Intestinal transport of aminopterin enantiomers in dogs and humans with psoriasis is stereoselective: evidence for a mechanism involving the proton-coupled folate transporter

Abstract

N-[4-[[(2,4-diamino-6-pterdinyl)methyl]amino]benzoyl]-L/D-glutamic acid (L/D-AMT) is an investigational drug in phase 1 clinical development that consists of the L-and D-enantiomers of aminopterin (AMT). L/D-AMT is obtained from a novel process for making the L-enantiomer (L-AMT), a potent oral antiinflammatory agent. The purpose of these studies was to characterize oral uptake and safety in the dog and human of each enantiomer alone and in combination and provide in vitro evidence for a mechanism of intestinal absorption. This is the first report of L /D-AMT in humans. In dogs (n = 40) orally dosed with L-AMT or D-AMT absorption was stereoselective for the L-enantiomer (6- to 12-fold larger peak plasma concentration after oral administration and area under the plasma concentration-time curve at 0-4 h; p < 0.001). D-AMT was not toxic at the maximal dose tested (82.5 mg/kg), which was 100-fold larger than the maximal nonlethal L-AMT dose (0.8 mg/kg). Dogs (n = 10) and humans with psoriasis (n = 21) orally administered L-AMT and L /D-AMT at the same L-enantiomer dose resulted in stereoselective absorption (absent D-enantiomer in plasma), bioequivalent L-enantiomer pharmacokinetics, and equivalent safety. Thus, the D-enantiomer in L/D-AMT did not perturb L-enantiomer absorption or alter the safety of L-AMT. In vitro uptake by the human proton-coupled folate transporter (PCFT) demonstrated minimal transport of D-AMT compared with L-AMT, mirroring the in vivo findings. Enantiomer selectivity by PCFT was attributable almost entirely to decreased binding affinity rather than changes in transport rate. Collectively, our results demonstrate a strong in vitro-in vivo correlation implicating stereoselective transport by PCFT as the mechanism underlying stereoselective absorption observed in vivo.

Trial registration: ClinicalTrials.gov NCT00937027.

Figures

Fig. 1.

Chemical structure of l-AMT (top; N-[4-[[(2,4-diamino-6-pterdinyl)methyl]amino]benzoyl]-l-glutamic acid) and d-AMT (bottom; N-[4-[[(2,4-diamino-6-pterdinyl)methyl]amino]benzoyl]-d-glutamic acid), each with a molecular weight of 440.4 g/mol.

Fig. 2.

Dog pharmacokinetics after the single-dose oral administration of l-AMT and d-AMT. Ten groups of four animals each received a single 2-ml oral dose of l-AMT or d-AMT solution in an escalating fashion from 0.02 to 2.5 mg/kg for the l-enantiomer and 0.8 to 82.5 mg/kg for the d-enantiomer. Blood samples were taken at specified intervals over 4 h after dosing. Concentrations of the l-and d-enantiomers in plasma were quantitated by using LC-MS/MS. Pharmacokinetic parameters Cmax and AUC(0–4 h) were calculated as described in the Materials and Methods. Values are mean ± S.D. (n = 4 per dose). A, concentration-time profiles after l-AMT dosing. B, concentration-time profiles after d-AMT dosing. C, change in Cmax as a function of increasing dose. D, change in AUC(0–4 h) as a function of increasing dose. At the same dose (0.8 and 2.5 mg/kg), the Cmax and AUC(0–4 h) of the l-enantiomer were significantly larger than the d-enantiomer. ***, p < 0.001.

Fig. 3.

Dog pharmacokinetics after the single-dose oral administration of l-AMT and l/d-AMT. Ten beagle dogs received l-AMT (0.77 mg of l-enantiomer) and l/d-AMT (0.7 mg of l-enantiomer and 0.3 mg of d-enantiomer) by gavage in a randomized, single-dose, two-way crossover design. Plasma samples were collected over a 12-h period and analyzed by LC-MS/MS as described under Materials and Methods. Pharmacokinetic parameters were derived from the concentration versus time profiles. There was no detectable d-enantiomer in the plasma after dosing of either drug product; values shown are only for the l-enantiomer in plasma. Shown are the mean plasma concentration-time profiles for l-AMT and l/d-AMT, where each concentration value is the mean ± S.D. There was no significant difference between l-AMT and l/d-AMT for any mean pharmacokinetic parameter (p > 0.05).

