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Pharmacokinetics of Hydroxyurea in Plasma and Cerebrospinal Fluid of HIV-1-Infected Patients

From the Colleges of Pharmacy (Dr. Gwilt, Dr. Manouilov, Dr. McNabb) and Medicine (Dr. Swindells), University of Nebraska Medical Center, Omaha, Nebraska.

Address for reprints: Peter R. Gwilt, PhD, College of Pharmacy, 986025 Nebraska Medical Center, Omaha, NE 68198-6025.

	ABSTRACT

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Hydroxyurea has been shown to potentiate the activity of the antiretroviral nucleoside analogs. A significant complication of AIDS is invasion of the virus into the CNS, resulting in HIV-associated dementia (HAD). Because of the polar nature of these nucleosides and the presence of efflux pumps in the blood-brain barrier, only low CNS drug concentrations are achieved. Introduction of hydroxyurea into the CNS may therefore increase the antiviral activity of these drugs. This study evaluates the accessibility of hydroxyurea to the CNS following oral drug administration. Twelve HIV patients received 800 mg, 1000 mg, or 1200 mg oral hydroxyurea. Cerebrospinal fluid (CSF) and plasma drug concentrations were measured over 8 hours and simultaneously fitted to a pharmacokinetic model. It was determined that CSF hydroxyurea concentrations, corresponding to those found to increase antiretroviral nucleoside activity in vitro, were achieved.

Key Words: Hydroxyurea • pharmacokinetics • HIV-associated dementia • antiretroviral drugs

For hydroxyurea to contribute to antiviral activity in the brain, it must achieve and sustain pharmacologically active levels in the CNS, which can only be measured by the appearance and disposition of hydroxyurea in cerebrospinal fluid (CSF), and the data available on this are limited.

In animals, whole-brain concentrations of hydroxyurea (not adjusting for blood associated with the tissue) appear to be about 25% of that in the blood.^13,14 Beckloff et al¹⁵ measured CSF hydroxyurea concentrations at single time points corresponding to the peak plasma concentration following oral administration of 20, 40, and 80 mg/kg of hydroxyurea. Using a colorimetric assay, no drug in the CSF was detected at 20 mg/kg, whereas 3.3 mg/L (12% of plasma concentration) was found upon administration of 40 mg/kg, and 38.3 and 8.2 mg/L (24% and 23% of plasma concentration) were observed following an 80-mg/kg dose in 2 individuals. These findings indicate that hydroxyurea does distribute to the CSF.

The purpose of this study was to extend these observations to evaluate CNS hydroxyurea concentrations in HIV-infected subjects following oral hydroxyurea administration of 800 mg, 1000 mg, and 1200 mg. Evaluation of CNS exposure included characterization of the concentration-time course of the drug in CSF and plasma for each dose.

An increased toxicity profile, including some incidences of pancreatitis that proved fatal, has been associated with certain antiretroviral drug combinations that include hydroxyurea.¹⁶ The abandonment of such regimens is prudent when alternative drug combinations will suffice. However, if CNS hydroxyurea exposure is found to be pharmacologically active enough to potentiate the activity of antiretroviral drugs, investigation of hydroxyurea and antiretroviral combinations may still be fruitful in selected populations such as patients with HAD, provided the associated toxicity remains minimal.

	SUBJECTS AND METHODS

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Following approval by the institutional review board, written informed consent was obtained from 12 subjects recruited from the University of Nebraska Medical Center (UNMC) HIV clinic. Major inclusion criteria included documented HIV infection, a Karnofsky score > 60, age 19 years or older, and ability and willingness to sign an informed consent form. Exclusion criteria included platelet count < 75,000 cells/µL, PT or PTT greater than 1 x upper limit of normal, active opportunistic infection, pregnancy, or breastfeeding. The characteristics of the patients and concomitant drug use are summarized in Table I.

