Critical gases for critical issues: CO2 technologies for oral drug delivery.

In recent years, CO2-based technologies have gained considerable interest in the pharmaceutical industry for their potential applications in drug formulation and drug delivery. The exploitation of peculiar properties of gases under supercritical conditions has been studied in the last 20 years with mixed results. Promising drug-delivery technologies, based on supercritical CO2, have mostly failed when facing challenges of industrial scaleability and economical viability. Nevertheless, a 'second generation' of processes, based on CO2 around and below critical point has been developed, possibly offering technology-based solutions to some of the current issues of pharmaceutical development. In this review, we highlight the most recent advancements in this field, with a particular focus on the potential of CO2-based technologies in addressing critical issues in oral delivery, and briefly discuss the future perspectives of dense CO2-assisted processes as enabling technologies in drug delivery.

A novel dense CO(2) supercritical fluid technology for the development of microparticulate delivery systems containing ketoprofen.

VarioSol® is an innovative, solvent-free technology able to produce microparticles exploiting near-critical CO(2) properties as spraying and cooling agent. The aim of the present work was to evaluate the feasibility to produce in a single processing step by VarioSol® technology, oral ketoprofen-loaded microparticles with gastro-protective properties. The obtained products were powders composed of regular in shape and small in diameter microparticles, characterized by high drug content (40%) and good flow properties. Microparticles were composed by anionic lipids scarcely soluble at acidic pH, blended with gastro-resistant polymers of the methacrylate type. In vitro drug release results indicated that the drug was rapidly delivered from the microparticulate systems in phosphate buffer at pH 6.8, while in acidic medium, the microparticles were able to retard the drug release process but without reaching complete gastro-resistance. However, the results obtained in this study, although non optimal, are not far from the specifications required for gastro-resistant release products (i.e., no more than 10% drug released after 1h at pH 1.0) according to EMA guidelines and represent a good starting point for future formulation development.

The use of thiolated polymers as carrier matrix in oral peptide delivery--proof of concept.

It was the aim of this study to develop an oral delivery system for the peptide drug antide. The stability of the therapeutic peptide towards gastrointestinal peptidases was evaluated. The therapeutic agent and the permeation mediator glutathione were embedded in the thiolated polymer chitosan-4-thio-butylamidine conjugate (chitosan-TBA conjugate) and compressed to tablets. Drug release studies were performed in the dissolution test apparatus according to the Pharmacopoeia Europea using the paddle method and demineralized water as release medium. In order to avoid mucoadhesion of these delivery systems already in the oral cavity and oesophagus tablets were coated with a triglyceride. These tablets were orally given to pigs (weight: 50+/-2 kg; Edelschwein Pietrain). Moreover, antide was administered intravenously, subcutaneously and orally in solution. Results showed stability of antide towards pepsin, trypsin and chymotrypsin. In contrast, antide was rapidly degraded by elastase. Consequently a stomach-targeted delivery system was designed. Drug release studies demonstrated an almost zero-order controlled release of antide over 8 h. In vivo studies demonstrated a relative bioavailability of 34.4% for the subcutaneous administration. Oral administration of antide in solution led to no detectable concentrations of the drug in plasma at all. In contrast, administering antide being incorporated in the thiolated polymer resulted in a significant uptake of the peptide. The absolute and relative bioavailability was determined to be 1.1% and 3.2%, respectively.

NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor, hGRF(1-29)-NH2.

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.

NMR conformational analysis of antide, a potent antagonist of the gonadotropin releasing hormone.

Antide is a decapeptide [(N-Ac-D-Nal(1)-D-Cpa(2)-D-Pal(3)-Ser(4)-Lys(Nic)(5)-D-Lys(Nic)(6)-Leu(7)-Ilys(8)-Pro(9)-D-Ala(10)-NH(2)] that acts in vivo as an antagonist of GnRH (gonadotropin-releasing hormone). The conformational behavior of antide has been studied in water, TFE, DMF, and DMSO solutions by means of 2D-NMR spectroscopy and molecular dynamics calculations. Antide adopts in aqueous solution a delta-shaped backbone conformation, which is characterized by an irregular turn around residues D-Pal(3)-Ser(4) and by the close spatial proximity of the side chains belonging to D-Nal(1) and Ilys(8) (as many as 17 NOE peaks were detected between these side chains). The side-chain protons of Ilys(8) (especially the H(gamma) ones) present remarkably upfield shifted resonances, because of ring current effects induced by the naphthyl moiety. The upfield shifted resonances of the Ilys(8) H(gamma) hydrogen atoms are strictly characteristic of the water delta-shaped conformation and can be considered as structure markers. The observation of ring current shifted Ilys(8) H(gamma) resonances under different conditions (temperature, pH, solvent) indicates a remarkable stability of the water delta-shaped conformation. Such a conformation is at least partially disrupted in solvent mixtures containing high percentages of organic solvents. TFE can induce a well-defined conformation, which is characterized by an S-shaped backbone conformation. In DMF and DMSO solution, the molecule is basically endowed with a random coil conformation and high fluxionality. Antide fulfills the conformational requirements that are known to play a crucial role in receptor recognition, namely (i) the presence of a turn in the backbone and (ii) the all-trans nature of peptide bonds. In addition, the structural rigidity of antide likely adds a further contribution to the receptor binding affinity.