The Early Preclinical Development Program for Locally Administered Investigational Medicinal Products in Ophthalmology: Preclinical Data Required for Starting a First-in-Human Clinical Trial in Europe-Basic Considerations and 2 Case Studies.

BACKGROUND: Although regulatory guidance defines which preclinical data are required in general before proceeding to first-in-human clinical trials, a certain level of flexibility exists in the actual planning, timing, and design of a drug development program. Developing an ophthalmic medicinal product adds additional challenges, since the eye is a complex organ with unique features and specialized ophthalmic guidance documents are sparse. METHODS: We analyzed the preclinical guidelines with a focus on European Union legislation and guidance documents provided by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). We elaborated the particularities specific to ophthalmic drug developments and deduced the preclinical knowledge needed to safely enter a first-in-human trial program. Two hypothetical medicinal products for ophthalmic indications were chosen and specificities for ophthalmic preclinical tests were elaborated. RESULTS AND CONCLUSION: We conclude that the preclinical program of ophthalmic medicines is flexible and differs, based on the intended use and the nature of the active substance.

Human duodenal proteome modulations by glutamine and antioxidants.

PURPOSE: Glutamine (Gln) has protective, anti-inflammatory effects in animal models and humans. Antioxidant nutrients may exert synergistic effects on intestinal functions. Therefore, these combined nutrients may have a therapeutic potential during intestinal inflammation. This study was designed to investigate in humans the effects of a supplement composed of Gln and high-dosed antioxidant micronutrients compared to isomolar Gln only, on duodenal proteome. EXPERIMENTAL DESIGN: Enteral perfusion of Gln (0.8 mmol x kg(-1) x h(-1)) or supplement was performed in two groups of six healthy volunteers during 5 h before taking endoscopic duodenal biopsies. Protein expression was analyzed by 2-DE and the relevant proteins identified by MS/MS. RESULTS: About 1500 protein spots were revealed in both supplement and Gln conditions. Comparative proteomics analysis indicated that 11 proteins were differentially and significantly (p≤0.05) expressed in response to the supplement. These proteins were essentially implicated in metabolism pathways, e.g. fatty acid binding protein-1 and 40S ribosomal protein SA expressions were downregulated while manganese superoxide dismutase and retinal dehydrogenase-1 expressions were upregulated. CONCLUSIONS AND CLINICAL RELEVANCE: This study provides new information on human duodenal proteome and its nutritional modulation, and supports further clinical investigations designed to evaluate the effects of Gln plus antioxidants during intestinal inflammation and cancer.

Novel RPLC stationary phases for lipophilicity measurement: solvatochromic analysis of retention mechanisms for neutral and basic compounds.

An RPLC was developed to rapidly determine lipophilicity of neutral and basic compounds using three base deactivated RPLC stationary phases particularly designed for the analysis of basic compounds, namely, Supelcosil ABZ(+)Plus, Discovery RP Amide C16, and Zorbax Extend C18. The work consisted of three sets of experiments. In the first log kw values of neutral compounds were extrapolated using hydroorganic mobile phases at different compositions. Good correlation between log kw and log Poct indicated that the method was appropriate for these supports, without adding a silanol masking agent. In the second set of experiments, isocratic log k values of neutral and basic compounds were measured with three different mobile phases. The best estimation of lipophilicity was obtained for neutral and basic compounds when the secondary interactions were strongly reduced (i. e., when basic compounds were under their neutral form). In the third set of experiments, isocratic retention factors of basic compounds (in their neutral form) were measured with a high-pH mobile phase, on a chemically stable support (Zorbax Extend C18). Under these chromatographic conditions, correlation between the isocratic retention factors and log Poct (log D10.5) for basic compounds was similar to that for neutral compounds.

Determination of pKa values by capillary zone electrophoresis with a dynamic coating procedure.

