Dispersible hydrolytically sensitive nanoparticles for nasal delivery of thyrotropin releasing hormone (TRH).

Nose-to-brain delivery of drugs is affected by nanoparticles (NPs) deposited on the olfactory surface and absorbed directly into the brain. Thyrotropin releasing hormone (TRH), a water soluble drug used for treating suicidal patients, was incorporated into a fast degrading poly(sebacic anhydride) (PSA) NPs. NPs were prepared by a solvent-antisolvent process under strict anhydrous environment to obtain high TRH loading and to avoid premature PSA degradation and TRH release. PSA and TRH were dissolved in a mixture of dichloromethane and ethanol and added dropwise to a dispersion of mannitol particles in heptane as an antisolvent. Mannitol powder was included in the antisolvent, so that formed NPs adhered to the mannitol microparticles for easy isolation and immediate dispersion in water prior to use. The size, surface charge, and morphology of the TRH-PSA NPs were determined using dynamic light scattering (DLS), zeta-potential, and Scanning Electron Microscopy (SEM), respectively. The NPs prepared were uniform and spherical of ~250 nm. Further, the in vitro release profile of TRH from NPs lasted for 12 h with most TRH released within the first hour in water. Concentration dependent cell toxicity studies revealed low toxicity level at low concentrations of the NPs. Surface adsorption of the NPs was also uniform on the cell surface as examined through the odyssey near infrared fluorescence (NIR) images using Indocyanine green (ICG). The NPs are designed to enable direct delivery to the olfactory epithelium using a refillable nasal atomizer that deposits mist onto the olfactory neuro-epithelium.

Penetration of thyroliberin in the blood and brain regions at intranasal or intravenous administration.

The maximum amounts of the thyroliberin in the blood and brain of rats at intranasal and intravenous administration were determined. It is found that rat hippocampal, cortical, and cerebellar membranes contain two types of specific binding sites (high- and low-affinity) for the labeled ligand. It was shown that, at intranasal and intravenous administration, maximum amounts of the thyroliberin were detected in the cerebellum and then in the cortex and hippocampus. The degradation of the thyroliberin in the rat brain and its regions at intranasal and intravenous administration was studied. It is shown that the degree of degradation and the formation of proteolytic products of the thyroliberin is different in different regions of the rat brain.

Intestinal transport of TRH analogs through PepT1: the role of in silico and in vitro modeling.

The present study involves molecular docking, molecular dynamics (MD) simulation studies, and Caco-2 cell monolayer permeability assay to investigate the effect of structural modifications on PepT1-mediated transport of thyrotropin releasing hormone (TRH) analogs. Molecular docking of four TRH analogs was performed using a homology model of human PepT1 followed by subsequent MD simulation studies. Caco-2 cell monolayer permeability studies of four TRH analogs were performed at apical to basolateral and basolateral to apical directions. Inhibition experiments were carried out using Gly-Sar, a typical PepT1 substrate, to confirm the PepT1-mediated transport mechanism of TRH analogs. Papp of the four analogs follows the order: NP-1894 < NP-2378 < NP-1896 < NP-1895. Higher absorptive transport was observed in the case of TRH analogs, indicating the possibility of a carrier-mediated transport mechanism. Further, the significant inhibition of the uptake of Gly-Sar by TRH analogs confirmed the PepT1-mediated transport mechanism. Glide docking scores of all the four analogues were in good agreement with their transport rates, suggesting the role of substrate binding affinity in the PepT1-mediated transport of TRH analogs. MD simulation studies revealed that the polar interactions with amino acid residues present in the active site are primarily responsible for substrate binding, and a downward trend was observed with the increase in bulkiness at the N-histidyl moiety of TRH analogs.

Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 µM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

Novel thyrotropin-releasing hormone analogs: a patent review.

