Franklin Okumu

Structural requirements for intestinal absorption of peptide drugs

The clinical development of orally active peptide drugs has been restricted by their unfavorable physicochemical characteristics, which limit their membrane permeation, and their lack of stability against enzymatic degradation. Successful oral delivery of peptides will depend, therefore, on strategies designed to alter the physicochemical properties of these potential drugs, without changing their biological activity, in order to circumvent the barrier properties of the intestinal epithelial cells. This manuscript will focus on the physical and metabolic barrier functions of the intestinal mucosa, the structural features of peptides which influence their passive diffusion and carrier-mediated transport, including efflux mechanisms, and various approaches used to prevent enzymatic degradation of the peptides and increase their permeability across the intestinal mucosa.

Effect of size and charge on the passive diffusion of peptides across Caco-2 cell monolayers via the paracellular pathway.

AbstractPurpose. To evaluate the effect of size and charge on the permeation characteristics of peptides across the intestinal mucosa. Methods. The lipophilicities of neutral, positively and negatively charged capped amino acids (Asn, Lys, Asp), tripeptides (Ac-Gly-X-Ala-NH2; X = Asn, Lys, Asp) and hexapeptides (Ac-Tip-Ala-Gly-Gly-X-Ala-NH2; X = Asn, Lys, Asp) were estimated using an immobilized artificial membrane. The diffusion coefficients used to calculate the molecular radii were measured by NMR. The transport characteristics of the model peptides were determined across Caco-2 cell monolayers. Results. When model compounds having the same charge were compared, permeation was highly size-dependent (capped amino acids > tripeptides > hexapeptides), suggesting transport predominantly via the paracellular route. For example, the flux of the negatively charged Asp amino acid (Papp = 10.04 ± 0.43 × 10−8 cm/s) was 3 times greater than that observed for the Asp-containing hexapeptide (Papp = 3.19 ± 0.27 × 10−8 cm/s). When model compounds of the same size were compared, permeation across the cell monolayer was charge-dependent (negative < positive ≤ neutral). For example, the neutral, Asn-containing tripeptide (Papp = 25.79 ± 4.86 × 10−8 cm/s) was substantially more able to permeate the Caco-2 cell monolayer than the negatively charged Asp-containing tripeptide (Papp = 7.95 ± 1.03 × 10−8 cm/s) and the positively charged Lys-containing tripeptide (Papp = 9.86 ± 0.18 × 10−8 cm/s). The permeability of the cell monolayer to peptides became less sensitive to net charge as the size of the peptides increased. Conclusions. A positive net charge of hydrophilic peptides enhances their permeation across the intestinal mucosa via the paracellular pathway. With increasing molecular size, molecular sieving of the epithelial barrier dominates the transport of peptides, and the effect of the net charge becomes less significant.

Effect of restricted conformational flexibility on the permeation of model hexapeptides across Caco-2 cell monolayers

AbstractPurpose. To determine how restricted conformational flexibility of hexapeptides influences their cellular permeation characteristics. Methods. Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asp, Asn, Lys) and cyclic (cyclo[Trp-Ala-Gly-Gly-X-Ala]; X = Asp, Asn, Lys) hexapeptides were synthesized, and their transport characteristics were assessed using the Caco-2 cell culture model. The lipophilicities of the hexapeptides were determined using an immobilized artificial membrane. Diffusion coefficients used to calculate molecular radii were determined by NMR. Two-dimensional NMR spectroscopy, circular dichroism, and molecular dynamic simulations were used to elucidate the most favorable solution structure of the cyclic Asp-containing peptide. Results. The cyclic hexapeptides used in this study were 2−3 times more able to permeate (e.g., Papp = 9.3 ± 0.3 × 10−8 cm/sec, X = Asp) the Caco-2 cell monolayer than were their linear analogs (e.g., Papp = 3.2 ± 0.3 × 10−8 cm/sec, X = Asp). In contrast to the linear hexapeptides, the flux of the cyclic hexapeptides was independent of charge. The cyclic hexapeptides were shown to be more lipophilic than the linear hexapeptides as determined by their retention times on an immobilized phospholipid column. Determination of molecular radii by two different techniques suggests little or no difference in size between the linear and cyclic hexapeptides. Spectroscopic data indicate that the Asp-containing linear hexapeptide exists in a dynamic equilibrium between random coil and β-turn structures while the cyclic Asp-containing hexapeptide exists in a well-defined compact amphophilic structure containing two β-turns. Conclusions. Cyclization of the linear hexapeptides increased their lipophilicities. The increased permeation characteristics of the cyclic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of these increased lipophilicities. Structural analyses of the cyclic Asp-containing hexapeptide suggest that its well-defined solution structure and, specifically the existence of two β-turns, explain its greater lipophilicity.

