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AZT distribution in the fetal and postnatal rat central nervous system
AZT Distribution in the Fetal and Postnatal Rat Central Nervous System YAMIT BUSIDAN, XIANDONG SHI, DIANA L. DOW-EDWARDS Laboratory of Cerebral Metabolism, Department of Physiology & Pharmacology, State University of New York, Health Science Center, Box 29, 450 Clarkson Avenue, Brooklyn, New York 11203 Received 21 July 2000; revised 22 January 2001; accepted 7 June 2001
ABSTRACT: The distribution of 30 -azido-30 -deoxythymidine (AZT, zidovudine), an antiviral drug used in the treatment of human immunode®ciency virus, was investigated in gestation day-20 (G-20) fetuses and in postnatal day-20 (PND-20) rats. At both ages, a single dose of 150 mg/kg (1.78 mmol/kg) AZT was administered orally along with tracer amounts of 14C-AZT, and rats were randomly killed at 15, 30, 60, 120, or 240 min after dosing. The fetuses, brains, and spinal cords were processed for autoradiography. The peak concentrations of AZT in plasma of G-20 and PND-20 rats were 92.2 mg/mL (0.345 mmol/mL) and 56.6 mg/mL (0.21 mmol/mL) at 15 and 30 min after intubation, respectively. The peak concentration of fetal tissue occurred in the colon at 60 min and was 205.8 mg/g tissue. In the G-20 rats, the brain showed higher levels of AZT than spinal cord only at the 30-min sample time, whereas in the PND-20 rats, greater radioactivity was found in the spinal cord up to the 240-min sample time. This pattern of AZT distribution in the central nervous system may hypothetically be attributed to the postnatal development of an organic anion carrier system believed to be responsible for transporting AZT from the brain to the blood, resulting in relatively greater overall exposure of the spinal cord to AZT than observed in the brain. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1964±1971, 2001
Keywords: antiretroviral drugs; CNS development; organic anion transporter; AZT pharmacokinetics; autoradiography
INTRODUCTION AZT has become the standard of care in the United States for reducing the maternal±fetal transmission of immunode®ciency virus type-1 (HIV-1).1±3 The long-term effects of transplacental 30 -azido-30 -deoxythymidine (AZT) exposure have been studied for tumorigenicity4±7 and behavioral effects,6,8±16 and the metabolism and distribution of AZT have been examined in adult rats, mice, rabbits, and nonhuman primates.17±29 Correspondence to: D.L. Dow-Edwards (Telephone: 718270-3987; Fax: 718-270-2241; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1964±1971 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
1964
AZT penetration into the central nervous system (CNS) is an essential requirement for its effectiveness in the treatment of acquired immunode®ciency syndrome (AIDS) dementia complex or AIDS encephalopathy. Although it is clear that AZT is effective in the treatment of AIDS encephalopathy, brain levels of the drug are lower than would be expected because of its transport out of the CNS by an active transport system.19,26 ±28,30,31 Several forms of this transport system, now named OAT, have been found with OAT3 showing substantial activity for AZT transport in brain.32 The expression of several OATs has been examined in the mouse embryo with OAT1 being expressed to a greater degree in fetal brain than in adult brain.33 However, heterologous transcripts of OAT1, isolated from
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embryonic kidney, are not functional.33 Because one cannot make inferences about the activity of other transporters such as OAT3 in fetal brain, it is useful to study the distribution/elimination of AZT from developing tissue. To address this question, we used gestation day-20 (G-20) rats to study the transplacental distribution of AZT and postnatal day-20 (PND-20) rats to study the distribution later in development. Because the state of maturation of the human brain at 19± 20 weeks' gestation is roughly equivalent with that of rat on the day of birth,34 the postnatal period in the rat approximates the later half of prenatal development in humans. Therefore, PND 20 in the rat approximates the peripartum period for man. The distribution of AZT was examined in various organs in the G-20 rats and in the plasma and CNS in the PND-20 rats 15, 30, 60, 120, and 240 min after dosing, to determine the exposure of the body and especially the CNS to the drug.
1965
After surgery, the rats were immediately returned to their cages and 3 h later, they were gastrically intubated with AZT at 150 mg/kg (1.78 mmol/kg) including 100 mCi 14C-AZT (2 mL of solution B and an adequate amount of solution A to equal 150 mg/kg AZT) using a 16-gauge straight-feeding needle. The dams were killed by intra-arterial sodium pentobarbital (1.5 mL of 50 mg/mL) at various times (15, 30, 60, 120, or 240 min). Arterial blood samples were collected for determination of plasma AZT levels before intubation and at 15, 30, 60, 90, 120, and 240 min after intubation, with the number of samples depending on the rat's assigned kill time. At kill time, the fetuses were counted and removed, and then rapidly frozen at ÿ708C. The most proximal fetus on the right was later sectioned at 20 mm in a cryostat. Autoradiographs were generated by apposition of the sections to Min-R ®lm (Kodak, Rochester, NY) in lead X-ray cassettes along with 14C-microscales (Amersham, Arlington Heights, IL).