INTRODUCTION: The potential therapeutic applications of thyrotropin-releasing hormone (TRH) have attracted attention, based on its broad-spectrum neuropharmacological action rather than its endocrine properties. These central nervous system (CNS)-mediated effects provide the rationale for use of TRH and its analogs in the treatment of brain and spinal injury, and CNS disorders like schizophrenia, Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis, Parkinson's disease, depression, shock and ischemia. AREAS COVERED: This review summarizes the patent literature and advances in the discovery and development of novel TRH analogs over the past 20 years. It provides a comprehensive overview of the development of new TRH analogs, giving emphasis to their pharmaceutical profile. EXPERT OPINION: The use of TRH in the treatment of various CNS disorders has been proven clinically. However, TRH itself is a poor drug candidate due to its short plasma half-life (5 min), poor biopharmaceutical properties (low intestinal and CNS permeability) and endocrine side effect. Nevertheless, researchers have come up with metabolically stable, more potent and selective TRH analogs and prodrugs. Taltirelin, one of the TRH analogs, has been approved under the trade name of Ceredist(®) in Japan for the treatment of spinocerebellar degeneration. Several other TRH analogs are in various stages of preclinical or clinical development.

Application of hydrogel polymers for development of thyrotropin releasing hormone-loaded adhesive buccal patches.

To utilize hydrogels for fabricating thyrotropin releasing hormone (TRH) adhesive buccal patches, type of hydrogels such as polyacrylic acids (Polycarbophil AA1, Carbopols 934P, 974P and 971P), celluloses (HPMC K4M, K4MCR and K15M), polysaccharide (sodium alginate) and polyacrylic acid combinations with either cellulose or polysaccharide were evaluated for adhesion force, water uptake and swelling capacity. Upon the characterization of hydrogel polymers, TRH-loading of patches fabricated from these hydrogels was evaluated at various polymer concentrations, combinations and ratios and then in vitro release kinetics of TRH from these patches were studied. Results indicated that maximum adhesion force was shown by polyacrylic acids. Adhesive force of polymer combination mainly resulted from combination of adhesive force, according to ratio proportion used, of each polymer without any superimposed effect of polymer combination. Polycarbophil AA1 showed highest water uptake and swelling capacity. Maximum TRH-loading was obtained with sodium alginate and Polycarbophil AA1 and sodium alginate combination. TRH release profiles revealed that release was sustained from Polycarbophil AA1 and its combination with celluloses or polysaccharide at 2:1 level of polymer ratio. Based on adhesion, loading and release characteristics, patches of Polycarbophil AA1 with K4M, K4MCR and sodium alginate were concluded to be suitable for further development.

Exploratory neuropharmacological evaluation of a conformationally constrained thyrotropin-releasing hormone analogue.

A conformationally constrained peptidomimetic derived from the endocrine and neuroactive tripeptide thyrotropin-releasing hormone (pGlu-His-Pro-NH(2)) was synthesized by convenient solid-phase organic chemistry and evaluated as a potential central nervous system agent. While this ethylene-bridged peptide analogue has been reported to lack the hormonal effect of the native peptide, we have shown in animal models that it possesses central nervous system activity characteristic of thyrotropin-releasing hormone. Compared to control, the peptidomimetic showed significant analeptic and antidepressant-like potencies. Moreover, an enhanced selectivity in antidepressant-like effect was measured when compared to that of the native peptide. Immobilized artificial membrane chromatography and in vitro metabolic stability studies also revealed that this constrained peptidomimetic has higher affinity to the blood-brain barrier than the native peptide and is metabolically stable. Consequently, this structure may be used as a template to design centrally selective and metabolically stable thyrotropin-releasing hormone analogues as potential neuropharmaceutical agents.

Endocrine function is altered in chronic migraine patients with medication-overuse.

OBJECTIVE: To evaluate the effects of analgesic overuse on endocrine function in patients with chronic migraine and medication-overuse headache (CM-MOH). BACKGROUND: Chronic migraine is frequently associated with an overuse of symptomatic medications. Drugs currently used in acute migraine attacks are associated with several endocrine effects. At present, the endocrine effects of medication overuse in chronic migraine patients are unknown. METHODS: Eighteen patients with CM-MOH, diagnosed according to the ICHD-II criteria, and 18 healthy controls received an intravenous administration of GHRH, hCRH, and TRH. Plasma concentrations of GH, TSH, ACTH, and cortisol were measured for a 90-minute period after administration of the specific releasing hormones. RESULTS: Hormonal basal concentrations were similar in both groups. GH response to GHRH was significantly reduced in patients with CM-MOH in comparison with controls. TRH induced a reduction of TSH concentrations only at the end of the test. After hCRH administration, ACTH and cortisol concentrations were significantly higher in cases than in controls. A significant correlation between duration of the disease and altered hormonal response was found. CONCLUSIONS: Our study shows that both corticotropic and somatotropic functions are significantly impaired in CM-MOH patients and suggests a role for hormones in the development of chronic migraine.