The effect of size, charge and cyclization of model peptides on their in vitro release from DL-PLGA microspheres

Abstract This study was designed to examine the effects of size, charge and conformation on the release of peptides from microspheres of poly ( d,l -lactic-co-glycolic) acid 50:50 ( dl -PLGA). These microspheres were prepared using a double emulsion technique and were characterized for size, morphology and peptide release kinetics. The influence of formulation variables such as water volume of the internal-phase and excipient loading were also investigated. The linear hexapeptides (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2, X=Asp, Asn, Lys) used in this study were chosen because of their ability to form β-turn structures in solution. To determine what effect restricting the conformational flexibility would have on the release of peptide, a series of cyclic hexapeptides (cyclo [Trp-Ala-Gly-Gly-X-Ala], X=Asp, Asn, Lys), which exist primarily in compact, β-turn solution conformations, were also studied. The capped-amino acids (Ac-X-NH2, X=Asp, Asn, Lys) were used to determine the effect that size (MW, hydrodynamic volume) and charge have on their release from dl -PLGA microspheres. The volume of water used as the internal-phase has a significant effect on the release of the linear hexapeptide Ac-Trp-Ala-Gly-Gly-Asp-Ala-NH2. For example, when no water is added, the release appears to be erosion-controlled. In contrast, when 150, 500 or 1000 μl of water is added, the release shifts to a diffusion-controlled mechanism. Changing the size, charge and conformational flexibility of the model peptides had no significant effect on their release kinetics. The differences in release kinetics that were observed for the neutral capped amino acid and the positively-charged cyclic hexapeptide are due to unique specific interactions between these compounds and dl -PLGA.

Evaluation of the AERx Pulmonary Delivery System for Systemic Delivery of a Poorly Soluble Selective D-1 Agonist, ABT-431

AbstractPurpose. ABT-431 is a chemically stable, poorly soluble prodrug that rapidly converts in vivo to A-86929, a selective dopamine D-1 receptor agonist. This study was designed to evaluate the ability of the AERx™ pulmonary delivery system to deliver ABT-431 to the systemic circulation via the lung. Methods. A 60% ethanol formulation of 50 mg/mL ABT-431 was used to prepare unit dosage forms containing 40 μL of formulation. The AERx system was used to generate a fine aerosol bolus from each unit dose that was collected either onto a filter assembly to chemically assay for the emitted dose or in an Andersen cascade impactor for particle size analysis. Plasma samples were obtained for pharmacokinetic analysis after pulmonary delivery and IV dosing of ABT-431 to nine healthy male volunteers. Doses from the AERx system were delivered as a bolus inhalation(s) (1, 2, 4, and 8 mg) and intravenous infusions were given over 1hr (5 mg). Pharmacokinetic parameters of A-86929 were estimated using noncompartmental analysis. Results. The emitted dose was 1.02 mg (%RSD = 11.0, n = 48). The mass median aerodynamic diameter of the aerosol was 2.9 ± 0.1 μm with a geometric standard deviation of 1.3 ± 0.1 (n = 15). Tmax (mean ± SD) after inhalation ranged from 0.9 ± 0.6 to 11.5 ± 2.5. The mean absolute pulmonary bioavailibility (as A-86929) based on emitted dose ranged from 81.9% to 107.4%. Conclusions. This study demonstrated that the AERx pulmonary delivery system is capable of reproducibly generating fine nearly monodisperse aerosols of a small organic molecule. Aerosol inhalation utilizing the AERx pulmonary delivery system may be an efficient means for systemic delivery of small organic molecules such as ABT-431.