Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

A series of novel 2,4-diamino-pyrrolo[2,3-d]pyrimidines were described as potent inhibitors of iron-dependent lipid peroxidation, and proved to be cytoprotective in cell culture models of oxidative injury and neuroprotective in brain injury and ischemia models (Bundy et al., 1995; Andrus et al., 1997; Hall et al., 1997). First-generation compounds like tirilazad mesylate (PNU-74006F), one member of a series of 21-aminosteroids that inhibit iron-dependent lipid peroxidation and are used for the treatment of subarachnoid hemorrhage (Hall et al., 1994), have poor oral bioavailability and limited brain access and consequently are administered parenterally (Laizure et al., 1993). Therefore, major considerations in the discovery of the pyrrolopyrimidines were oral activity and superior brain penetration given that the objective was to treat chronic neurodegenerative diseases associated with oxidative mechanisms of pathology (Lees, 1993). One a priori criterion for the neuroprotective activity of pyrrolopyrimidines was the ability to reach target cells deep within brain tissue in efficacious amounts. To aid the selection of lead compounds with this property, we measured the transcellular permeability and cellular partitioning of pyrrolopyrimidine analogs in a cell culture model designed for highly lipophilic molecules.

Structural determinants of permeability and partitioning are discussed for a series of structurally similar homologs. In addition, detailed studies were conducted concurrently with two radiolabled compounds from the pyrrolopyrimidine series representing different physicochemical, permeability, and cell partitioning attributes to discern the roles of protein binding and cell partitioning on permeation and to complement ongoing pharmacological and pharmacokinetic studies. The data proved useful in predicting which compounds were most likely to leave the blood and penetrate underlying tissue. In a companion paper, brain uptake dynamics and cellular penetration of these compounds are confirmed in vivo (Sawada et al., 1999).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents. Madin-Darby canine kidney (MDCK) epithelial cells at passage 52 were from the American Type Culture Collection (Rockville, MD). Tissue culture-grade dimethyl sulfoxide (DMSO) and BSA (fraction V, lot 129F0136) were from Sigma Chemical Co. (St. Louis, MO). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (free base was from Boehringer-Mannheim Corp. (Indianapolis, IN). PBS was purchased from GIBCO BRL (Grand Island, NY) and used in 1 mg/ml D-glucose and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid adjusted to pH 7.4. Polycarbonate filter inserts (Transwell) with a diameter of 24 mm and a 0.4-µm pore size were purchased from Costar (Cambridge, MA). Reagents for HPLC were of the highest purity available. [¹⁴C]Mannitol (56 mCi/mmol) and [¹⁴C]testosterone (50 mCi/mmol) were purchased from Amersham Corp. (Arlington Heights, IL). The 2,4-diamino-pyrrolo[2,3-d]pyrimidine analogs were supplied as solids by Medicinal Chemistry, Pharmacia & Upjohn (Kalamazoo, MI). [¹⁴C]PNU-87663 monomethanesulfonate salt (22.2 µCi/mg; 443.9 g/mol) and [¹⁴C]PNU-89843·HCl (30.8 µCi/mg; 322.2 g/mol) were obtained as >98% pure by HPLC from the Pharmacia & Upjohn Radiosynthesis Group (Kalamazoo, MI). These were reconstituted in DMSO to give 0.5 mCi/ml, and the solutions were stored at 20°C under argon without significant oxidation/degradation.

