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Purpose: To further characterize CYP3A4-transfected Caco-2 cells with regard to morphological, transport, and metabolic properties, and to evaluate a different Caco-2 cell strain transfected with both CYP3A4 and oxidoreductase (OR)., Methods: Transfected Caco-2 cells, Caco-2 TC7 cells, and wild-type Caco-2 cells grown onto Millicell were used. We determined the morphological characteristics of transfected cell monolayers using light and transmission electron microscope. We determined the transport and metabolic capabilities of the transfected cells, TC7 cells, and wild-type cells with a variety of drugs, nutrients, and marker compounds., Results: The transfected Caco-2 cells formed a tight monolayer with TEER values and mannitol transport similar to the untransfected parent cell strain (wild type). However, the transfected cells (grown onto Millicell) reached maturity approximately 33% faster than the wild-type cells. Permeabilities of propranolol, nifedipine, testosterone, linopirdine, mannitol, and cephalexin were similar in transfected and wild-type Caco-2 cells. On the other hand, the transfected cells of early passages were much more metabolically active, and metabolized standard CYP3A4 substrates (e.g., testosterone and nifedipine) as much as 100 times faster than untransfected cells. In addition, metabolism of standard substrates was inhibitable by ketoconazole and TAO. Using comparable data, the transfected cells metabolized testosterone the fastest, followed by linopirdine and nifedipine (approximate ratio: 10:6:2). The metabolites of standard substrates were generally preferably excreted to the apical membrane., Conclusion: The monolayers of newly transfected cells (CYP3A4 + OR) have a significantly increased level of CYP3A4 activities compared to untransfected cells. These cell monolayers also have desirable morphological and transport characteristics that are similar to untransfected cells.

Purpose: To validate our hypothesis that a bisphosphonate (BP) having a nitrogen-containing heterocyclic ring on the side chain, and with no hydroxyl on the geminal carbon would possess increased activity, and better oral bioavailability due to enhanced solubility of its calcium complexes/salts and weaker Ca chelating properties., Methods: A novel BP, 2-(2-aminopyrimidinio)ethylidene-1, 1-bisphosphonic acid betaine (ISA-13-1) was synthesized. The physicochemical properties and permeability were studied in vitro. The effects on macrophages, bone resorption (young growing rat model), and tumor-induced osteolysis (Walker carcinosarcoma) were studied in comparison to clinically used BPs., Results: The solubility of the Ca salt of ISA-13-1 was higher, and the log beta(Ca:BP) stability constant and the affinity to hydroxyapatite were lower than those of alendronate and pamidronate. ISA-13-1 exhibited effects similar to those of alendronate on bone volume, on bone osteolysis, and on macrophages, following delivery by liposomes. ISA-13-1 was shown to have 1.5-1.7 times better oral absorption than the other BPs with no deleterious effects on the tight junctions of intestinal tissue., Conclusions: The similar potency to clinically used BPs, the increased oral absorption as well as the lack of effect on tissue tight junction of ISA-13-1 warrant its further consideration as a potential drug for bone diseases.

Purpose: Grapefruit juice (GJ) is known to increase the oral bioavailability of many CYP3A-substrates by inhibiting intestinal phase-I metabolism. However, the magnitude of AUC increase is often insignificant and highly variable. Since we earlier suggested that CYP3A and P-glycoprotein (P-gp) form a concerted barrier to drug absorption, we investigated the role of P-gp in GJ-drug interactions., Methods: The transcellular bidirectional flux of drugs that are (i) CYP3A-and/or P-gp substrates (Vinblastine, Cyclosporine, Digoxin, Fexofenadine, Losartan) or that are (ii) primary CYP3A-substrates (Felodipine, Nifedipine) was evaluated across MDCK-MDR1 cell monolayers with or without GJ, verifying monolayer integrity at all times., Results: While both apical-to-basal (A-B) and basal-to-apical (B-A) fluxes of all CYP3A/P-gp substrates tested were increased in the presence of GJ, the resulting net efflux (B-A/A-B) was in all cases significantly greater with GJ than control (Vin, 28.0 vs. 5.1; CsA, 9.9 vs. 2.8; Dig, 22. 9 vs. 14.7, Fex, 22.3 vs. 11.1, Los, 39.6 vs. 26). In contrast, no such GJ flux effect was observed with Fel and Nif, substrates of CYP3A only (2 vs. 1.7 and 1.2 vs. 1.3)., Conclusions: GJ significantly activates P-gp-mediated efflux of drugs that are substrates of P-gp, potentially partially counteracting the CYP3A-inhibitory effects of GJ.

Purpose: To investigate the effect of ultrasound on the histological integrity and permeability properties of whole rat skin in vitro., Methods: A defined, field-free source of ultrasound was used to irradiate excised rat skin prior to in vitro transport studies in Franz-type cells using sucrose, mannitol, hydrocortisone, 5-fluorouracil and aminopyrine., Results: High intensity ultrasound irradiation (1 to 2 W cm-2) irreversibly damaged cutaneous structures and increased the percutaneous transport rate of permeants. In contrast, skin integrity was largely maintained with low intensity ultrasound (0.1 to 1 W cm-2) which merely discharged sebum from the sebaceous glands so as to fill much of the hair follicle shafts. This effect caused the transfollicular absorption pathway to be blocked for hydrophilic molecules that penetrate via this route and reduced the transport rate significantly., Conclusions: This phenomenon may be used as a probe to elucidate the relative follicular contribution to total penetration for hydrophilic permeants. It was demonstrated that the shunt pathway was responsible for virtually all mannitol and sucrose penetration, perhaps half of hydrocortisone transport but negligible aminopyrine and 5-fluorouracil penetration.

Purpose: Chitosan has recently been demonstrated to effectively enhance the absorption of hydrophilic drugs such as peptides and proteins across nasal and intestinal epithelia (1-3). In this study, the effect of the chemical composition and molecular weight of chitosans on epithelial permeability and toxicity was investigated using monolayers of human intestinal epithelial Caco-2 cells as a model epithelium., Methods: Eight chitosans varying in degree of acetylation (DA) and molecular weight were studied. The incompletely absorbed hydrophilic marker molecule 14C-mannitol was used as a model drug to assess absorption enhancement. Changes in intracellular dehydrogenase activity and cellular morphology were used to assess toxicity., Results: Chitosans with a low DA (1 and 15%) were active as absorption enhancers at low and high molecular weights. However, these chitosans displayed a clear dose-dependent toxicity. Chitosans with DAs of 35 and 49% enhanced the transport of 14C-mannitol at high molecular weights only, with low toxicity. One chitosan (DA = 35%; MW = 170 kD) was found to have especially advantageous properties such as an early onset of action, very low toxicity, and a flat dose-absorption enhancement response relationship., Conclusions: The structural features of chitosans determining absorption enhancement are not correlated with those determining toxicity, which makes it possible to select chitosans with maximal effect on absorption and minimal toxicity.