Steven G. Ayre (1), Brian Skaletski (2) and Aron D. Mosnaim( 2)
Research Communications in Chemical Pathology and Pharmacology JANUARY 1989 VOL.63, NO. 1
Departments of Family Medicine and Pharmacology and Molecular Biology , University of Health Sciences/The Chicago Medical School, North Chicago, IL 60064.
SUMMARY Azidothymidine (AZT) crosses the blood-brain barrier (BBB) of 6-8 week old Sprague-Dawley male white rats (Oldendorf technique, ipsilateral cerebral hemisphere) with a brain uptake index (BUI) of 5.4±0.8 (x±S.D., n=13, range 4.4-6-6) using C-antipryine as the diffusible standard. Pretreatment of the animals with the higher doses of insulin (0.6 or 1.0, but not 0.1, 0.2, or 0.3 units per rat, 3 or 10 min. before decapitation) resulted in higher values for the BUI of AZT in most individual animals. In the group of rats treated with 1.0 unit of insulin 10 min. before decapitation, a statistically significant increase in the BUI was observed. Some possible clinical applications of this pharmacologic strategy are discussed.
INTRODUCTION The human immunodeficiency virus (HIV) has been shown to be the causative agent of the acquired immunodeficiency syndrome (AIDS) and related conditions (Montagnier et al., 1984; Gallo et al., 1984). As well as affecting immune function, HIV has been shown to be neurotropic (Ho et al., 1985), emphasizing the need for antiviral agents able to cross the blood-brain barrier (BBB). Eradication of the central nervous system (CNS) HIV infection is important as this is considered the cause of AIDS dementia complex, reported to affect up to 40% of AIDS patients (Elder and Sever, 1988; Price et al., 1988). In addition, persistence of HIV within the brain may possibly act as a reservoir to continuously reinfect peripheral tissues in the host.
Zydovudine (3′-azido-3′-deoxythymidine, Retrovir, formerly azidothymidine or AZT) is the principal agent currently in clinical use for direct treatment of HIV in patients with AIDS. Based on the determination of drug levels in the cerebrospinal fluid (CSF), this agent has been reported as being able to penetrate the BBB (Yarchoan et al., 1986; Klecker et al., 1987), and there is clinical evidence to indicate that AZT has some efficacy in the treatment of HIV infection of the CNS (Yarchoan et al., 1988). Because of the importance of intra-CNS HIV infection, and because of the significant incidence of dose-related side effects produced with currently used doses of AZT (Richman et al., 1987), much would be gained if it were possible to enhance the BBB transport of AZT, thereby permitting a decrease in the total dose of AZT administered.
With these considerations in mind, we have determined the brain-uptake index (BUI) of AZT in rats using the carotid artery injection technique of Oldendorf (1970). We also studied the effect that insulin might have on these values, as there is some anecdotal evidence suggesting that this hormone may increase the BUI of certain medications in humans (Ayre et al., 1986).
METHODOLOGY Research was conducted using male Sprague-Dawley white rats (6-8 weeks old, 200-220 gm each) housed in our facilities for at least three days before the experiments (ad libitum feeding with standard laboratory food). The animals were anesthetized (IM injection of a ketamine-xylazine/sterile water solution), the right common carotid artery was surgically exposed and, after ligating its external branch, the artery was stabilized on a thin strip of firm paper. The left external jugular vein was then exposed and each rat was injected (IV, 0.1 ml volume) with either normal saline (controls) or an insulin solution (Humulin-R Lilly; 0.1, 0.2, 0.3, 0.6 or 1.0 unit per animal). The rats were positioned under the guillotine, the carotid artery was cannulated and injected at the appropriate times (e.g., 3 or 10 min. after the saline or insulin injection) with a bolus (0.1 ml) containing 0.05 ml of a 8.8 x 10-8 M solution (phosphate buffer, pH 7.4) of antipyrine-N-methyl-14C (Sigma Co., spec. act. 41.4 mCi/mmol; approx. 4 x 105 dpm) and 0.05 ml of a 4.7 x 1O-9 M solution (phosphate buffer, pH 7.4) of 3H-AZT (Burroughs Wellcome, spec. act. 12.5 Ci/mmol; approx. 6.5 x 106 dpm). After 15 seconds the rats were decapitated and the whole brain was immediately removed and quickly washed in ice-cold saline. After patting dry over gauze, the brain was stripped of its superficial vessels, cut in half longitudinally, and the right half (ipsilateral to the carotid injection) was weighed and prepared for determination of its 14C and 3H content. The brain tissue was homogenized (1:2 v:v, phosphate buffer, pH 7.4) in a Dounce homogenizer. Double isotope analysis was performed in a Packard Tri-Carb model 4530 counter, using external standard and automatic efficiency correction. Duplicate aliquots of tissue samples and injected solutions were counted and BUI values were calculated (Fig. 1), the BUI being the ratio of the averages of 3H dpm/14C dpm in the tissue over the averages of 3H dpm/14C dpni in the injected solution (x 100).