Unlabeled Compound Permeability Assay. Continuous monolayers of MDCK cells were cultured on Transwell filter inserts as described previously (Sawada et al., 1994). The apical (donor) and basolateral (receiver) chambers were rinsed for 10 min with PBS and equilibrated for 20 min with PBS containing 3.0% (w/v) and 0.5% (w/v) BSA, respectively. All incubations were done at room atmosphere at 37°C with continuous mixing by nutation (Nutator; Clay Adams, Parsippany, NJ). Donor solutions were prepared by slowly injecting a small volume of a 4 mg/ml stock solution of compound in DMSO into PBS-3% BSA while mixing vigorously to give 50 µM compound and <0.5% (v/v) DMSO. After mixing at 37°C for 15 min, the donor solution was passed through a 0.2-µm-pore filter, and 1.5 ml was added to the cells in the apical chamber. An aliquot was taken from the donor solution before addition to the cells to determine the initial concentration by reversed-phase HPLC. Thereafter, samples were taken every hour from both the donor (25 µl) and receiver (100 µl) solutions to follow the kinetics of disappearance and appearance during apical-to-basolateral flux of compound across the cell monolayer. The insert with cells was moved to 2.5 ml of fresh receiver solution at each time interval to maintain sink conditions. In cases where flux was fast, sampling times were adjusted so that <10% of the initial drug mass permeated per time interval. In all cases, the donor solutions were depleted by <10% of their initial volume over the course of the assay. After the last time point, each insert with cells was rinsed quickly three times with cold PBS, and the cells were collected by scraping. After extraction (Folch et al., 1957), the amount of compound in the organic phase was measured by reversed-phase HPLC to determine the amount of compound that had become cell associated and to obtain mass balance.

Uptake, Efflux, and Transmonolayer Flux of ¹⁴C Compound. The details of the procedures for these experiments are reported by Sawada et al. (1994). For measuring uptake and efflux kinetics at the apical membrane, MDCK cell monolayers were grown in plastic 6-well dishes. In uptake experiments, cells were incubated with 1 ml of PBS-BSA containing 0.1 µCi/ml (10.1 µM) [¹⁴C]PNU-87663 or 0.5 µCi/ml (50.7 µM) [¹⁴C]PNU-89843. For efflux experiments, cell monolayers were preloaded with either 7.8 µM [¹⁴C]PNU-87663 or 21.3 µM [¹⁴C]PNU-89843 in 1 ml of PBS-3% BSA for 45 min. After a brief rinse with PBS, 1 ml of PBS-BSA was added to each well, and the entire volume was replaced with fresh PBS-BSA at each time interval to maintain sink conditions.

Solubility. The solubility of the radiolabeled compounds was measured in PBS containing 0.05, 0.1, 0.5, or 3% (w/v) BSA and 9.1 µM [¹⁴C]PNU-87663 or 8.7 µM [¹⁴C]PNU-89843. Solutions were prepared as described above and assayed for total radioactivity. Aliquots (1 ml) were centrifuged for 10 min at 150,000g at 20°C, and the amount of radioactivity remaining in solution was determined. Donor solutions for all subsequent experiments were prepared in this way to ensure that the compound was soluble and not present as a microprecipitate.

Compound Stability. The stability of the ¹⁴C compounds was examined under experimental conditions to avoid measurements in which significant degradation or metabolism occurred. Donor solution and the aqueous and organic solvent extracts (Folch et al., 1957) of plastic-grown MDCK cell monolayers collected after 30, 90, and 180 min were subjected to reversed-phase HPLC. Control samples without cells and using [¹⁴C]PNU-89843 also were analyzed.