Centrally acting and metabolically stable thyrotropin-releasing hormone analogues by replacement of histidine with substituted pyridinium.

Metabolically stable and centrally acting thyrotropin-releasing hormone (TRH) analogues were designed by replacing the central histidine with substituted pyridinium moieties. Their analeptic and acetylcholine-releasing actions were evaluated to assess their potency as central nervous system (CNS) agents. A strong experimental connection between these two CNS-mediated actions of the TRH analogues was obtained in subject animals. The analogue 3-(aminocarbonyl)-1-(3-[2-(aminocarbonyl)pyrrolidin-1-yl]-3-oxo-2-[[(5-oxopyrrolidin-2-yl)carbonyl]amino]propyl)pyridinium (1a) showed the highest (TRH-equivalent) potency and longest, dose-dependent duration of action from a series of homologous compounds in antagonizing pentobarbital-induced narcosis when administered intravenously in its CNS-permeable prodrug form (2a) obtained via reduction of the pyridinium moiety to the nonionic dihydropyridine. The maximum change in hippocampal acetylcholine concentration upon perfusion of the pyridinium-containing tripeptides into the hippocampus of rats was also achieved with 1a. No binding to the endocrine TRH receptor was measured for the TRH analogues reported here; therefore, our design afforded a novel lead for centrally acting TRH analogues. We have also demonstrated the benefits of the prodrug approach on the pharmacokinetics and brain uptake/retention of pyridinium-containing TRH analogues (measured by in vivo microdialysis sampling) upon systemic administration.

Sistema GH/Prolactina y crecimiento prenatal / GH/Prolactine system and prenatal growth

Hasta muy recientemente se ha considerado que durante la vida fetal la hormona somatotropa (GH) no ejercía efecto alguno sobre el crecimiento prenatal, a consecuencia de la expresión tardía y progresiva maduración de los receptores de la hormona. Sin embargo estudios clínicos de déficit congénitos del crecimiento y el descubrimiento del perfil de expresión de los receptores de GH (GHR) y prolactina (PRLR) durante el desarrollo embrionario y fetal, ha impulsado la búsqueda de nuevos conocimientos sobre el papel que las hormonas del sistema GH/PROLACTINA (GH/PRL) tienen en el desarrollo de esta etapa temprana de la vida. Al crecimiento fetal contribuyen las hormonas del sistema GH/ PRL, en el que se incluye el eje GH-IGF-I, procedentes de los compartimientos materno, placentario y fetal. Las hormonas GH placentaria (PGH) y Lactógeno placentario (LP) son liberadas a la circulación materna y la hipófisis del feto secreta prolactina y GH a la circulación fetal, a la cual también se libera el lactógeno placentario procedente de la madre. Aunque su función es poco conocida, este sistema de péptidos hormonales parecen intervenir en el proceso del desarrollo desde la etapa de la preimplantación, en la diferenciación de órganos y tejidos embrionarios y en los mecanismos que ayudan al crecimiento, la maduración y la transición del feto a la vida extrauterina. Estudios de defectos congénitos del crecimiento han permitido proponer dos posibles modelos que explicarían la relación entre la formación del factor IGF-I (mediador unívoco y necesario en el crecimiento fetal) y el resto de componentes materno-fetales del eje GH/Prolactina: bien la producción del factor IGF-I, sería independiente de la influencia somatotropa durante el segundo trimestre de gestación; o bien existiría un mecanismo de cooperación redundante, entre todas las hormonas somatotropas del eje GH/Prolactina y de sus receptores. El hallazgo clínico de mutantes que eliminan la contribución de parte del potencial hormonal del sistema GH/PRL así como de un mutante antagonista de GH, que suprime la redundancia a nivel receptor y origina un profundo retardo del crecimiento intrauterino, sugiere que sería la acción concatenada de estas hormonas, actuando sobre los receptores somatógeno y lactógeno fetales, los que mediante la estimulación de la producción de IGF-I determinaría el crecimiento fetal. Ello implica, que la sensibilidad tisular a la GH podría iniciarse a partir del segundo tercio de la gestación, momento en que el eje somatotrópico sería activo sobre el crecimiento fetal (AU)

Thyrotropin-releasing hormone administration does not affect seizure threshold during electroconvulsive therapy.