Following an overnight fast, subjects ingested oral hydroxyurea doses of 800, 1000, or 1200 mg, with 4 subjects assigned to each dose level. Using a heparin-lock catheter, blood (5.0 mL) was drawn from a peripheral vein at 0, 0.5, 0.75, 1.0, 1.5, 3, and 8 hours following hydroxyurea administration. Blood was collected in heparinized tubes and centrifuged at 2000 x g for 10 minutes at room temperature to obtain plasma. The plasma was then stored at -70°C until the samples were assayed. CSF (3.0 mL) samples were obtained via a continuous spinal catheter inserted into the subarachnoid space at the L3-L4 or L4-L5 interspaces. CSF collection times were 0.5, 1.5, 2.5, and 5.0 hours following dose administration. The CSF samples were similarly stored at -70°C until assay.

Plasma and CSF samples were assayed using high-performance liquid chromatography.¹⁷ To 200 µL of plasma or CSF, methylurea 10 or 50 µg/mL (depending on the anticipated hydroxyurea concentration) was added as an internal standard. Plasma proteins were precipitated with methanol (1:2). Following centrifugation, the methanol was evaporated under vacuum, and 5 mg/mL urease was added. Then, 100 µL of the supernatant was mixed with the reagents of a commercial colorimetric assay kit for urea (Sigma Diagnostics). Specifically, 175 µL of blood urea nitrogen (BUN) acid reagent (No. 353-3) and 150 µL of color reagent (No. 535-5) were used. The tube was then placed in boiling water for 10 minutes to allow the color complexes of hydroxyurea and methylurea to form, and then it was cooled in iced water. An aliquot of the resulting colored solution was injected onto an Ultrasphere C₈ column (150 mm x 4.6 mm ID) with a flow rate of 1 mL/min. The injection volumes were between 10 and 50 µL, depending on the anticipated hydroxyurea concentration. The mobile phase consisted of 14% acetronitrile in water at a flow rate of 1 mL/min. Detection was by UV-Vis set at 449 nm. Calibration was linear in the 0.5 to 20 mg/L range, and intraday variability was less than 15%. The retention times for the hydroxyurea and methylurea peaks were 7.9 and 17.2 minutes, respectively.

Pharmacokinetic Analysis
The uptake and disposition of hydroxyurea by the CNS of patients with HIV disease were determined by simultaneously modeling CSF and plasma hydroxyurea concentrations using a two-compartment pharmacokinetic model with first-order absorption. The model was further refined by treating the CSF as an effect compartment, as proposed in two recent publications describing the CSF uptake of acetaminophen¹⁸ and rivastigmine.¹⁹ Pharmacokinetic analysis was performed using the population pharmacokinetic package NONMEM.²⁰ The equations used to describe hydroxyurea oral absorption and plasma disposition were as follows:

where X_g and X_p are amounts of hydroxyurea in the gut and plasma, respectively; C_p is the hydroxyurea plasma concentration; ka is the apparent first-order absorption rate constant; K is the apparent elimination rate constant for hydroxyurea; and V is the apparent volume of distribution of hydroxyurea in the body. The pharmacokinetics of hydroxyurea in the CSF were described using an effect model, that is,

where C_csf is the concentration of hydroxyurea in the CSF, k_eq is the plasma-CSF equilibration rate constant for hydroxyurea, and PC is the pseudo-partition coefficient.

Plasma and CSF hydroxyurea concentration-time profiles were modeled using subroutine ADVAN6 of NONMEM. The model accounts for both unexplainable inter- and intrasubject effects (random effects) as well as concomitant effects (fixed effects). The interindividual variability in model parameters was modeled by an exponential variance model. An additive term characterized the residual error.

