Introduction

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MAbs¹ directed against cell surface receptors (e.g. the transferrin receptor or the insulin receptor) on the brain capillary endothelium, which makes up the BBB in vivo, are potential brain drug-delivery vectors (Pardridge, 1997). The OX26 murine MAb to the rat transferrin receptor has been used to deliver peptide or antisense pharmaceutical agents, as well as polyethyleneglycol-conjugated liposomes, to the brain (Pardridge, 1997). The effects of repetitive administration of cell surface receptor-specific MAbs, such as the OX26 MAb, on the potential up-regulation or down-regulation of BBB transferrin receptors have not been investigated. Previous studies reported that transferrin receptors on cells in tissue culture are subject to up- or down-regulation by exogenous factors, such as the administration of lipopolysaccharide or interferon- (Lu et al., 1995) or chronic exposure to a cell surface-specific MAb. The T lymphoma cell line HUT78 up-regulates cell surface transferrin receptors when exposed to the J64 MAb (Keyna et al., 1994). Conversely, the lymphoma cell line AKR1 down-regulates cell surface transferrin receptors by as much as 10-fold when cells are exposed to the RI7-208 MAb (Lesley and Schulte, 1985). The down-regulation of cell surface transferrin receptors by receptor-specific MAbs is consistent with the inhibitory effect on cell proliferation of several transferrin receptor-specific MAbs (White et al., 1990). These observations raise the question of whether chronic administration of the OX26 MAb to rats would result in down-regulation of BBB transferrin receptors in vivo. Therefore, in the present studies a therapeutic concentration of the OX26 MAb (0.25 mg/kg sc daily for 1 week) was administered, followed by measurement of BBB transport of ¹²⁵I-labeled OX26 MAb. The dose of 0.25 mg/kg was chosen because previous studies (Kang et al., 1994) demonstrated that this concentration of OX26 results in partial saturation of the BBB transferrin receptors in vivo in rats.

	Materials and Methods

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Materials. Male Sprague-Dawley rats (190-220 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Na¹²⁵I (specific activity, 2050 Ci/mmol) was from Amersham Co. (Arlington Heights, IL). mIgG2a was purchased from Organon Teknika Corp. (West Chester, PA). The OX26 MAb was prepared from hybridoma cell-conditioned media by protein G affinity chromatography, as reported previously (Kang and Pardridge, 1994). All other chemicals were of analytical grade and were obtained from Fisher Scientific (Tustin, CA).

Chronic Treatment of Rats. Fed rats were randomly assigned into three groups. The first group received sc injections of saline (100 µl/rat), as controls. The second group received 0.25 mg/kg mIgG2a (dissolved in 100 µl of saline) by sc injection, whereas the third group received a sc dose of 0.25 mg/kg OX26. The animals were treated once each day for 5 consecutive days and, after a 1-day break, the last dose was given on day 7. Body weight was measured before each sc injection. After the injections, the animals had free access to water and rat chow.

Iodination of OX26. OX26 MAb (0.33 nmol) was dissolved in 20 µl of 0.2 M Na₂HPO₄ buffer (pH 7.4) and mixed with 1 mCi of Na¹²⁵I (0.53 nmol). The reaction was initiated by the addition of 8.4 nmol of chloramine-T, proceeded at room temperature for 2 min, and was stopped by the addition of 12.5 nmol of sodium metabisulfite. The iodinated OX26 was eluted from a 0.7- × 28-cm Sephadex G-25 column with 12 ml of 1 mM phosphate-buffered sodium chloride containing 0.1% bovine serum albumin (pH 7.4). The specific activity of ¹²⁵I-OX26 was 2.8 µCi/µg, and TCA-precipitability was >98%.

Intravenous Administration of ¹²⁵I-OX26. After an overnight fast, the rats were anesthetized with 100 mg/kg ketamine and 2 mg/kg xylazine ip on day 8. The left femoral vein was cannulated with a PE50 cannula, and 0.2 ml of Ringer/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid solution (pH 7.4) containing 5 µCi of ¹²⁵I-OX26 was injected. Blood samples (0.25 ml) were collected, via a heparinized PE50 cannula implanted in the left femoral artery, at 0.25, 0.5, 1, 2, 5, 10, 30, and 60 min after the injection. The blood volume was replaced with an equal volume of saline. All animals were sacrificed at 60 min after iv injection, for removal of the brain and liver. The plasma and organ samples were counted for ¹²⁵I using a Beckman -counter. Aliquots of the plasma samples were precipitated with TCA for examination of the metabolic stability of the labeled OX26. This research was conducted in compliance with the Principles of Laboratory Animal Care (National Institutes of Health, 1985).