RESULTS As can be seen from Fig. 1, when compared to antipyrine as the standard, AZT has a BUI of 5.4±0.8 (x±S.0.; 13 rats, range of 4.4 to 6.6). The time of the saline administration (3 or 10 min. before carotid injection) made no significant differences in the BUI averages or ranges (for 3 min., n=7; 5.5±0.7, range 4.4 to 6.5; and for 10 min., n=6; 5.6±0.7, range 4.4 to 6.6). Similar BUI values were obtained with pretreatment of the animals (3 or 10 min.) with the lower doses of insulin (0.1, 0.2 or 0.3 units per rat). While higher insulin doses (0.6 or 1.0 units acting for 3 min.) failed to produce a statistically significant difference between the experimental and saline treated groups, some animals did show BUI values above the control range (3 out of 4 rats for the 0.6 unit group, and 2 out of S for the 1.0 unit group). Whereas 0.6 units of insulin (10 min. prior to the carotid injection) produced similar results (3 out of 5 animals with BUI values above the control range), 1.0 unit per rat resulted not only in individual BUI values above the range of the control group (7 out of 7), but as a group the BUI was statistically significantly higher than the controls (7.8±1.3 versus 5.6±0.7. Newman-Keuls test, 99% confidence level p<0.01).
DISCUSSION HIV infection is accompanied in many cases by dementia and/or other neurologic symptomatology (Elder and Sever, 1988; Price et al., 1988), emphasizing the need for effective antiviral drugs able to penetrate the BBB in pharmacological concentrations. Despite the current use of AZT as an agent “at least temporarily effective in decreasing mortality and morbidity in some patients with AIDS or AIDS-related complex” (Medical Letter, 1986), there is no direct evidence for the presence of the drug in the brain after its oral or intravenous administration. Results from preliminary studies showing the presence of AZT in the CSF of patients suggests that the drug crosses the BBB (Yarchoan et al., 1986; Klecker et al., 1987), however one should keep in mind that all or substantial amounts of this agent could have reached the CSF via the blood-CSF barrier rather than by the BBB (Terasaki and Pardridge, 1988).
Using a slightly modified version of the Oldendorf technique (1970), we have shown that AZT crosses the BBB in rats with a BUI of 5.4±-0.8 (x±S.D.). This value was fairly similar for all of the animals studied (n=13; range 4.4 to 6.6). The time under anesthesia did not appear to significantly alter these values. We used 14C labeled antipyrine as the diffusible standard in this experiment which has a BUI of approximately 68% compared to tritiated water (Oldendorf, 1974). A recent concise communication, however, suggests that AZT crosses the BBB with great difficulty (Terasaki and Pardridge, 1988), underscoring the need for more work in this important area.
Inasmuch as the passage of chemicals through the BBB may occur via a variety of mechanisms (e.g., carrier-mediated penetration or facilitated diffusion), the BUI values for different drugs and/or nutrients may be significantly altered under certain conditions, including a variety of brain insults and drug treatments(Cornford and Oldendorf, 1980). Because of reports suggesting that insulin may enhance the passage of a number of chemicals through biological membranes (Alabaster et al., 1981; Schilsky et al., 1981; Poznansky et al., 1984; Yoshimasa et al., 1984; Gasparro et al., 1986), and of some anecdotal reports suggesting that the administration of this hormone to humans may result in an increased brain uptake of certain drugs (Ayre et al., 1986) we decided to study the possible effect of insulin on the BUI of AZT.
We now report that the administration of the lower insulin doses (at both times tested) failed to produce a significant change in the penetration of AZT into the brain, but that 0.6 and 1.0 unit of insulin per rat resulted in increased BUI values for AZT in some animals in the groups studied (3 and 10 minutes, and 3 minutes respectively). Pretreatment of the animals with 1.0 unit of insulin per rat during the 10 minute period prior to injecting the AZT resulted in statistically significant increases in the BUI values for all the rats tested (6 controls and 7 insulIn treated rats; Fig. 1).
It should be emphasized that these results are of a preliminary nature, and more work needs to be done before definitive conclusions may be drawn from these observations. While the insulin receptors on capillaries of the BBB described by Pardridge et al., (1985) may play some role, no attempts have been made thus far to elucidate the mechanism(s) for these observed insulin-associated increases in the BUI of AZT with this experimental model. The possibility that insulin can alter the rate of brain penetration of AZT, or other drugs, in humans should be considered. The potential benefits of such a therapeutic modality could prove valuable were the adverse effects of insulin safely controllable. One could speculate that AZT treatment, especially in AIDS patients with compromised neurologic function, could benefit from such an approach.
ACKNOWLEDGEMENTS We are grateful to the Burroughs Wellcome Company for the kind gift of tritiated AZT and valuable clinical information, and to Dr. Ann Snyder and Dr. Marion E. Wolf for helpful discussions.
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