could be accounted for using a model which includes rapid, saturable tissue binding. Dose dependence in the kinetics of uptake of ddI into brain tissue and cerebrospinal fluid (CSF) was also examined during and after iv infusions. Steady state concentrations of ddI in brain tissue and CSF varied linearly with steady state plasma concentrations over a plasma concentration range of greater than 30-fold. Mean tissue to plasma concentration ratios, expressed as percentages, were 2% in CSF, 5% in brain tissue, and 1-2% in brain parenchymal tissue (corrected for the contribution of the cerebral vascular space).

Pharmacokinetics of dideoxypurine nucleoside analogs in plasma and cerebrospinal fluid of rhesus monkeys.

The pharmacokinetics of 2',3'-dideoxyadenosine (ddA), didanosine, 2',3'-dideoxyguanosine (ddG), and 6-halogenated prodrugs of ddG, 6-chloro-ddG and 6-iodo-ddG, in plasma and cerebrospinal fluid (CSF) were studied in a non-human primate model. ddA was rapidly and completely deaminated to didanosine, such that didanosine concentration profiles in plasma and CSF were identical following administration of ddA and didanosine. The mean clearance of didanosine was 0.50 liters/h/kg, the terminal half-life was 1.8 h, and the CSF-to-plasma ratio was 4.8%. The disposition of ddG was similar, with a clearance of 0.70 liters/h/kg and a half-life of 1.7 h. The adenosine deaminase-mediated conversion of the 6-halogenated-ddG prodrugs to ddG was rapid but incomplete (48% for 6-chloro-ddG and 29% for 6-iodo-ddG). The CSF-to-plasma ratios of ddG with equimolar doses of ddG, 6-chloro-ddG, and 6-iodo-ddG were 8.5, 24, and 17%, respectively, but the actual ddG exposures in CSF (area under the CSF concentration-time curve) were comparable for ddG (12.1 microM.h) and the 6-halogenated-ddG prodrugs (18.8 microM.h for 6-chloro-ddG, 9.3 microM.h for 6-iodo-ddG).6-Chloro-ddG was not detectable in plasma or CSF, and the CSF-to-plasma ratio of 6-iodo-ddG was 9.4%, so the higher CSF-to-plasma ratios of ddG with the administration of the 6-halogenated-ddG prodrugs does not appear to be the result of enhanced penetration of the prodrug and subsequent dehalogenation to ddG. The penetration of ddG into CSF exceeds that of didanosine and is enhanced by administration of the 6-halogenated prodrugs, although the mechanism of this enhanced penetration is unclear.

Uptake kinetics of 2',3'-dideoxyinosine into brain and cerebrospinal fluid of rats: intravenous infusion studies.

The pharmacokinetics of 2',3'-dideoxyinosine (ddl) and its distribution to plasma, brain tissue and cerebrospinal fluid (CSF) were determined during and after 2-hr i.v. infusions of ddl (125 mg/kg/hr) in rats to define its specific pharmacokinetic parameters for subsequent studies of prodrugs designed to target this compound to the brain. Steady-state plasma concentrations of 50 micrograms/ml were obtained within 30 min after the start of infusions corresponding to a total clearance of 2.4 l/kg/hr. Postinfusion, ddl concentrations declined biphasically from plasma with alpha T1/2 = 3 min and beta T1/2 = 35 min. STeady-state concentrations of ddl in brain tissue and CSF were 2.6 micrograms/g in tissue and 0.81 microgram/ml in CSF, respectively. These values represent 4.7 and 1.5%, respectively, of the simultaneously determined plasma concentration. The estimated brain vascular space contribution to the observed brain uptake was 4.1%, obtained by least squares fitting of a compartmental pharmacokinetic model to the uptake data. Postinfusion, the elimination of ddl from the brain and CSF was significantly slower than from plasma, resulting in increased brain/plasma and CSF/plasma ratios after the infusions. The low steady-state brain/plasma or CSF/plasma ratios suggest rapid disappearance of ddl from the CNS relative to its rate of entry. These data indicate that ddl penetrates poorly into the brain. Thus, prodrugs with enhanced blood-brain barrier transport may improve the delivery of ddl to the brain.