Reversed-Phase HPLC. Donor and receiver solutions containing unlabeled pyrrolopyrimidines were diluted 4-fold with methanol or acetonitrile to yield the appropriate mobile phase. The organic phase of cell extracts was dried under nitrogen and reconstituted in mobile phase. After centrifugation for 5 min at 14,000g to remove precipitated protein, samples were injected in mobile phase. The mobile phase varied with compound and consisted of either 25% to 75% (v/v) methanol diluted with aqueous phase containing 0.5% (v/v) triethyamine and adjusted to pH 5 with glacial acetic acid or 20% to 40% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Conditions were optimized to obtain a capacity factor (k') of 2 to 3. Columns used were a 250 × 4.6 (i.d.)-mm Zorbax RX-C18 or SB-CN (5-µm particle size) column (DuPont, Wilmington, DE) or a 150 × 4.6 (i.d.)-mm Inertsil-C8 (5-µm particle size) column (Metachem, Torrance, CA), with a 1-cm guard column. The samples were chromatographed with an isocratic method at a flow rate of 1.0 to 1.5 ml/min. Detection was at 242 nm or using fluorescence with excitation at 320 nm and an emission cutoff filter of 389 nm. Five-point standard curves were generated with 0.05 to 10 µg/ml in mobile phase without protein present. The ¹⁴C compounds were chromatographed with an isocratic method using a 250 × 4.6 (i.d.)-mm Zorbax SB-CN (5-µm particle size and 80-Å pore size; Mac-Mod Analytical, Chadds Ford, PA) column, at ambient temperature with a constant injection volume of 45 µl and a Flo-One/Beta model CR radioactive flow detector equipped with a 2500-µl flow cell (Radiomatic Instruments, Tampa, FL). The mobile phases consisting of 0.5% (v/v) triethylamine in distilled, deionized water at pH 5.0 (acetic acid) and methanol were mixed on line at organic-to-aqueous ratios of 80:20 (v/v) (PNU-87663) and 75:25 (v/v) (PNU-89843). Detection was at 242 nm with retention times of 6.3 min (PNU-87663) and 5.7 min (PNU-89843) at a flow rate of 1.0 ml/min. The liquid scintillation fluid (Flo-Scint II, Radiomatic Instruments, Meriden, CT) was added to the column effluent at a rate of 4 ml/min.

Data Analyses. Apparent permeability coefficients (P_e) were obtained from the slope of the rate of appearance of unlabeled parent compound into the receiver solution, after donor disappearance reached equilibrium, using:
where A is area of filter, 4.71 cm²; V_D is volume of the donor solution, 1.5 cm³; M_R is change in mass of compound that had accumulated in the receiver solution over a time interval, t (s), when flux was linear and sink conditions were met; and M_D(0) is the initial amount of compound in the donor solution. The permeability coefficient of the monolayer (P_m) was calculated using:
where P_ABL is the permeability coefficient for the aqueous boundary layer of the cell and filter under these hydrodynamic conditions, and P_f is the permeability coefficient for the filter without cells. The P_ABL + P_f was 1.58 × 10⁴ cm/s determined from the flux of [¹⁴C]testosterone. The leakage of membrane-impermeable [¹⁴C]mannitol between the cells in the monolayer gave a P_e of 3 to 5 × 10⁷ cm/s.

3

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Permeability and Lipophilicity. A congener series of 26 pyrrolopyrimidines with discrete additions, deletions, and substitutions (Tables 1 and 2) were chosen for this study from hundreds of compounds to derive a qualitative structure-permeability relationship to understand and predict tissue partitioning and permeation. Flux of compound from donor to receiver through a continuous cell monolayer occurred with a variety of different kinetics from relatively unrestricted permeability, where appearance nearly equals disappearance (Fig. 1A), to membrane-restricted permeability, where the rate of disappearance is nonlinear and exceeds the rate of appearance to different degrees (Fig. 1, B-D). In all cases, permeability of these lipophilic compounds was controlled by the aqueous boundary layer (Raub et al., 1993).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Transcellular flux kinetics of different pyrrolopyrimidine compounds. Disappearance of compound from the donor solution () and appearance in the receiver solution () bathing the basolateral surface of the cell monolayer were measured by HPLC with time. The results were plotted as mass fractions relative to the mass in the donor at time zero or MD(0). A, strongly ionized compounds or those that are highly protein bound are poorly permeable, and the cell associated fraction is low. B, as cell accumulation increases, both the rates of disappearance and appearance increase and disappearance kinetics become hyperbolic. C and D, marked and rapid cell accumulation results in distinctly different kinetics of loss and appearance with relatively slow (C) or fast (D) appearance kinetics.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Correlation between the transmonolayer permeability coefficient (Pm) and lipophilicity of the weakly basic pyrrolopyrimidines. Pm values are the mean of triplicate determinations obtained from the slope of the kinetics of appearance of compound in the receiver solution measured by reversed-phase HPLC, defined by eq. 1, and corrected for the unstirred water layer described in Materials and Methods. Lipophilicity is an estimate using the calculated log Poct/water or CLOGP based on a computational fragmental method. Apparent CLOGP values for the piperazinyl analogs () are adjusted for ionization at pH 7.4. The data points are identified by number according to the list of compounds in Table 1. The two unidentified compounds (*) are examples that are extensively metabolized.