	RESULTS

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Patient demographic data are shown in Table I. Mean parameter values for hydroxyurea pharmacokinetic parameters, with associated standard errors, are shown in Table II. Hydroxyurea plasma and CSF concentration-time curves for the 1200-mg dose are shown in Figure 1. A number of potential covariates were evaluated to account for interpatient variability, including patient weight, age, administration of antiretroviral drugs, and viral load. However, none proved to significantly (p < 0.05) reduce the minimum value of the objective function (MVOF). The parameters characterizing the plasma disposition of hydroxyurea are similar to those described in previous reports.²¹ The hydroxyurea elimination rate constant was independent of the dose of drug administered, consistent with a Km of 25 mg/L hydroxyurea in plasma reported previously.²² The pseudo-partition coefficient (PC) is a measure of the penetration of the CSF by the drug. Its value, 0.329, predicts that the concentration of hydroxyurea in the CSF under steady-state conditions would be 33% of that in plasma. However, a comparatively long hydroxyurea CSF equilibration time (teq) of 4.15 hours was estimated where teq = 0.693/Keq. Nevertheless, at the doses studied, over the period of measurement, hydroxyurea achieves CSF concentrations reported to potentiate antiretroviral activity in vitro (i.e., 0.76-7.6 mg/L).²³

View larger version (24K): [in this window] [in a new window]   Figure 1. Observed mean ± SEM hydroxyurea plasma (•) and cerebrospinal fluid (CSF) () concentrations following oral administration of 1200 mg hydroxyurea.

	DISCUSSION

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The potentiation of some antiretroviral nucleosides by hydroxyurea is attributed to increased production of the intracellular triphosphate form of these drugs. Hydroxyurea achieves this through two mechanisms. In one of these mechanisms, the de novo pathway, hydroxyurea inhibits ribonuclease reductase, thereby reducing cellular adenosine triphosphate (ATP). This increases incorporation of adenosine phosphate analogs, such as dideoxinosine triphosphate, into DNA under synthesis by reverse transcriptase.¹¹ A second pathway, the salvage pathway, also leads to elevated concentrations of triphosphate analogs but, in this case, by increasing the activity of intracellular phosphorylating enzymes such as deoxycytidine and thymidine kinase.¹² Incorporation of the nucleoside analog triphosphate inhibits DNA chain elongation.

The hydroxyurea concentration required to inhibit HIV-1 in vitro in activated PBMC is 0.4 mM (30.4 mg/L).²³ In combination with the nucleoside analogs, hydroxyurea concentrations of only 0.01 to 0.1 mM (0.76-7.6 mg/L)²⁴ will suffice. In the present study, the highest concentrations of hydroxyurea in CSF and plasma were 0.04 mM and 0.26 mM, respectively. Thus, hydroxyurea may have minimal effect on HIV titers in plasma and CSF when administered alone. However, used in combination with nucleoside analogs, hydroxyurea may significantly contribute to a reduction in HIV levels in both plasma and CSF.

The pseudo-partition coefficient for hydroxyurea was estimated to be 0.329. This is similar to the figure of 25% reported for hydroxyurea: CSF plasma ratios determined in man and animals.^13,14 It also compares with values of 1.18 and 0.398 for acetaminophen¹⁸ and rivastigimine,¹⁹ estimated by similar means to those used in this study. The plasma-CSF equilibration time (4.15 h) is long compared with those of acetaminophen (0.72 h)¹⁸ and rivastigmine (0.23 h).¹⁹ The precise relationship between the in vivo partition coefficients, equilibration times, and drug uptake and disposition in the CSF has yet to be determined. However, the extensive work of Rapaport and coworkers²⁵ has established the log partition coefficient as a reliable predictor of the CSF/unbound plasma drug concentration ratio for drugs passively crossing the blood-brain barrier.

In conclusion, it appears that oral administration of hydroxyurea produces CSF drug concentrations equivalent to those shown in vitro to potentiate the activity of antiretroviral nucleoside analogs. This may support further investigation of hydroxyurea in combination chemotherapy for the treatment of HIV-associated dementia.

	FOOTNOTES

 
This study was supported by Bristol-Myers Squibb Company. Abstract presented at the XIV International AIDS Conference, Barcelona, Spain, July 2002.

DOI: 10.1177/0091270003256144

Submitted for publication February 22, 2003; Revised version accepted May 10, 2003.

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