METHODS PND-20 Rats
Drugs Solution A: AZT (30 azido-30 deoxy-thymidine; Sigma, St. Louis, MO) was dissolved in sterile water (Baxter, Deer®eld, IL) with sonication and some warming, and administered at 20 mg/mL. Solution B: AZT (30 azido-30 deoxy-thymidine14 2- C; speci®c activity: 55 mCi/mmol (Sigma) at 0.15 mCi/mL water/ethanol (98:2) was dried under a nitrogen stream and then reconstituted with solution A at 0.05 mCi/mL. G-20 Rat Fetuses SUNY Institutional Animal Care and Use Committee approved all procedures. Adult virgin female Sprague-Dawley rats (VAF strain; Charles River, Wilmington, ME) were mated with a male of the same strain in our AAALAC-accredited animal facility (20±228C with controlled humidity; 12-h light±dark cycle, lights turned on at 0700 hours). Detection of a sperm-positive smear on the following morning was referred to as G 1. The females were then weighed, housed individually, and left undisturbed until G 20 in 44 24 20 cm plastic cages containing wood chip bedding with ad libitum food and water. On G 20, polyethylene tubing was inserted into the femoral artery under light anesthesia using a mixture of halothane, nitrous oxide, and oxygen.
Adult virgin female Sprague-Dawley rats were mated as previously described and left undisturbed until day of birth. On the day of birth (PND 1), litters were culled to ten, maintaining equal sex representation, if possible. On PND 20, the pups were weighed and gastrically intubated with AZT at 150 mg/kg (with 20 mCi/per rat) using 200 PE 10 tubing. The rats were decapitated at 15, 30, 60, 120, or 240 min after intubation and trunk blood was collected for determination of plasma AZT levels. The brain and spinal cord were removed, frozen, and stored at ÿ708C until sectioning at 20 mm. Autoradiographs were generated by apposition of the sections to Min-R ®lm as described above. Concentration of AZT in Plasma All blood samples were collected in heparinized tubes (Beckman, Palo Alto, CA) and centrifuged for 2 min (Beckman Microfuge F). After centrifugation, 0.02 mL of plasma was withdrawn for the determination of radioactivity (counts per minute, cpm) of 14C using the Beckman LS 1800 Scintillation counter. Also, 0.01 mL of solution B was tested for cpm of 14C. The cpm in the plasma was converted to weight of AZT by the following formulas:
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Statistical Analyses
cpm/1 mg AZT cpm of solution B=0:01 mL 20 mg AZT=mL 1000 mg=mg
2. The concentration of AZT in plasma (mg/mL) was calculated by: cpm=mL plasma cpm=1 mg AZT
AZT Distribution in the CNS of G-20 and PND-20 Rats The distribution of AZT in the tissues of G-20 fetuses and the brain and spinal cord of G-20 fetuses and PND-20 rats was quanti®ed using computer-aided analysis of autoradiographs. Optical densities for different organs in these autoradiographs were determined with an image analyzer (MCID; Imaging Research Inc., Brock University, St. Catherine, Canada). Radioactivity levels in tissues (nCi/g) were computed from readings of optical densities using precalibrated standards. Values for radioactivity detected in each organ were the means of all sections (at least ®ve) analyzed. To calculate the AZT concentration, the nCi 14C/g tissue was then converted to mg/g tissue according to the amount of 14C-AZT that was given to each rat. The nCi/g tissue was converted to cpm/g tissue by multiplying 2220 dpm/nCi 0.9 (ef®ciency, cpm/dpm). The cpm/g tissue was divided by the cpm/1 mg AZT that was intubated to each rat as described above.
Fetal levels of AZT were analyzed using General Linear Models in SAS to examine the differences between maternal plasma and fetal levels in brain, liver, and spinal cord across sample times (a 4 region by 5 sample time analysis). Pairwise comparisons were made using tests of within subjects contrasts adjusted for multiple comparisons using the least signi®cant difference. Similarly, levels of AZT in plasma, brain, and spinal cord of pups were examined across sample times using General Linear Models in SAS (5 regions by 5 sample times) and then tests of within subjects contrasts used to determine whether plasma or high or low levels in brain and spinal cord differed signi®cantly at each sample time.