Permeability and Cell Partitioning. At the end of each flux experiment, the cells are collected and extracted with organic solvents, and the amount of compound recovered in the organic phase or that became cell associated is quantified and expressed as the percentage of the initial concentration of compound that was in the donor solution. In most, but not all cases (see Fig. 1, A and B), a pseudoequilibrium was reached (see Fig. 1, C and D), and this is especially true for the more lipophilic compounds. A plot of lipophilicity versus percent of compound that became cell associated suggests that separate optima exist for the weak base and N-7-piperazinyl compounds where an increase in cell partitioning is independent of the apparent CLOGP value (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Relationship between lipophilicity and the fraction of compound that is cell associated at the end of the flux experiment. Lipophilicity is an estimate using the calculated log Poct/water or CLOGP based on a computational fragmental method. The cell-associated fraction is an estimate of partitioning of weakly basic () and piperazinyl analogs () from buffer-albumin to the cell monolayer at pseudoequilibrium. The data points are the mean of triplicate samples and are identified by number according to the list of compounds in Table 1.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Influence of structural determinants of a homologous series of weakly basic pyrrolopyrimidine analogs on permeability and cell partitioning. Pm and the fraction cell associated were obtained as described in the legends to Figs. 2 and 3. The data points are the mean of triplicate samples and are identified by number according to the list of compounds in Table 1.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Kinetic model of passive diffusion of a protein (P)-interacting drug (D) across a cell within a confluent monolayer. Lateral diffusion into neighboring cells and from the apical to basolateral membranes is limited by the tight junction. Free and bound species diffuse across the aqueous boundary layer, and cell uptake is the free drug partitioning into the apical membrane in a thermodynamic step across the water-lipid interface. The free drug concentration is determined by the operational dissociation constant (Kd), and its membrane insertion is dictated by the intrinsic partition coefficient (Kintr). Once in the hydrocarbon core or inner leaflet of the lipid membrane bilayer, drug can partition into and diffuse through the cytoplasm (pathway 1) or diffuse laterally throughout the cell plasma membrane (pathway 2), reaching equilibrium. Efflux from the cell into the aqueous receiver solution is the mirror image of the uptake kinetic steps. The effective permeability coefficient is a summation of all of these steps (Raub et al., 1993; Sawada et al., 1994).

Transmonolayer Diffusion of PNU-87663 and PNU-89843. The amount of radioactivity associated with the cells and both solutions was measured to obtain mass balance and confirmed that metabolism and/or degradation was negligible. Reversed-phase HPLC analysis showed that [¹⁴C]PNU-87663 chromatographed as a single peak after 4 h of continuous incubation with confluent MDCK cell monolayers. In contrast, metabolism of [¹⁴C]PNU-89843 begins within 1 h and continued slowly with time. Both compounds were stable for 4 h in buffer alone, confirming that [¹⁴C]PNU-89843 was metabolized by the cells. Except for transmonolayer flux where the rate of diffusion superseded metabolism, the kinetic data for [¹⁴C]PNU-89843 were collected within 1 h.

4

4

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Transmonolayer flux of (A) [14C]PNU-87663 and (B) [14C]PNU-89843. Cumulative mass fractions (mean and S.D. of triplicate samples) relative to the total mass in the donor at t0 are shown as a function of time for disappearance from the donor () and appearance in the receiver (). In all cases, the donor solution at pH 7.4 contained 3% BSA. The BSA concentration in the receiver solution at pH 7.4 was either 0.5% (dashed line) or 3% (solid line). At the end of the experiment, the amount of radioactivity recovered in the cells was 39 ± 2% (PNU-87663) and 5.1 ± 1.1% (PNU-89843), giving mass balances of 97 ± 5% and 111 ± 10%, respectively. The curves are the best fit of nonlinear least-squares regression analysis according to the model of Sawada et al. (1994).

