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Lipid-based drug delivery systems have been vastly investigated as a pharmaceutical method to enhance oral absorption of lipophilic drugs. However, these vehicles not only affect drug bioavailability but may also have an impact on gastric emptying, drug disposition, lymphatic absorption and be affected by lipid digestion mechanisms. The work presented here compared the pharmacokinetic (PK) behavior of the non-intoxicating cannabinoid cannabidiol (CBD) in sesame oil

Myeloid differentiation primary response 88 (MyD88) is an intracellular adaptor protein central to the signaling of multiple receptors involved in inflammation. Since innate immune inflammation promotes autoimmunity, MyD88 is an attractive target in autoimmune disease. We previously developed c(MyD 4-4), a novel cyclic peptide competitive inhibitor of MyD88 dimerization that is metabolically stable. Parenteral administration of c(MyD 4-4) reduces disease severity in a mouse model of the human autoimmune disease multiple sclerosis. We now show that N-terminal myristoylation of c(MyD 4-4) enhances the competitive inhibition of MyD88 dimerization in living cells, leading to improved inhibition of the Toll-like receptor and IL-1 receptor signaling. Importantly, myristoylation converts c(MyD 4-4) to an orally bioavailable inhibitor of MyD88. Oral administration of c(MyD 4-4) significantly lowered the inflammatory cytokines secreted by peripheral autoimmune T cells in mice immunized with myelin antigens and ameliorated disease severity in the mouse model of multiple sclerosis. Taken together, we show the conversion of a protein active region to a metabolically stable, selective cyclic peptide that is orally bioavailable.

Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the P

Phase II biotransformation reactions have been gaining more attention due to their acknowledged significance in drug bioavailability, drug development, and drug-drug interactions. However, the predominant role of phase I metabolism has always overshadowed phase II metabolism, resulting in insufficient data regarding its mechanisms. In this paper, we investigate the effect of an advanced lipid based formulation on the phase II metabolism process of glucuronidation, occuring in the enterocytes monolayer. The investigated formulation is a self-emulsifying drug delivery system, termed pro-nano lipospheres, which contains the natural absorption enhancer piperine. To evaluate the effect of this formulation on direct glucuronidation we chose the model molecule raloxifene. First, glucuronidation is the main clearance pathway of this compound without involvement of preceding mechanisms. Second, raloxifene's extensive glucuronidation site is primarily at the intestine. Raloxifene's oral bioavailability was determined in a series of pharmacokinetic experiments using the freely moving rat model. In order to test the effect of the formulation on the relevant UGT enzymes reported in the clinic, we used the in vitro method of UGT-Glo Assay. Coadministration of raloxifene and piperine pro-nano lipospheres to rats resulted in a 2-fold increase in the relative oral bioavailability of raloxifene. However, coadministration of raloxifene with blank pro-nano lipospheres had no effect on its oral bioavailability. In contrast to the difference found in vivo between the two vehicles, both formulations extended an inhibitory effect on UGT enzymes in vitro. Ultimately, these findings prove the ability of the formulation to diminish intestinal direct phase II metabolism which serves as an absorption obstacle for many of today's marketed drugs. Pro-nano lipospheres is a formulation that serves as a platform for the simultaneous delivery of the absorption enhancer and a required drug. The discrepancy found between the in vivo and in vitro models demonstrates that the in vitro method may not be sensitive enough to distinguish the difference between the formulations.

Recent progress in peptide synthesis simplified the synthesis of multiple N-methylation of peptides. To evaluate how multiple N-methylation affects the bioavailability of peptides, a poly alanine cyclic hexapeptide library (n = 54), varying in the number of N-methyl (N-Me) groups (1-5 groups) and their position, was synthesized. The peptides were evaluated for their intestinal permeability in vitro using the Caco-2 model. Further evaluation of the transport route of chosen analogues was performed using rat excised viable intestinal tissue, a novel colorimetric liposomal model and the parallel artificial membrane permeability assay (PAMPA). While most members were found to have poor permeability (permeability coefficient, P(app)1 x 10⁻⁶ cm/s, lower than mannitol, the marker for paracellular permeability), 10 analogues were found to have high Caco-2 permeability, (P(app)1 x 10⁻⁵ cm/s, similar to testosterone, a marker of transcellular permeability). No correlation was found between the number of N-methylated groups and the enhanced permeability. However, 9/10 permeable peptides in the Caco-2 model included an N-Me placed adjacently to the D-Ala position. While the exact transport route was not fully characterized, the data suggests a facilitated diffusion. It can be concluded that multiple N-methylation of peptides may improve intestinal permeability, and therefore can be utilized in the design of orally available peptide-based therapeutics.

Tetraphenylphosphonium (TPP), a phosphonium cation, is a promising means for tumor imaging. A major contributor to the pharmacokinetics of phosphonium cations is the efflux transporter P-glycoprotein (P-gp). For this application it is important to ascertain the influence of the multidrug resistance system on TPP. Therefore, our aim was to characterize the interaction of TPP with P-gp, in vitro and in in vivo models. P-gp-mediated transport of [3H]-TPP was assessed in Caco-2 cells and ex vivo in rat intestinal wall by the use of a diffusion cell system. The distribution of [3H]-TPP across the blood-brain barrier (BBB) was studied in rats and mice treated with P-gp modulators and in Mdr-1a/b((-/-)) knockout mice. The in vitro permeability coefficient of basolateral-to-apical transfer (PappB-A) of [3H]-TPP was 8-fold greater than apical-to-basolateral (PappA-B) coefficient, indicative of net mucosal secretion. A concentration dependent decrease of this secretion was obtained by the P-gp substrate verapamil, while no effect was evident by the MRP2 inhibitor MK-571 and the BCRP inhibitor FTC. [3H]-TPP transfer across rat jejunum wall was directional and concentration-dependent. 2,4-Dinitrophenol, cyclosporin A (CsA), verapamil and PSC-833 enhanced A-B transport of TPP 3.6-fold, 4-fold, 4.6-fold and 5.3-fold respectively. Likewise, PappA-B of [3H]-TPP was 5-fold greater in P-gp knockout mice than in controls. In vivo, PSC-833, P-gp inhibitor, significantly increased the uptake of [3H]-TPP in the liver, heart, small intestine and the lungs but not the brain. Similar results were obtained in P-gp knockout mice. Our study demonstrates that P-gp mediates TPP efflux in vitro and in vivo; however, the consistently poor BBB permeation of TPP in all in vivo studies including P-gp knockout animals indicates that it is most likely mediated by other mechanisms. These findings are important for optimized clinical application of TPP as an imaging agent in cancer.