RESULTS Maternal Administration Maternal plasma and fetal tissue (administered at G 20) distribution are summarized in Table 1. The AZT was absorbed rapidly (tmax 15 min, 92.2 mg/mL maternal plasma) and the clearance half-life was approximately 62 min. To determine whether the exposure patterns in plasma, brain, liver, and spinal cord were similar, levels of AZT across time points were subjected to a two-way analysis (region sample time). Overall, the analysis indicated that there was a signi®cant region time interaction such that the differences between plasma and liver, brain, or spinal
Table 1. Distribution of 14C-AZT (mg AZT/g tissue or mL of plasma) in Rat Fetal Tissues After a Single Oral Intubation at 150 mg/kg on Gestation Day 20a
Maternal Plasmab Placenta Fetal Brainb Spinal cordb Liverb Heart Kidney Lung Intestine Colon
150 (2)
300 (3)
92.2 14.8 90.0 23.1
70.7 10.8 116.5 9.6
22.9 3.3 29.7 11.6 132.5 42.3 103.1 48.1 78.7 20.6 80.2 30.0 90.9 25.8 90.6 33.9
40.2 5.7 30.4 3.9 160.2 31.4 69.8 12.4 86.6 10.3 65.9 12.4 118.2 24.6 150.6 31.2
a
600 (3)
1200 (3)
2400 (4)
62.4 13.9 82.5 6.9
34.9 5.9 35.9 6.4
5.9 1.0 9.6 1.3
44.7 2.1 41.4 6.5 178.3 7.8 89.4 14.5 90.1 4.7 63.6 7.1 134.8 17.0 205.8 13.7
20.0 4.0 13.6 0.6 63.7 21.7 33.8 8.8 36.6 8.3 37.8 4.9 51.0 14.2 97.3 50.0
6.5 1.5 4.5 1.1 26.2 8.4 8.2 2.2 12.6 2.1 9.8 1.8 16.6 4.4 23.5 4.6
The data are means SEM for 2±4 rats/time point indicated in parentheses. Region selected for statistical analysis. See Results section for signi®cances.
b
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periventricular area contains relatively greater amounts of AZT. It is important to note that metabolites of AZT such as GAZT and AMT cannot be differentiated from AZT itself in the autoradiographs because the 14C label will remain in these metabolic products. Pup (PND 20) Administration
Figure 1. Illustration of the distribution of 14Clabeled AZT or metabolites in fetal tissues 15, 30, and 60 min after maternal administration. Representative sections through the cranium (upper) and trunk (lower) are shown. B indicates brain, SC indicates spinal cord, P indicates placenta, L indicates liver. The arrow indicates the periventricular area which contains relatively high levels of AZT compared with brain. Original magni®cation 1.2.
cord varied according to sample time. For example, maternal plasma versus liver generated an F(4,10) 4.6, p 0.023 for region time. Post hoc pairwise comparisons indicated that liver had signi®cantly more AZT than maternal plasma, fetal brain, and spinal cord at 30 and 60 min (Table 1). At other sample times, maternal plasma and fetal liver were not signi®cantly different. Fetal brain and spinal cord did not differ from each other except at minute 30, when the brain had slightly but signi®cantly higher levels (p 0.042) of AZT than the spinal cord. The distribution of labeled AZT at selected fetal levels is shown in Figure 1. Although the brain and spinal cord clearly contain less AZT than surrounding tissue at all sample times, the distribution of AZT in brain is heterogenous such that the
The AZT was absorbed rapidly (tmax 30 min, 56.6 mg/mL plasma) after gastric intubation in PND-20 rat pups. The clearance half-life of plasma AZT was approximately 48 min. The distribution of AZT in plasma, brain, and spinal cord is shown in Table 2. An initial analysis indicated that there were no gender differences in AZT distribution and subsequently the data for the genders were combined. Analyzed across all time points, within subjects comparisons indicate that all regions of brain and spinal cord differ signi®cantly from plasma [e.g., the area of spinal cord showing maximal AZT has signi®cantly less AZT than plasma; F (4,35) 15.1, p < 0.001], whereas pairwise comparisons indicate that all regions contain signi®cantly less AZT than plasma ( p values < 0.001) up to 120 min when the spinal cord areas showing the greatest concentrations of AZT were not signi®cantly different from plasma. At 15, 30, and 60 min, brain had signi®cantly less AZT than spinal cord ( p values < 0.001). For all sample times up to 240 min, the brain region showing the maximal concentration had signi®cantly less AZT than did the spinal cord region showing the maximal concentration (p values < 0.01). At 240 min, there were no differences in AZT levels for any of the regions analyzed. Figure 2 qualitatively illustrates the distribution of 14C-labeled AZT (and potential metabolites) in PND-20 brain and spinal cord for each sample time. Initially, AZT distributes to the periventricular areas of the brain. At 30 min, although the brain parenchyma
Table 2. Distribution of 14C-AZT (mg AZT/g tissue or mL of plasma) in PND-20 Rats' Plasma and CNS at Various Times After a Single Oral Intubation at 150 mg/kga
Plasma Brain Brain-max Spinal cord Spinal cord-max
150 (12)
300 (13)
600 (12)
1200 (10)
2400 (11)
42.0 4.4 3.8 0.4 10.4 0.9 9.0 1.2 18.6 2.7
56.6 2.8 9.8 0.6 18.0 1.0 17.8 1.5 28.6 2.4
48.3 2.0 9.0 0.7 14.8 1.2 13.6 1.2 22.4 2.4
15.3 3.2 4.1 0.5 7.3 0.7 5.6 0.9 12.8 3.0
6.1 1.2 1.7 0.3 3.3 0.7 2.4 0.5 4.0 0.7
a
The data are means SEM for the numbers of rats shown in parentheses. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 12, DECEMBER 2001
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BUSIDAN, SHI, AND DOW-EDWARDS
Figure 2. Illustration of the concentrations of 14Clabeled AZT in PND-20 brain and spinal cord for each sample time. The periventricular area in brain contains maximal AZT levels at all time points except 2400 when the compound is only seen in what appears to be the subarachnoid space surrounding the brain. Spinal cord (right side of ®gure) also shows AZT in the subarachnoid space except at 300 when the gray matter contains relatively greater amounts of the compound. Original magni®cation: brain 1.5; spinal cord 3.0.
appears to contain a large amount of AZT, areas of the spinal cord contain signi®cantly greater amounts of AZT than the highest concentration in brain ( p < 0.001). This pattern continues until 240 min when there are no signi®cant differences between any of the regions analyzed, including the plasma.