7

7

Cell Uptake Kinetics of PNU-87663 and PNU-89843. The uptake kinetics involve the diffusion of free and bound compound across the aqueous boundary layer, followed by partitioning of free compound into the apical membrane, diffusion throughout the cell, and attainment of equilibrium (Fig. 5). The initial rates of uptake for both compounds were minimally affected by BSA concentration because uptake of both the free and the BSA-bound compound was aqueous boundary layer controlled, and a linear correlation between the uptake P_e values and the free drug concentration supported this (data not shown). A dependence on BSA concentration was realized with differences in the equilibrium reached after 10 min (Fig. 7). Cell uptake increases as the concentration of BSA decreases due to the increase in free or unbound compound. These data were analyzed using nonlinear least-squares regression analysis per the biophysicokinetic model described to calculate an apparent distribution coefficient (K_e) between total compound (free and protein bound) in the aqueous solution and the cell. By using eq. 4 from Raub et al. (1993), an intrinsic distribution coefficient (K_intr) and an apparent dissociation binding constant (K_d) were calculated. The K_d value was operational because the stoichiometry of the interaction between compound and BSA was not known. PNU-87663 had a significantly greater K_intr than PNU-89843, or 1750 ± 100 versus 750 ± 50. Accordingly, PNU-87663 was less bound to BSA with a K_d of 130 µM compared with a K_d of 20 µM for PNU-89843.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Accumulation of (A) [14C]PNU-87663 and (B) [14C]PNU-89843 at the apical membrane domain of plastic-grown cell monolayers is measured as disappearance from the donor solution as a function of BSA concentration. The data are the mean and S.D. of triplicate samples expressed as the fraction of mass remaining in the donor solution relative to the total mass in the donor at t0. The curves are the best fit of nonlinear least-squares regression analysis according to the model of Sawada et al. (1994).

Efflux Kinetics of PNU-87663 and PNU-89843 from Cell. The appearance of compound in the receiver during transmonolayer flux was limited by desorption or efflux of compound from the basolateral cell membrane into the receiver bathing solution. Therefore, this event was governed by the membrane partition coefficient of the compound and its affinity for BSA (Fig. 5). Efflux of PNU-87663 and PNU-89843 was dependent on the concentration of BSA (Fig. 8), but PNU-89843 was 40- to 70-fold faster than PNU-87663. In all cases, the cell-associated compound desorbed with first order kinetics, suggesting that there was a single kinetic pool.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Efflux kinetics of cell-associated (A) [14C]PNU-87663 and (B) [14C]PNU-89843 from the apical membrane of plastic-grown cell monolayers into buffer containing different BSA concentrations. The cell monolayers were allowed to accumulate 14C compound to equilibrium in 1% BSA and quickly rinsed, and the cumulative mass fraction that appeared in the bathing solution (receiver) with time is measured relative to the total mass that was cell associated at t0. The mean and S.D. of triplicate samples are shown and fit by nonlinear least-squares regression analysis according to the model of Sawada et al. (1994).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Passive transcellular or transmembrane permeation of hydrophobic molecules is governed by a number of physicochemical and thermodynamic properties, including solubility, lipophilicity, polarity, and serum/protein interactions. Biophysicokinetic models are one approach to separate and quantify these components to recognize and understand their relative importance in the design of compounds for optimal delivery and, thus, efficacy. In this report, we surveyed a homologous series of novel, lipophilic antioxidants by using cultured cell monolayers and a previously developed kinetic model (Raub et al., 1993; Sawada et al., 1994). The objective was to understand the structural dependence of permeance of pyrrolopyrimidines and to use this information to predict tissue penetration, especially brain, and residence time to aid in the selection of lead candidate compounds.

