Preclinical absorption, distribution, metabolism and excretion (ADME) characterization of ICAM1988, an LFA-1/ICAM antagonist, and its prodrug.

Intercellular adhesion molecule (ICAM)-1988 is a small molecule lymphocyte function-associated antigen-1 (LFA-1) antagonist being considered for its anti-inflammatory properties. Following intravenous administration of ICAM1988, clearances in mice, rats, dogs, and monkeys were 17.8, 3.31, 15.4, and 6.85 ml min(-1) kg(-1), respectively. In mass balance studies using [(14)C]-ICAM1988 in rats dosed intravenously, unchanged ICAM1988 contributed to 25.1% of the dose. In rats, the systemic bioavailability of ICAM1988 was improved to 0.28 when the drug was administered orally as its isobutyl ester, ICAM2660. In rats, this was consistent with the complete in vitro conversion of ICAM2660 to ICAM1988 in plasma, and liver and intestinal S9. In dogs and monkeys, ICAM2660 did not improve the bioavailability of ICAM1988. This is consistent with limited in vitro conversion of ICAM2660 to ICAM1988 in plasma and liver S9. In human in vitro studies, ICAM2660 conversion to ICAM1988 in liver was similar to rats while no conversion in plasma and intestinal S9 fractions were observed. Based on the in vitro metabolism similarities of human and rat, it would be anticipated that in human oral administration of ICAM2660 would improve the systemic exposure of ICAM1988.

Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues.

This study sought to identify the spatial patterns of expression of peptide transporter 1 (PepT1), peptide transporter 3 (PTR3), peptide/histidine transporter 1 (PHT1), and the human peptide transporter 1 (HPT-1) mRNA in complementary DNA (cDNA) libraries of the human and rat gastrointestinal tracts (GIT), Caco-2 in vitro cell culture model, and in a human multiple tissue panel. Human PTR3 and PHT1 are putative peptide transporters recently discovered. Using sequence-specific primers designed to amplify regions of PepT1, PTR3, PHT1, and HPT-1, we were able to identify the expression of mRNA for each of these transporters in human cDNA panels (Clontech, Palo Alto, CA), the rat GIT, and in Caco-2 cDNA libraries by the polymerase chain reaction (PCR) and Southern Blot analysis. These studies suggest that in the human GIT, PepT1 appears to be localized predominantly in the duodenum, with decreasing expression in the jejunum and ileum. In contrast, PTR3 and HPT-1 were widely expressed in the human GIT, with predominant expression in the different regions of the colon. PHT1 appeared to be expressed in low levels throughout the human GI tract. Interestingly, the mRNAs for all 4 peptide transporters were expressed in Caco-2 cells throughout 30 days of culture. PepT1, PTR3, PHT1, and HPT-1 were also widely expressed in the rat GIT. Human tissue cDNA panel screening suggests that PTR3 and PHT1 are more uniformly expressed, whereas PepT1 and HPT-1 demonstrated site-specific expression. These results suggest that PepT1, PTR3, PHT1, and HPT-1 all may act to facilitate the diffusion of peptides and peptide-based pharmaceuticals in the GIT. PTR3, PHT1, and HPT-1 expressions in Caco-2 cell monolayers strongly suggest that their function needs to be further elucidated and their contribution to peptide transport not ignored. Taken together, these results demonstrate the potential for molecular biological characterization in localizing active transporter systems that can potentially be targeted for enhancing the absorption of peptide-based pharmaceuticals.

The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the coumarinic-based (3 and 4) and the phenylpropionic acid-based (5 and 6) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu5]-enkephalin (1, H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of the cyclic prodrugs, we have determined the possible conformations of these cyclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A and B) in solution. Conformer A exhibited a type I beta-turn at Tyr1-D-Ala2-Gly3-Phe4. The presence of a turn was supported by ROE cross-peaks between the NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II beta-turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs have solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well-defined secondary structures in cyclic prodrugs 3-6, but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker.

The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu5]-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C- and N-terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N- and C-terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes-Einstein molecular radii similar to those of [Leu5]-enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco-2 cell monolayer than [Leu5]-enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P-glycoprotein, which significantly reduced their ability to permeate across Caco-2 cell monolayers. When P-glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.

The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu5]-enkephalin(1, H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE(2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3:2 where both conformers exhibit gamma and beta-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4. It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4, with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.

Coumarinic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeability.