DISCUSSION The distribution of AZT was examined after a single oral dose in G-20 and PND-20 rats. The results indicate that in the G-20 rat fetus, AZT distributed almost equally between brain and spinal cord, but in the pups (PND 20), the distributions of AZT in brain and spinal cord were signi®cantly different from each other, with the spinal cord containing greater concentrations,
and the magnitude of the difference depending on the sample time. Our ®ndings of AZT distribution during gestation agreed with other studies that showed that maternal blood levels were lower than fetal liver within a few hours after maternal dosing and that AZT levels were lowest in the brain.18,22,24 The relatively great exposure of liver, intestine, and colon to AZT undoubtedly relates to the metabolism and excretion of AZT which occurs in these tissues in the adult rodent and in the primate fetus.18,24 In addition, the relatively great exposure of the liver to AZT may relate to the hepatotoxicity previously observed.35±37 It should be noted that autoradiographic images such as Figure 1 do not discriminate between various metabolic forms of AZT such as GAZT or AMT and that particularly in liver and gastrointestinal tract, where rapid metabolism and excretion of AZT occurs, a signi®cant contribution of the metabolites to the quanti®ed AZT content is expected. Several studies have shown that there is an organic anion transporter responsible for limiting the brain's exposure to AZT; the transporter is inhibited by probenecid and is located in the choroid plexus and brain microvasculature.19,26 ±28, 30,31 This carrier system transports AZT across the blood-brain barrier and the bloodcerebrospinal ¯uid (CSF) barrier with a predominant ef¯ux from brain to blood. Because our study showed that there is less exposure of the brain in the postnatal rat than at G 20, we hypothesize that in the G-20 fetuses, the transporter has either not developed or is not yet ef®cient at removing AZT from the brain, resulting in essentially equal distribution between the brain and spinal cord. The organic anion transporter (OAT3) has recently been identi®ed in rodent brain and is active in transporting AZT from the brain to the blood in the adult.32 However, the ontogeny of OAT3 has not been studied. Heterologous transcripts of OAT1, a transporter similar to OAT3 and isolated from the fetal kidney, have been found to be inactive for anion transport.33 Therefore, it is possible that even if OAT3 transcripts were identi®ed in fetal brain, the resulting proteins may not possess their adult-like transporter functions. Studies in adult mice using radiolabeled AZT found that both brain and spinal cord contained no radioactivity after the administration of 273 mCi/kg AZT intravenously.17 Perhaps the organic anion carrier matures later in spinal cord than in brain so that in the adult, all CNS tissues have low AZT concentrations.
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The relatively greater exposure of the spinal cord to AZT compared with brain in the postnatal period is consistent with other data. A study conducted by Blaney et al.38 supports our ®ndings, because they found that CSF/plasma ratio of lamivudine, a cytidine analog, was signi®cantly higher in lumbar CSF than ventricular CSF, suggesting that the transport mechanism in spinal cord of the adult is not as effective as in brain. The relatively greater exposure of the spinal cord is consistent with behavioral data in rats treated with AZT throughout development showing an altered acoustic startle re¯ex, a re¯ex which involves brain stem and spinal cord circuits.6 Hypothetically, AZT induces functional alterations in the circuits that mediate the acoustic startle re¯ex, perhaps through effects on mitochondrial oxidation-phosphorylation.36,37 Because our results at PND 20 suggest that during the third trimester in humans, the spinal cord is exposed to a relatively greater concentration of AZT than brain, children exposed to AZT prenatally should be examined for alterations in spinal-cord-mediated functions such as re¯exes.
CONCLUSIONS
2.
3. 4.
5.
6.
7.
AZT rapidly distributes to fetal tissues after maternal administration, with brain and spinal cord levels being signi®cantly lower than fetal liver and maternal plasma at most times examined. Later in development, exposure of spinal cord is relatively greater than brain although quanti®able AZT or metabolites are found in both CNS tissues at all time points studied.
8.
9.
ACKNOWLEDGMENTS These studies were supported by Grant HD 35035 from the NICHD, NIH. The technical assistance of April Jackson and Mariya Kreymerman is gratefully acknowledged.
10.
11.
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38. Blaney SM, Daniel MJ, Harker AJ, Godwin K, Balis FM. 1995. Pharmacokinetics of lamivudine and BCH-189 in plasma and cerebrospinal ¯uid of nonhuman primates. Antimicrob Agents Chemother 39:2779±2782.