Contrary to what is often assumed generally, permeability of pyrrolopyrimidines in vitro is inversely related to lipophilicity or to increased cell partitioning. An inverse relationship between permeability and lipophilicity is not uncommon and has been described in many studies as a parabolic relationship (Hansch and Clayton, 1973; Nook et al., 1988; Grieg, 1989; Bernards and Hill, 1992; Ridout et al., 1992; Saji et al., 1992, Nau et al., 1994). Such data have generated controversy because it is widely accepted that permeability increases as lipophilicity increases, reaching a maximum. The way in which permeability is measured contributes to the discrepancy. Some studies rely solely on a rate of disappearance from the donor solution. This rate of disappearance increases with increased lipophilicity, reaching a plateau value limited only by the hydrodynamics of the system. A decrease in permeability under such circumstances has been attributed to a decrease in free solute concentration caused by lowered aqueous solubility or micelle formation. Here, we measured both disappearance and appearance and found that the disappearance kinetics were rapid and of the same order of magnitude for all compounds consistent with unstirred water layer-limited diffusion. The free solute concentration was accounted for by removing precipitates from the donor solution through ultracentrifugation before use. Although independent physical studies indicate that the pyrrolopyrimidines are surface active, they were relatively weak surfactants and do not form micelles at the concentrations used here in the presence of albumin (Epps and McCall, 1997).

The addition of albumin, while decreasing adsorption to noncellular surfaces and increasing solubility, complicated data interpretation such that the observed P_m value included a binding equilibrium effect to account for the change in free solute concentration. However, the effect of protein binding on decreased permeability in the present dataset for pyrrolopyrimidines was minimal and inconsistent with the observed weak binding constants for PNU-89843 and PNU-87663. The K_d value for PNU-89843 was 6.5-fold lower than that for PNU-87663, yet PNU-89843 was, according to the linear relationship between permeability and lipophilicity, significantly more permeable. These data imply that although protein interactions contribute to the observed P_m values, it was not the cause of the inverse relationship between permeability and lipophilicity.

Other factors that have been attributed to the parabolic effect and that have been accounted for here include measuring permeability under nonequilibrated conditions, the principle of bulk (molecular size) tolerance, metabolism, and active transport. All permeability coefficients are calculated from appearance kinetics after disappearance kinetics reach steady state or when the cell concentration is at steady state and mass balance is achieved. For this homologous series, molecular size was not correlated with increased lipophilicity; however, it was clearly shown that partitioning and, hence, permeability were consistent with Overton's rules of steric influence on diffusion of a molecule through a lipid membrane (Wright and Diamond, 1969). Metabolism was negligible under the conditions of the assay. We also ruled out that decreased flux can be attributed to active efflux, which can markedly decrease net apical-to-basolateral flux.

Our results were similar to those reported by Ridout et al. (1992) that percutaneous absorption of phenolic analogs was negatively controlled by increased partitioning. Using intestinal epithelial cell monolayers, Wils et al. (1994a) also showed that highly lipophilic (log P_oct-buffer, pH 7.4 > 3.5) molecules have lowered transcellular permeability. They proposed that this observed slow permeability in vitro predicts low oral absorption. Others have claimed that a threshold P_e value of 1 × 10⁶ cm/s is obligatory for maximal oral absorption (Artursson and Karlsson, 1991; Wils et al., 1994b). This may occur only when buffer-to-membrane partitioning in the donor is the rate-controlling mechanism for diffusion, which was not valid in the present situation. The pyrrolopyrimidines have greater than 50% oral bioavailability in rats (Bundy et al., 1995). We contend that the slow permeability of highly lipophilic molecules was a consequence of these in vitro assays, which have an aqueous receiving solution of limited volume. This study and previous data showed that desorption from the receiver-side membrane into the buffer is the limiting step and that the in vitro models did not adequately mimic the in vivo situation (Raub et al., 1993; Sawada et al., 1994). In illustrating the caveats of relying solely on disappearance kinetics for predicting oral absorption, Nook et al. (1988) noted the importance of blood extraction, or the "receiver" equivalent. This explanation can be nicely demonstrated in vitro by adding to the receiver solution more protein or protein that binds compound with high affinity (Sawada et al., 1994). Attempts to improve this transfer were unsuccessful, and none of the alternate approaches outperformed serum proteins. We suggest this is due to their inability to readily diffuse through the 0.4-µm-pore filter to access the basolateral membrane and act as efficient acceptors. Lipophiles still have to partition from the membrane into the underlying aqueous solution. Additional cell monolayers underneath the filter simply enlarge the receiver compartment and may mimic the in vivo situation better if situated more closely to the cell monolayer barrier separated only by a basement membrane.