Efflux of zidovudine and 2',3'-dideoxyinosine out of the cerebrospinal fluid when administered alone and in combination to Macaca nemestrina.

To determine if there is active efflux of zidovudine (ZDV) and 2',3'-dideoxyinosine (ddI) out of the cerebrospinal fluid (CSF), and if this efflux is saturable, we investigated the steady-state CSF/plasma concentration ratio of the two drugs when administered alone or in combination. Constant-rate infusions of ZDV, ddI or both were administered to seven macaques (Macaca nemestrina) through a chronic venous catheter for a minimum of 28 hr. Antipyrine, a marker of passive diffusion, was coinfused in all experiments. Blood (5 mL) and CSF samples (0.5-1 mL) were collected by venous and lumbar/thoracic punctures, respectively, at 24 and 28 hr after beginning the infusion. When ZDV and ddI were administered alone, the steady-state CSF/plasma concentration ratios were significantly different from unity (ZDV, 0.20 +/- 0.08; ddI, 0.09 +/- 0.04) and were independent of the plasma concentration (P > 0.05). In contrast, the CSF/plasma concentration ratio of antipyrine (0.82 +/- 0.19) was close but significantly smaller than unity (P > 0.05). The CSF/plasma concentration ratios after simultaneous administration of ZDV and ddI were not significantly different (P > 0.05) from those obtained after administration of the drugs alone. These results suggest that ZDV and ddI are actively transported out of the CSF; however, within the concentration range studied, this efflux is neither saturable nor mutually competitive. Concomitant administration of ZDV and ddI did not produce a systemic interaction in the animals, indicating that the pharmacokinetics of either drug is unaffected by the presence of the other.

Probenecid enhances central nervous system uptake of 2',3'-dideoxyinosine by inhibiting cerebrospinal fluid efflux.

The effects of probenecid on the pharmacokinetics of 2',3'-dideoxyinosine (ddl) and on the distribution of ddl to cerebrospinal fluid (CSF) and brain tissue were determined in rats during and after a 2-hr i.v. infusion of ddl, 125 mg/kg/hr. Probenecid-treated rats received a loading dose of probenecid followed by an i.v. infusion of probenecid initiated 1 hr before and continued during and for 2 hr after termination of the ddl infusion. Plasma concentrations of probenecid averaged 221 +/- 34 micrograms/ml upon termination of the ddl infusion and 258 +/- 34 micrograms/ml (mean +/- S.D., n = 4) 1 hr later. In the probenecid-treated animals, ddl concentrations were higher in plasma (1.5-fold), brain (1.5-fold) and CSF (5.4-fold) at the termination of the ddl infusion and postinfusion concentrations declined more slowly compared to controls. Postinfusion, the CSF/plasma and brain/plasma ratios steadily increased to a greater extent in the probenecid-treated rats compared to control animals. The time course of plasma, CSF and brain tissue concentrations were analyzed by nonlinear least-squares regression using two different compartmental models, one which neglected the direct exchange of drug between the CSF and brain parenchyma, whereas the other allowed for such exchange to occur and neglected direct vascular transfer of drug to brain tissue. Allowing exchange between the CSF and brain tissue gave slightly improved fitting of the data from both probenecid-treated and control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Study on didanosine concentrations in cerebrospinal fluid. Implications for the treatment and prevention of AIDS dementia complex.

It has been hypothesized that didanosine has a low efficacy in the prevention and treatment of patients with the dementia complex of acquired immunodeficiency syndrome (AIDS) because "... the drug has not been detected in the cerebrospinal fluid". We investigated didanosine concentrations in cerebrospinal fluid (CSF) and plasma of four patients with AIDS who were using didanosine chronically. Didanosine levels, 4 h after the last drug administration, averaged 0.16 (+/- 0.03) mumol/l in CSF and 0.70 (+/- 0.27) mumol/l in plasma. When compared with historical data from patients using zidovudine, didanosine concentrations in CSF appeared to be approximately half (on a molar base) those of zidovudine concentrations in the CSF. Whether this difference in CSF levels is the explanation for the presumed lower efficacy of didanosine in the prevention and treatment of AIDS dementia complex remains to be proven. However, it is clear from this study, in contrast with earlier suggestions, that didanosine is able to pass the blood-CSF barrier in human immunodeficiency virus-infected individuals.