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ABSTRACT: The distribution of 30 -azido-30 -deoxythymidine (AZT, zidovudine), an antiviral drug used in the treatment of human immunode®ciency virus, was investigated in gestation day-20 (G-20) fetuses and in postnatal day-20 (PND-20) rats. At both ages, a single dose of 150 mg/kg (1.78 mmol/kg) AZT was administered orally along with tracer amounts of 14C-AZT, and rats were randomly killed at 15, 30, 60, 120, or 240 min after dosing. The fetuses, brains, and spinal cords were processed for autoradiography. The peak concentrations of AZT in plasma of G-20 and PND-20 rats were 92.2 mg/mL (0.345 mmol/mL) and 56.6 mg/mL (0.21 mmol/mL) at 15 and 30 min after intubation, respectively. The peak concentration of fetal tissue occurred in the colon at 60 min and was 205.8 mg/g tissue. In the G-20 rats, the brain showed higher levels of AZT than spinal cord only at the 30-min sample time, whereas in the PND-20 rats, greater radioactivity was found in the spinal cord up to the 240-min sample time. This pattern of AZT distribution in the central nervous system may hypothetically be attributed to the postnatal development of an organic anion carrier system believed to be responsible for transporting AZT from the brain to the blood, resulting in relatively greater overall exposure of the spinal cord to AZT than observed in the brain. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1964±1971, 2001
Keywords: antiretroviral drugs; CNS development; organic anion transporter; AZT pharmacokinetics; autoradiography
INTRODUCTION AZT has become the standard of care in the United States for reducing the maternal±fetal transmission of immunode®ciency virus type-1 (HIV-1).1±3 The long-term effects of transplacental 30 -azido-30 -deoxythymidine (AZT) exposure have been studied for tumorigenicity4±7 and behavioral effects,6,8±16 and the metabolism and distribution of AZT have been examined in adult rats, mice, rabbits, and nonhuman primates.17±29 Correspondence to: D.L. Dow-Edwards (Telephone: 718270-3987; Fax: 718-270-2241; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1964±1971 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
1964
AZT penetration into the central nervous system (CNS) is an essential requirement for its effectiveness in the treatment of acquired immunode®ciency syndrome (AIDS) dementia complex or AIDS encephalopathy. Although it is clear that AZT is effective in the treatment of AIDS encephalopathy, brain levels of the drug are lower than would be expected because of its transport out of the CNS by an active transport system.19,26 ±28,30,31 Several forms of this transport system, now named OAT, have been found with OAT3 showing substantial activity for AZT transport in brain.32 The expression of several OATs has been examined in the mouse embryo with OAT1 being expressed to a greater degree in fetal brain than in adult brain.33 However, heterologous transcripts of OAT1, isolated from
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embryonic kidney, are not functional.33 Because one cannot make inferences about the activity of other transporters such as OAT3 in fetal brain, it is useful to study the distribution/elimination of AZT from developing tissue. To address this question, we used gestation day-20 (G-20) rats to study the transplacental distribution of AZT and postnatal day-20 (PND-20) rats to study the distribution later in development. Because the state of maturation of the human brain at 19± 20 weeks' gestation is roughly equivalent with that of rat on the day of birth,34 the postnatal period in the rat approximates the later half of prenatal development in humans. Therefore, PND 20 in the rat approximates the peripartum period for man. The distribution of AZT was examined in various organs in the G-20 rats and in the plasma and CNS in the PND-20 rats 15, 30, 60, 120, and 240 min after dosing, to determine the exposure of the body and especially the CNS to the drug.
1965
After surgery, the rats were immediately returned to their cages and 3 h later, they were gastrically intubated with AZT at 150 mg/kg (1.78 mmol/kg) including 100 mCi 14C-AZT (2 mL of solution B and an adequate amount of solution A to equal 150 mg/kg AZT) using a 16-gauge straight-feeding needle. The dams were killed by intra-arterial sodium pentobarbital (1.5 mL of 50 mg/mL) at various times (15, 30, 60, 120, or 240 min). Arterial blood samples were collected for determination of plasma AZT levels before intubation and at 15, 30, 60, 90, 120, and 240 min after intubation, with the number of samples depending on the rat's assigned kill time. At kill time, the fetuses were counted and removed, and then rapidly frozen at ÿ708C. The most proximal fetus on the right was later sectioned at 20 mm in a cryostat. Autoradiographs were generated by apposition of the sections to Min-R ®lm (Kodak, Rochester, NY) in lead X-ray cassettes along with 14C-microscales (Amersham, Arlington Heights, IL).