SUMMARY: Despite the fact that a role for thyrotropin-releasing hormone (TRH) in seizure modulation has been consistently hypothesized, the exact nature of this role remains unclear. In this study, we investigated the effects of TRH administration on seizure threshold and seizure duration in 13 depressed inpatients undergoing electroconvulsive therapy (ECT). In a balanced order crossover design, an intravenous bolus of 0.4 mg TRH or placebo was administered immediately before anesthesia, during the first two sessions, in a series of bilateral ECT. In both of these sessions, a threshold titration procedure was applied by using gradual increments of the electrical charge delivered until seizure elicitation, a procedure that has been safely used in the past. Seizure threshold was defined as the lowest energy level required for induction of a grand mal seizure, by use of this titration procedure. Seizure duration was estimated both by simultaneous EEG recording and by the cuff method. Results showed that neither seizure threshold, nor seizure duration (either by cuff or by EEG) differed between the TRH and the placebo conditions, regardless of the order in which TRH or placebo were administered in the two ECT sessions. This was the case regardless of whether the patients had at baseline a blunted TSH response to TRH or not. Our findings do not support a role for TRH on seizure modulation, at least when TRH is administered exogenously. Such an effect, if it exists, could be obscured, however, by several factors, including pharmacokinetics.

Studies on the transport of thyrotropin-releasing hormone (TRH) analogues in Caco-2 cell monolayers.

The transport mechanisms of thyrotropin-releasing hormone (TRH) and its pharmacologically active analogues ((3-methyl-His(2))TRH (MeTRH), taltirelin, montirelin, azetirelin) across Caco-2 cell monolayers were characterized. The results of kinetic analysis showed a linear relationship between the concentration (over the range 0.5-10 mM) and apical-to-basolateral transport rate of these agents. The permeability coefficients (P(app)) of these agents were not substantially different from each other, and their P(app) ratios of the basolateral-to-apical over the apical-to-basolateral transport were close to one (0.73-1.23). The cellular transport of [(3)H]MeTRH at low concentrations (3-15 nM) showed a linear relationship between the concentration and transport rate. The transport of [(3)H]MeTRH in Caco-2 cell monolayers was neither affected by TRH nor TRH analogues, and there was little difference in P(app) values between [(3)H]MeTRH and [(14)C]mannitol. The cell-per-medium ratio of [(3)H]MeTRH in the cellular uptake experiment was similar to the value of [14C]mannitol. A large excess of TRH and MeTRH did not significantly influence cell-per-medium ratios of [(3)H]MeTRH in Caco-2 cell monolayers. The k'(IAM) value, which represents lipophilicity, was decreased in the following order: montirelin > taltirelin > TRH > azetirelin, and the values varied from 0.234 to 1.028. These results indicate that a paracellular passive diffusion may be the major route for the transport of TRH and its analogues in Caco-2 cell monolayers.

Prodrugs to enhance central nervous system effects of the TRH-like peptide pGlu-Glu-Pro-NH2.

Potential prodrugs for the TRH-like tripeptide pGlu-Glu-Pro-NH(2) were synthesized either by esterifying the Glu side-chain of the parent peptide in solution with alcohols in the presence of resin-bound dicyclohexylcarbodiimide or by solid-phase peptide chemistry. Affinities of these ester prodrugs to lipid membranes as predictors of the transport across the blood-brain barrier were compared by immobilized artificial membrane chromatography, and prodrug activation was tested in the brain tissue of experimental animals. Esters of pGlu-Glu-Pro-NH(2) with long-chain primary alcohols emerged as potentially useful prodrugs to improve the central nervous system activity of pGlu-Glu-Pro-NH(2) upon systemic administration, as revealed by the enhancement of analeptic activity in mice.