Fig. 4.

Bioequivalence of l-AMT and l/d-AMT in psoriatic subjects (n = 21) after single-dose oral administration of l-AMT (0.7 mg of l-enantiomer) and l/d-AMT (0.7 mg of l-enantiomer and 0.3 mg of d-enantiomer) in a phase 1, two-arm randomized, open-label, two-period crossover trial. Plasma samples were collected over a 12-h period and analyzed by LC-MS/MS as described under Materials and Methods. Pharmacokinetic parameters were derived from concentration versus time profiles. There was no detectable d-enantiomer in the plasma after dosing of either drug product; values shown are only for the l-enantiomer in plasma. A, plasma concentration-time profiles for l-AMT and l/d-AMT, where each concentration value shown is the mean ± S.D. B and C, the pharmacokinetic parameters Cmax (B) and AUC∞ (C) derived for plasma l-AMT are shown as a function of administered l-AMT and l/d-AMT. Lines connect data points for the same subject, and horizontal arrows point to the group mean. The Cmax and AUC∞ for l-AMT and l/d-AMT were bioequivalent (i.e., the 90% CI of the Cmax and AUC∞ ratios for l-AMT and l/d-AMT within 0.8–1.25).

Fig. 5.

Comparison of l-AMT and l/d-AMT pharmacokinetic parameters in subjects with psoriasis (n = 21) against dose (of the l-enantiomer) and GFR. A, Cmax versus dose. B, AUC∞ versus dose. C, AUC∞ versus GFR. There was a significant linear correlation for Cmax versus dose (l-AMT, p = 0.002; l/d-AMT, p = 0.001), AUC∞ versus dose (l-AMT, p = 0.0002; l/d-AMT, p = 0.003), and AUC∞ versus GFR (l-AMT, p = 0.02; l/d-AMT, p = 0.001).

Fig. 6.

Comparison of pooled l-AMT and l/d-AMT pharmacokinetic parameters (n = 42) in psoriatic subjects with no AEs (−AE) versus psoriatic subjects with at least one AE (+AE). A, Cmax versus AE. B, AUC∞ versus AE. C, t1/2 versus AE. Horizontal bars are the mean. There was no significant difference between the means in the −AE and +AE groups for either Cmax, AUC∞, or t1/2 (p > 0.05).

Fig. 7.

Transport of l-AMT, d-AMT, and PMX by the human PFCT in vitro. A to C, CHO cells transfected with human PFCT (R2/hPCFT4 cells) or transporter-null R2 cells were incubated with 0.5 μM [3H]l-AMT or [3H]d-AMT at 37°C for 1, 2, and 5 min at pH 5.5 (A), pH 6.5 (B), or pH 6.8 (C). D, 0.5 μM [3H]PMX was incubated with R2/hPCFT4 or transporter-null R2 cells at 37°C for 1 min at pH 5.5, 6.5, and 6.8. Data are the averages from duplicate experiments performed on the same day.

Fig. 8.

Competition of l-AMT transport by the human PCFT. CHO cells transfected with human PFCT (R2/hPCFT4 cells) or transporter-null R2 cells were incubated with 0.5 μM [3H]l-AMT at 37°C for 2 min at pH 5.5 (A), pH 6.5 (B), or pH 6.8 (C), and the transport of [3H]l-AMT competed with 0.5% DMSO (vehicle for compound 1; Wang et al., 2010), 10 μM l-AMT, 10 or 30 μM d-AMT, 10 μM PMX, or 10 μM compound 1. A no-addition (NA) incubation served as a negative competition control. Values are averages ± S.D. (n = 4).