METHODS PND-20 Rats
Drugs Solution A: AZT (30 azido-30 deoxy-thymidine; Sigma, St. Louis, MO) was dissolved in sterile water (Baxter, Deer®eld, IL) with sonication and some warming, and administered at 20 mg/mL. Solution B: AZT (30 azido-30 deoxy-thymidine14 2- C; speci®c activity: 55 mCi/mmol (Sigma) at 0.15 mCi/mL water/ethanol (98:2) was dried under a nitrogen stream and then reconstituted with solution A at 0.05 mCi/mL. G-20 Rat Fetuses SUNY Institutional Animal Care and Use Committee approved all procedures. Adult virgin female Sprague-Dawley rats (VAF strain; Charles River, Wilmington, ME) were mated with a male of the same strain in our AAALAC-accredited animal facility (20±228C with controlled humidity; 12-h light±dark cycle, lights turned on at 0700 hours). Detection of a sperm-positive smear on the following morning was referred to as G 1. The females were then weighed, housed individually, and left undisturbed until G 20 in 44 24 20 cm plastic cages containing wood chip bedding with ad libitum food and water. On G 20, polyethylene tubing was inserted into the femoral artery under light anesthesia using a mixture of halothane, nitrous oxide, and oxygen.
Adult virgin female Sprague-Dawley rats were mated as previously described and left undisturbed until day of birth. On the day of birth (PND 1), litters were culled to ten, maintaining equal sex representation, if possible. On PND 20, the pups were weighed and gastrically intubated with AZT at 150 mg/kg (with 20 mCi/per rat) using 200 PE 10 tubing. The rats were decapitated at 15, 30, 60, 120, or 240 min after intubation and trunk blood was collected for determination of plasma AZT levels. The brain and spinal cord were removed, frozen, and stored at ÿ708C until sectioning at 20 mm. Autoradiographs were generated by apposition of the sections to Min-R ®lm as described above. Concentration of AZT in Plasma All blood samples were collected in heparinized tubes (Beckman, Palo Alto, CA) and centrifuged for 2 min (Beckman Microfuge F). After centrifugation, 0.02 mL of plasma was withdrawn for the determination of radioactivity (counts per minute, cpm) of 14C using the Beckman LS 1800 Scintillation counter. Also, 0.01 mL of solution B was tested for cpm of 14C. The cpm in the plasma was converted to weight of AZT by the following formulas:
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BUSIDAN, SHI, AND DOW-EDWARDS
Statistical Analyses
cpm/1 mg AZT cpm of solution B=0:01 mL 20 mg AZT=mL 1000 mg=mg
2. The concentration of AZT in plasma (mg/mL) was calculated by: cpm=mL plasma cpm=1 mg AZT
AZT Distribution in the CNS of G-20 and PND-20 Rats The distribution of AZT in the tissues of G-20 fetuses and the brain and spinal cord of G-20 fetuses and PND-20 rats was quanti®ed using computer-aided analysis of autoradiographs. Optical densities for different organs in these autoradiographs were determined with an image analyzer (MCID; Imaging Research Inc., Brock University, St. Catherine, Canada). Radioactivity levels in tissues (nCi/g) were computed from readings of optical densities using precalibrated standards. Values for radioactivity detected in each organ were the means of all sections (at least ®ve) analyzed. To calculate the AZT concentration, the nCi 14C/g tissue was then converted to mg/g tissue according to the amount of 14C-AZT that was given to each rat. The nCi/g tissue was converted to cpm/g tissue by multiplying 2220 dpm/nCi 0.9 (ef®ciency, cpm/dpm). The cpm/g tissue was divided by the cpm/1 mg AZT that was intubated to each rat as described above.
Fetal levels of AZT were analyzed using General Linear Models in SAS to examine the differences between maternal plasma and fetal levels in brain, liver, and spinal cord across sample times (a 4 region by 5 sample time analysis). Pairwise comparisons were made using tests of within subjects contrasts adjusted for multiple comparisons using the least signi®cant difference. Similarly, levels of AZT in plasma, brain, and spinal cord of pups were examined across sample times using General Linear Models in SAS (5 regions by 5 sample times) and then tests of within subjects contrasts used to determine whether plasma or high or low levels in brain and spinal cord differed signi®cantly at each sample time.
RESULTS Maternal Administration Maternal plasma and fetal tissue (administered at G 20) distribution are summarized in Table 1. The AZT was absorbed rapidly (tmax 15 min, 92.2 mg/mL maternal plasma) and the clearance half-life was approximately 62 min. To determine whether the exposure patterns in plasma, brain, liver, and spinal cord were similar, levels of AZT across time points were subjected to a two-way analysis (region sample time). Overall, the analysis indicated that there was a signi®cant region time interaction such that the differences between plasma and liver, brain, or spinal
Table 1. Distribution of 14C-AZT (mg AZT/g tissue or mL of plasma) in Rat Fetal Tissues After a Single Oral Intubation at 150 mg/kg on Gestation Day 20a
Maternal Plasmab Placenta Fetal Brainb Spinal cordb Liverb Heart Kidney Lung Intestine Colon
150 (2)
300 (3)
92.2 14.8 90.0 23.1
70.7 10.8 116.5 9.6
22.9 3.3 29.7 11.6 132.5 42.3 103.1 48.1 78.7 20.6 80.2 30.0 90.9 25.8 90.6 33.9
40.2 5.7 30.4 3.9 160.2 31.4 69.8 12.4 86.6 10.3 65.9 12.4 118.2 24.6 150.6 31.2
a
600 (3)
1200 (3)
2400 (4)
62.4 13.9 82.5 6.9
34.9 5.9 35.9 6.4
5.9 1.0 9.6 1.3
44.7 2.1 41.4 6.5 178.3 7.8 89.4 14.5 90.1 4.7 63.6 7.1 134.8 17.0 205.8 13.7
20.0 4.0 13.6 0.6 63.7 21.7 33.8 8.8 36.6 8.3 37.8 4.9 51.0 14.2 97.3 50.0
6.5 1.5 4.5 1.1 26.2 8.4 8.2 2.2 12.6 2.1 9.8 1.8 16.6 4.4 23.5 4.6
The data are means SEM for 2±4 rats/time point indicated in parentheses. Region selected for statistical analysis. See Results section for signi®cances.
