The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [(14)C]glycylsarcosine ([(14)C]Gly-Sar) up to 50.2 +/- 3.6% (n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux (V(max)) of transepithelial transport decreased from 3.00 +/- 0.17 nmol x cm(-2) x min(-1) in control cells to 0.50 +/- 0.07 nmol x cm(-2) x min(-1) in cells treated with 5 ng/ml EGF (n = 6, P < 0.01). The apparent Michaelis-Menten constant (K(m)) was 2.71 +/- 0.31 mM (n = 6) in control cells and 1.89 +/- 0.28 mM (n = 6, not significantly different from control) in EGF-treated cells. Similarly, apical uptake of [(14)C]Gly-Sar decreased in cells treated with EGF, with an ED(50) value of 0.36 +/- 0.06 ng/ml (n = 6) EGF and a maximal inhibition of 80 +/- 0.02% (n = 6). V(max) decreased from 2.61 +/- 0.4 to 1.06 +/- 0.1 nmol x cm(-2) x min(-1) (n = 3, P < 0.05), whereas K(m) remained constant. Basolateral Gly-Sar uptake showed no changes in V(max) or K(m) after EGF treatment (n = 3). RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane.

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P-glycoprotein inhibitors, like zosuquidar, have widely been used to study the role of P-glycoprotein in oral absorption. Still, systematic studies on the inhibitor dose-response relationship on intestinal drug permeation are lacking. In the present study, we investigated the effect of 0.79 nM-2.5 μM zosuquidar on etoposide permeability across Caco-2 cell monolayers. We also investigated etoposide pharmacokinetics after oral or IV administration to Sprague Dawley rats with co-administration of 0.063–63 mg/kg zosuquidar, as well as the pharmacokinetics of zosuquidar itself. Oral zosuquidar bioavailability was 2.6–4.2%, while oral etoposide bioavailability was 5.5 ± 0.9%, which increased with increasing zosuquidar doses to 35 ± 5%. The intestinal zosuquidar concentration required to induce a half-maximal increase in bioavailability was estimated to 180 μM. In contrast, the IC 50 of zosuquidar on etoposide permeability in vitro was only 5–10 nM, and a substantial in vitro-in vivo discrepancy of at least four orders of magnitude was thereby identified. Overall, the present study provides valuable insights for future formulation development that applies fixed dose combinations of P-glycoprotein inhibitors to increase the absorption of poorly permeable P-glycoprotein substrate drugs.

Conjugation of pleuromutilin is an attractive strategy for development of novel antibiotics and the fight against multi-resistant bacteria as the class is associated with low rates of resistance and cross-resistance development. Herein, the preparation of 35 novel (+)-pleuromutilin conjugates is reported. Their design was based on a synthetically more efficient benzyl-adaption of a potent lead, but still relied on the Cu(I)-catalysed alkyne-azide [3+2] cycloaddition for conjugation onto pleuromutilin. Their antibacterial activity was evaluated against the multi-resistant Staphylococcus 2 aureus strain USA300 for which they displayed moderate to excellent activity. Compound 35, bearing a para-benzyladenine substituent proved particularly potent against USA300 as well as additional strains of MRSA and displayed as importantly no cytotoxicity in four mammalian cell lines. Structure-activity relationship analysis revealed that the purine 6-amino is essential for high potency, likely due to strong hydrogen bonding with the RNA backbone of C2469 as suggested by a molecular model based on the MM-GBSA approach. 9 Alkylation of the nucleobases (thymine, adenine, 6-chloropurine and 2-amino-chloropurine) was achieved in the presence of an inorganic base (K2CO3 or NaH) in DMF. The yields for 68-78 were mainly deteriorated by lack of regioselectivity, i.e. formation of the N3 isomer (thymines) or N7 isomer (purines), which in turn also complicated their subsequent separation during Flash Chromatography. The strategy was overall still more efficient and straightforward than our previous approach.The chloropurins 74-78 were converted via nucleophilic aromatic substitution with various amines in anhydrous EtOH to grant 79-86, generally in excellent yields. Compound 87 was prepared via guanidination in accordance with a procedure optimized by Česnek and coworkers. 35 The primary benzylamines 89 and 96-97 were synthesized via two strategies (Scheme 3); the para analogue 89 via standard Gabriel synthesis, which proved highly efficient, simultaneously also granting the phthalimide 88. The second strategy involved reduction of ethynylbenzonitriles with LiAlH4, 36 which also proved efficient, granting the nitriles 94 and 95 as well. Scheme 3.Reagents and conditions: (a) K-phthalimide, DMF, rt.; (b) H2N-NH2  H2O 50-60% w/w, EtOH, 78 o C; (a) Trimethylsilylacetylene, Pd(PPh3)4, CuI, Anh. Piperidine 80 o C; (d) K2CO3, MeOH, rt.; (e) i) 1 M LiAlH4 in THF, THF, -10 o C → rt. ii) H2O, NaOH (sat.).

Short-term stimulation with EGF and insulin caused an increase in hPepT1-mediated uptake of Gly-Sar in Caco-2 cell monolayers, which could not be accounted for by changes in hPepT1 mRNA or proton-motive driving force.