Synthesis and biological evaluations of brain-targeted chemical delivery systems of [Nva2]-TRH.

Various chemical delivery systems for [Nva2]-TRH were synthesized and their CNS activity was investigated and compared with that of a similar chemical delivery system of [Leu2]-TRH, previously studied. Sequential metabolism of the chemical delivery system delivered to the brain, starting with the conversion of the dihydrotrigonellyl (DHT) to the trigonellyl (T+) moiety, will provide the lock-in to the brain of the T+-chemical delivery system, which will undergo hydrolysis of the cholesteryl ester, formation of the Pr-amide and cleavage of the spacer-T+ part, allowing ultimately the sustained release of the active [Nva2]-TRH. The CNS activity was assessed by measuring the extent of antagonizing barbiturate-induced sleeping time in mice. The fully packaged DHT-Pro-Pro-Gln-Nva-Pro-Gly-OCh produced robust antagonism, reducing sleeping time from 89 min to 48 min, similar to the Leu2-analogue (49 min). However, the partially substituted [Nva2]-TRH analogues showed little or no CNS activity. The results indicate that the fully packaged delivery system is necessary to produce the successful brain targeting of the precursor construct and effective release of the Gln-Nva-ProNH2.

Effect of 2-hydroxypropyl-beta-cyclodextrin on the solubility, stability, and pharmacological activity of the chemical delivery system of TRH analogs.

To improve the aqueous solubility and stability of the chemical delivery system (CDS) of the thyrotropin-releasing hormone (TRH) analogs, 2-hydroxypropyl-beta-cyclodextrin (HPBCD) has been attempted. TRH analogs were [Leu2]-TRH, [Nva2]-TRH and [Nva2, Pip3]-TRH. Excess amount of CDS was added in various HPBCD in water solutions (0%-50%, pH 6.5). The mixture was saturated by ultra-sonication for 1 h at 15 degrees C and filtered. The concentration of CDS in the filtrate (solubility) was determined with UV detector, and subsequently the stability was investigated. By HPBCD complexation, the aqueous solubility and stability (half-life) of CDS were significantly improved from undetectable levels to about 15 mg/ml and 30 h, respectively. In pH 6.5 and 7.4 HPBCD solution, the degradation of CDS was mainly via acid catalyzed water addition reaction, thus, e.g. [Leu2]-TRH-CDS was more stable in pH 7.4 than in pH 6.5 aqueous solutions. After lyophilizing the saturated CDSs in 50% HPBCD complex solutions, the amount of CDS in the complex was determined as 26.22, 26.79, and 30.34 mg/g for [Leu2]-TRH, [Nva2]-TRH and [Nva2, Pip3]-TRH, respectively. The half-life of [Leu2]-TRH-CDS/HPBCD solid complex at 25 degrees C, 4 degrees C and -15 degrees C was about 100 days, 440 days and no detectable change in three months, respectively. Argon protected condition did not improve the stability of lyophilized [Leu2]-TRH-CDS/HPBCD complex. Dimethyl sulfoxide although increased the solubility of [Leu2]-TRH-CDS in the 50% HPBCD solution by 1.3 times, significantly decreased its stability by 6.6 times. After intravenous administration of CDS (in 30% HPBCD) at a dose of 10 mumole/kg in mice, compared to the vehicle control or the same dose of [Leu2]-TRH (in 30% HPBCD), a significant increase in pharmacological effect (decrease in barbiturate-induced sleeping time) was observed. These results demonstrate the usefulness of cyclodextrin in the formulation of the CDSs of TRH analogs.

Brain receptor binding characteristics and pharmacokinetic-pharmacodynamic analysis of thyrotropin-releasing hormone analogues.