CZE allows to measure the acidic dissociation constant (pKa) of many drug substances. However, determining the EOF intensity may be time-consuming, especially at a low pH. In order to overcome this drawback, a dynamic coating procedure of the capillary was carried out to increase microEOF, and thus to reduce the analysis time. In addition, this coating procedure enhanced migration time stability. The effective mobilities of 15 compounds were measured at different pH, producing pK'a values dependent on BGE ionic strength. The latter values were corrected with the activity coefficient to obtain a "true" pKa value. The 15 investigated compounds were (i) five acids: namely, salicylic acid, benzoic acid, ketoprofen, phenobarbital, and phenol, (ii) four bases: lidocaine, propafenone, propranolol, and quinine, (iii), five ampholytes: sulfanilamide, sulfabenzamide, sulfadimethoxine, sulfadoxine, and sulfisoxazole, and (iv) one zwitterion: cetirizine. The range of determined pKa values was between 1.2 and 11.2, and close to the pKa values available from the literature.

Partitioning of halofantrine hydrochloride between water, micellar solutions, and soybean oil: Effects on its apparent ionization constant.

Recent studies in a conscious dog model demonstrated intestinal lymphatic transport to be a significant contributor to the bioavailability of the highly lipid-soluble free-base of halofantrine (Hf), and surprisingly, also the poorly lipid-soluble hydrochloride salt (Hf. HCl). Partial conversion of solubilized Hf. HCl to Hf base within the intestinal lumen prior to the lymphatic uptake seemed to be the most likely explanation for these results. This hypothesis was supported by studies exploring the partitioning behavior of Hf. HCl between soybean oil (SBO) and aqueous micellar solutions containing different ionic and nonionic surfactants. Mixed micelles prepared from sodium taurodeoxycholate (NaTC) and lecithin (PC) were chosen to represent fed-state intestinal fluids. The apparent ionization constants derived from the partitioning versus pH profiles showed marked shifts when compared with the likely aqueous pK(a) value. In the present paper, the apparent pK(a) values of Hf in aqueous micellar phases, without a coexisting oil phase, were investigated to further probe the mechanisms underlying the effect of micellar media on the apparent ionization equilibrium, and subsequently, on its partitioning behavior in the triphasic systems. Another aim of this study was to further evaluate the aqueous pK(a) value of Hf. The results indicate that the aqueous pK(a) of Hf is most probably in the range approximately 8-9, and that the ionization equilibrium is highly dependent on the solution environment. For example, marked pK(a) shifts of several units were observed for Hf in the presence of different micellar species and SBO. The apparent ionization equilibrium depends not only on interaction of Hf with the micelles, but also on its partitioning into the oil phase.

Immobilized artificial membrane liquid chromatography: proposed guidelines for technical optimization of retention measurements.

The objectives of this study were to establish guidelines for the proper measurement of capacity factors (log k(IAMw) on immobilized artificial membrane (IAM) stationary phases. In this context, some aspects related to the extrapolation of log(kIAMw) values, the stability and properties of IAM.PC.DD2 stationary phases and the column-to-column variability are discussed. No significant difference was observed when using either acetonitrile or methanol for the linear extrapolation of log k(IAM) values. However, methanol seems more appropriate when working with ionized compounds. Plotting isocratic capacity factors against the percentage (v/v) of co-solvent instead of the mole fraction leads to more reliable log k(AMW) values. Furthermore, our results with a YMC ODS-AQ and an IAM.PC.DD2 HPLC column indicate that only small differences arise between extrapolated capacity factors when using the (w(w))pH or the (s(w))pH operational scale and correcting or not the ionic strength for dilution caused by the co-solvent. The use of the (s(w))pH scale is recommended when working with ionized compounds in order to avoid parabolic relationships during linear extrapolation. The pH-dependent retention of three ionizable drugs on an IAM.PC.DD2 phase showed that secondary interactions with the charged moieties of the chromatographic surface affect the retention of ionized compounds around physiological pH. Finally, it was shown that column ageing occurs also with IAM.PC.DD2 stationary phases and that it depends on the column as well as on the investigated analyte. The intra-batch variability for IAM.PC.DD2 phases was small, whereas a marked and solute-dependent batch-to-batch variability was apparent.