Evaluation of the Permeation Characteristics of a Model Opioid Peptide, H-Tyr-d-Ala-Gly-Phe-d-Leu-OH (DADLE), and Its Cyclic Prodrugs across the Blood-Brain Barrier Using an In Situ Perfused Rat Brain Model

Abstract

The permeation characteristics of a model opioid peptide, HTyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs [acyloxyalkoxy-based cyclic prodrug of DADLE (AOA-DADLE), coumarinic acid-based cyclic prodrug of DADLE (CA-DALE), and oxymethyl-modified coumarinic acid-based cyclic prodrug of DADLE (OMCA-DADLE)] across the blood-brain barrier (BBB) were determined using an in situ perfused rat brain model. The rat brains were perfused with Krebs-bicarbonate buffer containing test compounds in the absence or presence of a specific P-glycoprotein inhibitor (GF-120918). Brain samples were collected after perfusion and processed by a capillary depletion method. After liquid phase extraction with acetonitrile, samples were analyzed using high-performance liquid chromatography with tandem mass spectrometric detection. Linear uptake kinetics of DADLE and its cyclic prodrugs was observed within the range of 60 to 240 s of perfusion. The apparent permeability coefficient (P app ) of DADLE across the BBB was very low (Ͻ10 Ϫ7 cm/s), probably due to its unfavorable physicochemical properties (e.g., charge, hydrophilicity, and high hydrogen-bonding potential). All three cyclic prodrugs, however, also exhibited low membrane permeation (P app Ͻ10 Ϫ7 cm/s) in spite of their more favorable physicochemical properties (e.g., no charge, high hydrophobicity, and low hydrogen-bonding potential). Inclusion of GF-120918 (10 M) in the perfusates fully inhibited the P-gp activity in the BBB and dramatically increased the P app values of AOA-DADLE, CA-DADLE, and OMCA-DADLE by approximately 50-, 460-, and 170-fold, respectively. In contrast, GF-120918 had no effect on the P app value of DADLE. In addition, the observed bioconversions of the prodrugs to DADLE in the rat brains after 240-s perfusion were very low (5.1% from AOA-DADLE, 0.6% from CA-DADLE, and 0.2% from OMCA-DADLE), which was consistent with the in vitro bioconversion rates determined previously in rat brain homogenates.In an attempt to improve the blood-brain barrier (BBB) permeation of H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), an opioid peptide (Hill and Pepper, 1978;Iyengar et al., 1987;Prokai-Tatrai et al., 1996), our laboratory has synthesized cyclic prodrugs of this peptide using an acyloxyalkoxy (AOA) linker (Bak et al., 1999b), a coumarinic acid (CA) linker (Wang et al., 1999), and an oxymethyl-modified coumarinic acid (OMCA) linker (Ouyang et al., 2002a) (Fig. 1). Unlike DADLE, which is hydrophilic and charged, AOA-DADLE (Bak et al., 1999b), CA-DADLE (Wang et al., 1999), and OMCA-DADLE (Ouyang et al., 2002a) are lipophilic and uncharged. The physicochemical properties of these cyclic prodrugs of DADLE are indicative of solutes that have good cell membrane permeation characteristics (Pauletti et al., 1997).However, when the cell membrane permeation of these cyclic prodrugs of DADLE was evaluated using various in vitro cell culture models (e.g., Caco-2 cells and Madin-Darby canine kidney cells) (Bak et al., 1999a;Ouyang et al., 2002b; Tang and Borchardt, 2002a,b...

ABC and SLC Transporter Expression and Pot Substrate Characterization across the Human CMEC/D3 Blood−Brain Barrier Cell Line

“…[8] There are a number of options to potentially formulate around gastrointestinal efflux transporters, however current studies demonstrate very little success in bypassing these transporters in the BBB. [24,25] There is also arguably greater significance in delineating the factors that contribute to influx transport through these cells, particularly with respect to POT members, as they could potentially serve as targets for effective CNS delivery.…”

P-glycoprotein deficient mouse in situ blood–brain barrier permeability and its prediction using an in combo PAMPA model

“…indicates intravenous injection method, for compounds not likely to be strongly bound to serum proteins, and thought to be weak substrates for transporters. b This work. c Aoyagi et al 1988. d Fukui et al 1991. e Summerfield et al 2007 (please note that the original publication has an error in Table 1 in situ units: cm/min are correct, not cm/s - Summerfield S., private correspondence). f Gratton et al 1997. g Takasato et al .1984. h Liu et al 2004. i Obradovic et al 2007. j Dagenais et al 2004. k Murakami et al 2000. l Cisternino et al 2003. m Dagenais 2000. n Cisternino et al 2001. o Cisternino et al 2004. p Levin 1980. q Dagenais et al 2000. r Dagenais et al 2001. s Fernstermacher 1989. t Bickel et al 1996. u Youdim et al 2004. v Cisternino et al 2004. w Chen et al 2002. x Cisternino et al 2003. y Parepally et al 2006. z Tamai et al 2000. aa Murata et al 1999. bb Bihorel et al 2007. cc Pardridge et al 1990. dd Momma et al 1987. ee Smith et al 1986. ff Greig et al 1990. gg Bourasset et al 2003. hh Greig et al 1987. ii Wu et al . 1998.…”

Significant differences in the disposition of cyclic prodrugs of opioid peptides in rats and guinea pigs following IV administration

“…Previous in vivo studies in rats showed high biliary clearance and low brain penetration of OMCA‐DADLE 18,19. Additionally, in vitro studies demonstrated that while the conversion of this prodrug to DADLE in the target organ brain was too slow, its conversion in plasma was too fast 19.…”

“…In an attempt to improve the oral bioavailability and BBB permeation of peptides and peptidomimetics, our laboratory has synthesized cyclic prodrugs of opioid peptides 13–16. Compared to the opioid peptides, these cyclic prodrugs exhibited improved stability to hydrolytic pathways of metabolism and better permeation across various cell membranes in the presence of inhibitors of efflux transporters 17,18. However, efflux transporters such as P‐gp and MRP2 adversely affected the cell membrane permeation of the cyclic prodrugs by producing net basolateral‐to‐apical flux.…”