Role of brain tissue localized purine metabolizing enzymes in the central nervous system delivery of anti-HIV agents 2'-beta-fluoro-2',3'-dideoxyinosine and 2'-beta-fluoro-2',3'-dideoxyadenosine in rats.

PURPOSE: This study examines the central nervous system (CNS) delivery of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) and 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), acid stable analogues of dideoxyadenosine (ddA) and dideoxyinosine (ddI) having reduced susceptibility to purine salvage pathway enzymes important in the metabolism of ddA and ddI, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), respectively. Their CNS delivery compared to that for ddI provides insight into the role of brain tissue ADA and PNP in these processes. METHODS: Brain and cerebrospinal fluid (CSF) concentration-time profiles were obtained for F-ddI during and after intravenous infusions of F-ddI, and for both F-ddA and F-ddI after F-ddA infusions in normal rats or rats pre-treated with the ADA inhibitor 2'-deoxycoformycin (DCF). Rate constants for CNS entry, efflux and metabolism were estimated by computer fits using plasma concentration-time profiles as the driving force functions. RESULTS: The CNS delivery of F-ddI did not differ significantly from that for ddI. F-ddA, which is more lipophilic than F-ddI, provided higher brain (approximately 8x) and CSF (approximately 11x) concentrations of total dideoxynucleoside (F-ddA and F-ddI) compared to F-ddI. Deamination by brain tissue ADA to form F-ddI reduced CNS levels of intact F-ddA but provided higher brain parenchyma (5x) and CSF/plasma (3x) ratios of F-ddI relative to F-ddI controls. Thus, F-ddA functions in part as a CNS-activated prodrug of F-ddI. DCF pre-treatment inhibited brain tissue ADA, abolishing the prodrug effect, and enhancing F-ddA concentrations in both brain parenchyma (5x) and CSF (6x). CONCLUSIONS: PNP metabolism does not appear to play a role in the low CNS delivery of ddI. On the other hand, deamination of F-ddA by brain tissue ADA is an important process, such that F-ddA functions in part as a CNS-activated prodrug of F-ddI. Enhanced CNS uptake of intact F-ddA can be achieved with ADA inhibition.

Mechanisms by which 2',3'-dideoxyinosine (ddI) crosses the guinea-pig CNS barriers; relevance to HIV therapy.

The influence of transport mechanisms at the blood-brain barrier (BBB) and blood-CSF barrier (choroid plexus) on the CNS distribution of anti-human immunodeficiency virus (HIV) drugs was examined using guinea-pig brain perfusion and incubated choroid plexus models. 2',3'-dideoxyinosine (ddI) passage across the BBB was demonstrated to be via non-saturable (Kd = 0.22 +/- 0.3 microL/min/g) and saturable (Km = 20.1 +/- 15.0 microm, Vmax = 6.5 +/- 2.1 pmol/min/g) processes. Cross competition studies implicated an equilibrative nucleoside transporter in this influx. The brain distribution of ddI was unchanged in the presence of additional nucleoside reverse transcriptase inhibitors (NRTIs). ddI transport from blood into choroid plexus was demonstrated to involve an organic anion transporting polypeptide 2-like transporter. The NRTIs, abacavir, 3'-azido 3'-deoxythymidine and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine, competed with ddI for transporter binding sites at the choroid plexus, altering the tissue concentration of ddI. This has clinical implications as the choroid plexus is a site of HIV replication, and suboptimal CNS concentrations of anti-HIV drugs could result in neurological complications. Furthermore, this may promote the selection of drug resistant variants of HIV within the CNS, which could re-infect the periphery and lead to HIV therapy failure. This study indicates that understanding drug interactions at the transporter level could prove valuable when selecting drug combinations to treat HIV within the CNS.