Pharmacokinetic Analysis. Pharmacokinetic parameters were calculated by fitting the plasma TCA-precipitable radioactivity data to a biexponential equation, where A(t) = % ID/ml of plasma. The biexponential equation was fit to plasma data using a derivative-free, nonlinear regression analysis (PARBMDP, Biomedical Computer P-Series, developed at the University of California, Los Angeles, Health Sciences Computing Facilities). The data were weighted using the equation weight = 1/(concentration)², where concentration = % ID/ml of plasma. The organ volume of distribution of ¹²⁵I-OX26 at 60 min after iv injection was determined from the dpm per gram of tissue/dpm per microliter of terminal plasma ratio. The pharmacokinetic parameters of plasma clearance, central compartment volume, steady-state volume of distribution, AUC_0-, and mean residence time were determined from A₁, A₂, K₁, and K₂, as described previously (Kang and Pardridge, 1994). The organ clearance or PS product was determined as follows: where A(T) is the terminal plasma concentration, V_d is the organ volume of distribution, and V_O is the plasma volume for the respective organ. Previous studies showed that V_O = 11 ± 1 and 87 ± 4 µl/g for brain and liver, respectively, for a mIgG2a isotype control antibody (Pardridge et al., 1991). The organ uptake, expressed as percentage of ID per gram of organ, was calculated from the following equation: where the units of PS are microliters per minute per gram and the units of AUC are percentage of ID per minute per milliliter. Previous studies showed that brain and liver uptake of the OX26 MAb is linear for 6 and 1 hr, respectively (Pardridge et al., 1991).

	Results and Discussion

Top Abstract Introduction Materials & Methods Results & Discussion References

The animals demonstrated normal activity and weight gain in all three groups, and there were no significant differences in the body weights. The metabolic stability of ¹²⁵I-labeled OX26 was high during the 60 min of observation, inasmuch as TCA precipitation was 98% for samples at time 0 and was 96.8 ± 0.1, 96.5 ± 0.5, and 96.3 ± 0.2% for the terminal plasma samples (obtained at 60 min) from rats treated with saline, mIgG2a, and OX26, respectively. The rates of clearance of ¹²⁵I-OX26 from the plasma compartment in vivo in the three groups of rats are shown in fig. 1; these data were subjected to pharmacokinetic analysis, to yield the parameters listed in table 1. The data show that, although there was no significant change in the plasma clearance of ¹²⁵I-OX26 in the rats treated with the mIgG2a isotype control, compared with the animals treated with saline, there was a 45% increase in the plasma clearance of ¹²⁵I-OX26 in the animals treated chronically with OX26 (table 1). Parallel reductions in the plasma AUC values and mean residence times were also observed in the OX26-treated animals (table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Plasma profiles of 125I-OX26 in three groups of rats after pretreatment with daily doses of either saline, mIgG2a, or OX26 for 1 week. Points, mean ± SE of three rats in each group.

The availability of the plasma AUC data, in conjunction with the brain or liver volume of distribution values, allowed for computation of the organ clearance or PS products for brain and liver, as well as the percentage of ID per gram for these two organs (table 2). These data show there was no significant change in the hepatic PS product for ¹²⁵I-OX26 in the animals treated with the mIgG2a isotype control, compared with the saline-treated animals, but there was a 41% increase in the hepatic PS product for ¹²⁵I-OX26 in the OX26-treated animals (table 2). Although there was no change in the hepatic PS product for ¹²⁵I-OX26 in the mIgG2a isotype control-treated animals, there was a 27% increase in the percentage of ID per gram for liver uptake of ¹²⁵I-OX26 in the mIgG2a isotype control-treated animals (table 2). This is attributed to a somewhat elevated plasma AUC value observed at 60 min in these animals (table 1), although the AUC_0- values were not significantly different between the saline- and mIgG2a-treated animals (table 1).

The data reported in table 2 provide evidence for modest up-regulation of transferrin receptors and OX26 MAb clearance sites on the cell surface of rat liver cells in vivo. However, there was no significant change in the BBB PS product for ¹²⁵I-OX26 in the mIgG2a isotype control-treated animals or in the animals treated with OX26 (table 2). Similarly, there was no significant change in the percentage of ID per gram for ¹²⁵I-OX26 uptake in brain in any of the three groups of animals. The downward trend in the brain percentage of ID per gram for ¹²⁵I-OX26 in the OX26-treated animals (table 2) is attributed to the decreased plasma AUC values (caused by the increased hepatic clearance) in these animals (table 1).

The data in table 2 show that the PS product for OX26 transport into liver is approximately 4-fold greater than the PS product for OX26 transport across the BBB. This suggests that the cell densities of transferrin receptors in the two tissues are comparable. The density of transferrin receptors in rat liver cells is 129,000 receptors/cell (Rudolph et al., 1988). The surface density of transferrin receptors has been measured for isolated human brain capillaries and is 0.1 pmol/mg of protein (Pardridge et al., 1987). Assuming 1 mg of protein/10⁶ cells, this is equivalent to 60,000 receptors/cell, or approximately one half the value estimated for rat liver (Rudolph et al., 1988). The density of transferrin receptors is somewhat reduced when brain capillary endothelial cells are grown in tissue culture, where the transferrin receptor density is approximately 35,000 receptors/cell (Descamps et al., 1996). The comparable estimates for hepatic and BBB OX26 PS products and for hepatic and BBB transferrin receptor densities suggest that the principle factor affecting the magnitude of the PS product for a given MAb vector is the receptor density per cell.

In conclusion, these studies describe an initial chronic treatment schedule for a transferrin receptor-specific MAb that is a BBB drug-delivery vector. These results show that daily injections of therapeutic concentrations of the MAb result in no up- or down-regulation of the BBB PS product for the OX26 MAb. Because the PS product for OX26 is proportional to the transferrin receptor density on the cell surface, it is inferred that there is no change in the density of the transferrin receptors at the BBB caused by this treatment schedule for the OX26 MAb.