Characterization of Recombinantly Expressed Rat and Monkey Intestinal Alkaline Phosphatases: In Vitro Studies and In Vivo Correlations

Abstract

Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiologic role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from preclinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify, and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, recombinant IALP (rIALP)-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate, and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl-linked prodrugs. Overall, the rat rIALPderived data were qualitatively consistent with in vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and no in vivo parent exposure. Finally, the apparent K m values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses.

Design, Synthesis, and Pharmacokinetic Evaluation of Phosphate and Amino Acid Ester Prodrugs for Improving the Oral Bioavailability of the HIV-1 Protease Inhibitor Atazanavir

“…As a potential approach to solving the pharmaceutical issues associated with the inherently poor aqueous solubility of 1 that result in dissolution-limited and pH-dependent absorption, the initial focus was directed toward the synthesis of phosphate derivatives, with the direct phosphate prodrug 2 selected as the first iteration. − This prodrug was designed to enhance the solubility of the drug in the gastrointestinal tract, with the release of the parent drug and nontoxic inorganic phosphate as a byproduct through a presystemic activation mechanism mediated by alkaline phosphatases (ALPs) that are abundantly expressed on the brush border membranes of enterocytes. − While direct phosphate derivatives of 1 have not been described, phosphate monoesters of structural analogues of 1 , which are covalently modified by the attachment of water-soluble nonpeptidic oligomers, have been reported . Phosphate prodrugs have been investigated for other marketed PIs including 3 , amprenavir (APV; 5 ), and lopinavir (LPV, 7 ), which are compounds 4 , 6 , and 8 , respectively, with fosamprenavir ( 6 ) approved by the FDA (Figure ).…”

Differences in the In Vivo and In Vitro Metabolism of Imrecoxib in Humans: Formation of the Rate-Limiting Aldehyde Intermediate

“…In vitro metabolism incubations (e.g., subcellular fractions) occur in a static system, representing only a few of several dimensions of a complex in vivo system (Wang et al, 2010;Subramanian et al, 2013). Several drugs, such as linezolid and ziprasidone, are metabolized through multiple sequential reactions in vivo, whereas they can only undergo one or two sequential reactions in in vitro systems (Wang et al, 2010), especially when a metabolic intermediate is generated.…”

Pharmacokinetic and pharmacodynamic study of a phospholipid-based phase separation gel for once a month administration of octreotide

“…The correlation between in vitro release and in vivo absorption was evaluated by regression coefficient (R 2 ) [38]. Rabbits showed the best correlation, which was possibly due to its animal size, injection dose, gene homology, routes of absorption, digestion, metabolism and elimination [52]. The differences observed in burst release of OCT-PPSG in vitro and in vivo were largely due to the setup of the in vitro release study, which might not be sufficient to mimic the in vivo conditions.…”