UNLABELLED: To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of coumarinic acid-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. METHODS: The rates of conversion of the cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporus membranes and monitored by HPLC. RESULTS: In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded chemically to intermediates that further degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma and rat liver homogenate, the disappearance of cyclic prodrugs 1 and 2 was significantly faster than in HBSS, pH 7.4. The half-lives in 90% human plasma and in rat liver homogenate were substantially longer after pretreatment with paraoxon, a known inhibitor of serine-dependent esterases. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 and DADLE exhibited similar stability when applied to the AP side of the Caco-2 cell monolayer. Prodrug 1 was 665-fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 31 fold more able to permeate a Caco-2 cell monolayer than was DADLE. CONCLUSIONS: Cyclic prodrugs 1 and 2, prepared with the coumarinic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]-enkephalin. In various biological media, the opioid peptides were released from the prodrugs by an esterase-catalyzed reaction, which is sensitive to paraoxon inhibition.

Acyloxyalkoxy-based cyclic prodrugs of opioid peptides: evaluation of the chemical and enzymatic stability as well as their transport properties across Caco-2 cell monolayers.

PURPOSE: To evaluate the chemical and enzymatic stability, as well as the cellular permeation characteristics, of the acyloxyalkoxy-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. METHODS: The rates of conversion of 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, were measured by HPLC in HBSS, pH = 7.4, and in various biological media (e.g., human plasma and Caco-2 cell and rat liver homogenates) having measurable esterase activity. The cellular permeation and metabolism characteristics of [Leu5]-enkephalin, DADLE and the cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporous membranes and monitored by HPLC. RESULTS: Cyclic prodrugs 1 and 2 degraded slowly but stoichiometrically to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH = 7.4. In homogenates of Caco-2 cells and rat liver, as well as 90% human plasma, the rates of disappearance of the cyclic prodrugs were significantly faster than in HBSS. The stabilities of the cyclic prodrugs 1 and 2 were increased significantly in 90% human plasma and Caco-2 cell homogenates when paraoxon, a potent inhibitor of serine-dependent esterases, was included in the incubation mixtures. A similar stabilizing effect of paraoxon was not observed in 50% rat liver homogenates, but was observed in 10% homogenates of rat liver. When applied to the AP side of a Caco-2 cell monolayer, DADLE and cyclic prodrugs 1 and 2 exhibited significantly greater stability than [Leu5]-enkephalin. Based on their physicochemical properties (i.e., lipophilicity), cyclic prodrugs 1 and 2 should have exhibited high permeation across Caco-2 cell monolayers. Surprisingly, the AP-to-BL apparent permeability coefficients (P(App)) for cyclic prodrugs 1 and 2 across Caco-2 cell monolayers were significantly lower than the P(App) value determined for the metabolically stable opioid peptide DADLE. When the P(App) values for cyclic prodrugs 1 and 2 crossing Caco-2 cell monolayers in the BL-to-AP direction were determined, they were shown to be 36 and 52 times greater, respectively, than the AP-to-BL values. CONCLUSIONS: Cyclic prodrugs 1 and 2, prepared with an acyloxyalkoxy promoiety, were shown to degrade in biological media (e.g., 90% human plasma) via an esterase-catalyzed pathway. The degradation of cyclic prodrug 1, which contained an ester formed with an L-amino acid, degraded more rapidly in esterase-containing media than did prodrug 2, which contained an ester formed with a D-amino acid. Cyclic prodrugs 1 and 2 showed very low AP-to-BL Caco-2 cell permeability, which did not correlate with their lipophilicities. These low AP-to-BL permeabilities result because of their substrate activity for apically polarized efflux systems.

Phenylpropionic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeation.

PURPOSE: To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of phenylpropionic acid-based cyclic prodrugs 1 and 2 of opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. METHODS: The rates of conversion of cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE, and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporus membranes and monitored by HPLC. RESULTS: In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma, the rates of disappearance of cyclic prodrugs 1 and 2 were slightly faster than in HBSS, pH 7.4. These accelerated rates of disappearance in 90% human plasma could be reduced to the rates observed in HBSS, pH 7.4, by pretreatment of the plasma with paraoxon, a known inhibitor of serine-dependent esterases. In homogenates of Caco-2 cells and rat liver, accelerated rates of disappearance of cyclic prodrugs 1 and 2 were not observed. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 and DADLE exhibited stability similar to prodrug 1 when applied to the AP side of the Caco-2 cell monolayers. Prodrug 1 was 1680 fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 77 fold more able to permeate a Caco-2 cell monolayer than was DADLE. CONCLUSIONS: Cyclic prodrugs 1 and 2, prepared with the phenylpropionic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]-enkephalin. In 90% human plasma but not in Caco-2 cell and rat liver homogenates, the opioid peptides were released from the cyclic prodrugs by an esterase-catalyzed reaction that is sensitive to paraoxon inhibition. However, the rate of this bioconversion appears to be extremely slow.