b
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1967
periventricular area contains relatively greater amounts of AZT. It is important to note that metabolites of AZT such as GAZT and AMT cannot be differentiated from AZT itself in the autoradiographs because the 14C label will remain in these metabolic products. Pup (PND 20) Administration
Figure 1. Illustration of the distribution of 14Clabeled AZT or metabolites in fetal tissues 15, 30, and 60 min after maternal administration. Representative sections through the cranium (upper) and trunk (lower) are shown. B indicates brain, SC indicates spinal cord, P indicates placenta, L indicates liver. The arrow indicates the periventricular area which contains relatively high levels of AZT compared with brain. Original magni®cation 1.2.
cord varied according to sample time. For example, maternal plasma versus liver generated an F(4,10) 4.6, p 0.023 for region time. Post hoc pairwise comparisons indicated that liver had signi®cantly more AZT than maternal plasma, fetal brain, and spinal cord at 30 and 60 min (Table 1). At other sample times, maternal plasma and fetal liver were not signi®cantly different. Fetal brain and spinal cord did not differ from each other except at minute 30, when the brain had slightly but signi®cantly higher levels (p 0.042) of AZT than the spinal cord. The distribution of labeled AZT at selected fetal levels is shown in Figure 1. Although the brain and spinal cord clearly contain less AZT than surrounding tissue at all sample times, the distribution of AZT in brain is heterogenous such that the
The AZT was absorbed rapidly (tmax 30 min, 56.6 mg/mL plasma) after gastric intubation in PND-20 rat pups. The clearance half-life of plasma AZT was approximately 48 min. The distribution of AZT in plasma, brain, and spinal cord is shown in Table 2. An initial analysis indicated that there were no gender differences in AZT distribution and subsequently the data for the genders were combined. Analyzed across all time points, within subjects comparisons indicate that all regions of brain and spinal cord differ signi®cantly from plasma [e.g., the area of spinal cord showing maximal AZT has signi®cantly less AZT than plasma; F (4,35) 15.1, p < 0.001], whereas pairwise comparisons indicate that all regions contain signi®cantly less AZT than plasma ( p values < 0.001) up to 120 min when the spinal cord areas showing the greatest concentrations of AZT were not signi®cantly different from plasma. At 15, 30, and 60 min, brain had signi®cantly less AZT than spinal cord ( p values < 0.001). For all sample times up to 240 min, the brain region showing the maximal concentration had signi®cantly less AZT than did the spinal cord region showing the maximal concentration (p values < 0.01). At 240 min, there were no differences in AZT levels for any of the regions analyzed. Figure 2 qualitatively illustrates the distribution of 14C-labeled AZT (and potential metabolites) in PND-20 brain and spinal cord for each sample time. Initially, AZT distributes to the periventricular areas of the brain. At 30 min, although the brain parenchyma
Table 2. Distribution of 14C-AZT (mg AZT/g tissue or mL of plasma) in PND-20 Rats' Plasma and CNS at Various Times After a Single Oral Intubation at 150 mg/kga
Plasma Brain Brain-max Spinal cord Spinal cord-max
150 (12)
300 (13)
600 (12)
1200 (10)
2400 (11)
42.0 4.4 3.8 0.4 10.4 0.9 9.0 1.2 18.6 2.7
56.6 2.8 9.8 0.6 18.0 1.0 17.8 1.5 28.6 2.4
48.3 2.0 9.0 0.7 14.8 1.2 13.6 1.2 22.4 2.4
15.3 3.2 4.1 0.5 7.3 0.7 5.6 0.9 12.8 3.0
6.1 1.2 1.7 0.3 3.3 0.7 2.4 0.5 4.0 0.7
a
The data are means SEM for the numbers of rats shown in parentheses. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 12, DECEMBER 2001
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BUSIDAN, SHI, AND DOW-EDWARDS
Figure 2. Illustration of the concentrations of 14Clabeled AZT in PND-20 brain and spinal cord for each sample time. The periventricular area in brain contains maximal AZT levels at all time points except 2400 when the compound is only seen in what appears to be the subarachnoid space surrounding the brain. Spinal cord (right side of ®gure) also shows AZT in the subarachnoid space except at 300 when the gray matter contains relatively greater amounts of the compound. Original magni®cation: brain 1.5; spinal cord 3.0.
appears to contain a large amount of AZT, areas of the spinal cord contain signi®cantly greater amounts of AZT than the highest concentration in brain ( p < 0.001). This pattern continues until 240 min when there are no signi®cant differences between any of the regions analyzed, including the plasma.