The interplay of hydrophilic and hydrophobic characteristics and their position on the molecule illustrate what determines the permeability of pyrrolopyrimidines. The importance of the pyrrole C-6 position for cell partitioning was shown. These data are consistent with the proposed role for hydrogen-bonding groups in membrane permeability of nonelectrolytes (Wright and Diamond, 1969) and peptides (Burton et al., 1992) and in brain penetration (van de Waterbeemd and Kansy, 1992), but the net effect was positive rather than negative. Hydrophilic substitutions that increased aqueous solubility had a lesser affect on cell accumulation if made on positions other than the hydrophobic moiety at position C-6. This assignment was consistent with film balance data indicating that amphiphilic PNU-87663 partitions to the aqueous-phospholipid interface oriented with the protonated pyrrolidine groups in the aqueous phase and the hydrophobic pyrrole buried in the fatty acyl chains of the membrane (Epps and McCall, 1997).

Delineation of transcellular flux into uptake by the apical membrane, cell partitioning, and efflux from the basolateral membrane showed that efflux of PNU-89843 from the basolateral membrane (efflux P_bl) (Table 3) was 17-fold faster than that for PNU-87663. These results with PNU-89843 and PNU-87663 could be compared with data for earlier-generation antioxidants PNU-78517F (Table 3) and PNU-74006F (data not shown) because identical studies were performed using the same biophysicokinetic analyses and hydrodynamic conditions (Raub et al., 1993). The marked difference between these compounds was the rate of efflux from the basolateral membrane. However, efflux P_bl was not simply a cell-to-buffer partitioning event because it was derived by including the kinetics of uptake on the donor side and therefore is influenced by the concentration of compound in the cell. Free PNU-89843 immediately partitioned into the apical membrane and, given its lower K_intr, repartitions into the receiver solution down the concentration gradient created by sink conditions. Consequently, marked cell concentrations were never accumulated. The same result could be achieved with those compounds that have a large K_intr by shifting the equilibrium in favor of the receiver solution and inducing a gradient as likely occurs in vivo. Evidence for this was obtained in vivo where efflux of PNU-89843 from brain was greater than 5-fold faster than PNU-87663 (Sawada et al., 1999).

We predict from these results that the pyrrolopyrimidines permeate the brain (and other organs) beyond the blood-brain barrier faster than PNU-74006F or PNU-78517F. Laser scanning fluorescence microscopy showed that PNU-87663 penetrates deep into the brain soon after i.v. injection (Sawada et al., 1999). Evidence against a similar distribution for PNU-74006F and PNU-78517F was indirect and less convincing, yet the results suggest that these compounds reside mostly within the endothelium before they are rapidly cleared (Epps and McCall, 1997). The equilibrium amount of compound and its residence time were dependent on the rate of clearance from the blood. As blood levels fall, tissue levels of PNU-89843 will also decline immediately, whereas tissue levels of PNU-87663E decline more slowly. That this occurs in vivo was confirmed by measuring brain and plasma drug levels in mice 5 and 60 min after i.v. administration. Although plasma levels for PNU-87663 and PNU-89843 decreased (84-86%) similarly within ~1 h, the brain/plasma ratio for PNU-87663 was ~5-fold greater than that for PNU-89843 (Sawada et al., 1999).

In conclusion, in vitro assays showed that pyrrolopyrimidines were a more permeable structural class of antioxidants, in support of oral activity and superior brain penetration. A qualitative structure-permeability assessment indicated that substitutions at the pyrrole C-6 position influence membrane partitioning and that permeability was inversely related to lipophilicity or cell partitioning. These results indicated that the assessment of tissue penetration of highly lipophilic compounds using these in vitro assays involved more than a series of connected aqueous partitioning steps and that addition of serum proteins, or as yet unidentified endogenous carriers, to the receiving compartment can improve this assessment.