DISCUSSION The distribution of AZT was examined after a single oral dose in G-20 and PND-20 rats. The results indicate that in the G-20 rat fetus, AZT distributed almost equally between brain and spinal cord, but in the pups (PND 20), the distributions of AZT in brain and spinal cord were signi®cantly different from each other, with the spinal cord containing greater concentrations,
and the magnitude of the difference depending on the sample time. Our ®ndings of AZT distribution during gestation agreed with other studies that showed that maternal blood levels were lower than fetal liver within a few hours after maternal dosing and that AZT levels were lowest in the brain.18,22,24 The relatively great exposure of liver, intestine, and colon to AZT undoubtedly relates to the metabolism and excretion of AZT which occurs in these tissues in the adult rodent and in the primate fetus.18,24 In addition, the relatively great exposure of the liver to AZT may relate to the hepatotoxicity previously observed.35±37 It should be noted that autoradiographic images such as Figure 1 do not discriminate between various metabolic forms of AZT such as GAZT or AMT and that particularly in liver and gastrointestinal tract, where rapid metabolism and excretion of AZT occurs, a signi®cant contribution of the metabolites to the quanti®ed AZT content is expected. Several studies have shown that there is an organic anion transporter responsible for limiting the brain's exposure to AZT; the transporter is inhibited by probenecid and is located in the choroid plexus and brain microvasculature.19,26 ±28, 30,31 This carrier system transports AZT across the blood-brain barrier and the bloodcerebrospinal ¯uid (CSF) barrier with a predominant ef¯ux from brain to blood. Because our study showed that there is less exposure of the brain in the postnatal rat than at G 20, we hypothesize that in the G-20 fetuses, the transporter has either not developed or is not yet ef®cient at removing AZT from the brain, resulting in essentially equal distribution between the brain and spinal cord. The organic anion transporter (OAT3) has recently been identi®ed in rodent brain and is active in transporting AZT from the brain to the blood in the adult.32 However, the ontogeny of OAT3 has not been studied. Heterologous transcripts of OAT1, a transporter similar to OAT3 and isolated from the fetal kidney, have been found to be inactive for anion transport.33 Therefore, it is possible that even if OAT3 transcripts were identi®ed in fetal brain, the resulting proteins may not possess their adult-like transporter functions. Studies in adult mice using radiolabeled AZT found that both brain and spinal cord contained no radioactivity after the administration of 273 mCi/kg AZT intravenously.17 Perhaps the organic anion carrier matures later in spinal cord than in brain so that in the adult, all CNS tissues have low AZT concentrations.
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The relatively greater exposure of the spinal cord to AZT compared with brain in the postnatal period is consistent with other data. A study conducted by Blaney et al.38 supports our ®ndings, because they found that CSF/plasma ratio of lamivudine, a cytidine analog, was signi®cantly higher in lumbar CSF than ventricular CSF, suggesting that the transport mechanism in spinal cord of the adult is not as effective as in brain. The relatively greater exposure of the spinal cord is consistent with behavioral data in rats treated with AZT throughout development showing an altered acoustic startle re¯ex, a re¯ex which involves brain stem and spinal cord circuits.6 Hypothetically, AZT induces functional alterations in the circuits that mediate the acoustic startle re¯ex, perhaps through effects on mitochondrial oxidation-phosphorylation.36,37 Because our results at PND 20 suggest that during the third trimester in humans, the spinal cord is exposed to a relatively greater concentration of AZT than brain, children exposed to AZT prenatally should be examined for alterations in spinal-cord-mediated functions such as re¯exes.
CONCLUSIONS
2.
3. 4.
5.
6.
7.
AZT rapidly distributes to fetal tissues after maternal administration, with brain and spinal cord levels being signi®cantly lower than fetal liver and maternal plasma at most times examined. Later in development, exposure of spinal cord is relatively greater than brain although quanti®able AZT or metabolites are found in both CNS tissues at all time points studied.
8.
9.
ACKNOWLEDGMENTS These studies were supported by Grant HD 35035 from the NICHD, NIH. The technical assistance of April Jackson and Mariya Kreymerman is gratefully acknowledged.
10.
11.
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myopathy, lactic acidosis, and mitochondrial DNA depletion. J Hepatol 30:156±160. 37. de la Asuncion JG, del Olmo ML, Sastre J, Pallardo FV, Vina J. 1999. Zidovudine (AZT) causes an oxidation of mitochondrial DNA in mouse liver. Hepatology 29:985±987.
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38. Blaney SM, Daniel MJ, Harker AJ, Godwin K, Balis FM. 1995. Pharmacokinetics of lamivudine and BCH-189 in plasma and cerebrospinal ¯uid of nonhuman primates. Antimicrob Agents Chemother 39:2779±2782.
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