Thyrotropin-releasing hormone (TRH) receptor binding in the rat brain after intravenous (i.v.) injections of novel TRH analogues, taltirelin and montirelin, was examined and the data were analyzed in relation to their plasma concentrations which were simultaneously determined. Taltirelin and montirelin inhibited specific [3H]-Me-TRH binding in the rat brain and their Ki values were 311 and 35.2 nM, respectively. The i.v. injection of taltirelin and montirelin (0.1-3 mg/kg) produced a significant reduction in [3H]-Me-TRH binding sites (Bmax values) in the rat brain. The reduction by both agents tended to reach a maximum after 60 min and lasted up to at least 120 min. On the other hand, the i.v. injection of both agents had little significant effect on the apparent dissociation constant (Kd) for [3H]-Me-TRH in the rat brain. Plasma concentrations of taltirelin and montirelin in rats peaked immediately after i.v. injection, and thereafter they decreased with t 1/2 of 23.0 and 14.1 min, respectively. Counter-clockwise hysteresis between the plasma concentration and receptor occupancy of these agents was observed after the i.v. injection of taltirelin and montirelin, and the temporal delay between plasma concentration and brain receptor occupancy was successfully minimized using the "effect compartment" model in combination with the "linear-effect" model. We concluded that taltirelin and montirelin exert a fairly potent effect following sustained occupation of brain TRH receptors under in vivo condition. Thus, both agents could be clinically useful for the treatment of CNS disorders.

Effects of sodium glycocholate and protease inhibitors on permeability of TRH and insulin across rabbit trachea.

The permeabilities of thyrotropin-releasing hormone (TRH) and insulin as model peptides were examined to characterize the tracheal epithelial barrier in in vitro experiments using excised rabbit trachea. TRH was not metabolized during 150 min duration of tracheal permeation and the apparent permeability coefficient (Papp) for TRH was about 3 x 10(-7) cm/s. The tracheal permeability of TRH was increased about three times by 10 mM glycocholate as a permeation enhancer. Insulin showed a slight degradation during 150 min duration of tracheal permeation, the Papp for insulin was 7 x 10(-9) cm/s. The tracheal permeability of insulin was significantly increased by 10 mM glycocholate, 1 mM bestatin (aminopeptidase B and leucine aminopeptidase inhibitor), and 10,000 KIU/ml aprotinin (trypsin and chymotrypsin inhibitor). The peptidase activities of rabbit tracheal epithelium were found to be the following; di-peptidyl-aminopeptidase IV (DPP IV) > Leu-aminopeptidase > cathepsin-B > trypsin. These activities were significantly lower than those of jejunal mucosal tissues. These results suggest that the tracheal absorption of peptide drugs through the respiratory tract may contribute to the systemic delivery of these drugs following the pulmonary administration of these drugs by intratracheal insufflation and instillation.

Modification of ocular permeability of peptide drugs by absorption promoters.

The purpose of this study was to investigate the effect of absorption promoters on the ocular membrane permeability of thyrotropin-releasing hormone (TRH) and luteinizing hormone-releasing hormone (LHRH) as model peptides. The permeabilities of TRH and LHRH were measured using a two-chamber glass diffusion cell mounted with isolated ocular membranes of albino rabbits. Saponin, EDTA, benzalkonium chloride and paraben were used as absorption promoters. These promoters enhanced the permeability of hydrophilic molecules through the cornea and conjunctiva. The promoting effects of the absorption promoters on the conjunctival drug penetrations were not as strong as those on the corneal penetrations. The different responses of the corneal and conjunctival drug penetrations to these promoters may be useful in controlling the extent and pathway of the ocular and systemic absorptions of instilled drugs. The promotional effects of absorption promoters on the corneal drug penetration apparently increased with an increase in penetrant molecular weights, although those on the conjunctival drug penetrations did not depend on the molecular weights.

Evaluation of the Caco-2 monolayer as a model epithelium for iontophoretic transport.

PURPOSE: To assess the Caco-2 monolayer as a model for iontophoresis of drugs across a model epithelium. METHODS: The apparent permeability co-efficient (Papp) of mannitol, thyrotrophin releasing hormone (TRH), dexamethasone and a range of sizes of fluorescein isothiocyanate (FITC) dextrans across Caco-2 monolayers was measured under passive and electrically stimulated conditions. Trans-epithelial electrical resistance (TEER) was determined throughout. Transmission electron micrographs (TEM) of the monolayers were taken. Confocal laser scanning microscopy (CLSM) was used to visualize the iontophoretic transport route of FITC-Dextran (MW = 20 kDa) across a Caco-2 monolayer. RESULTS: Application of 14.3 micro-Eq x cm(-2) across the monolayer evoked a transient drop in TEER. The drop in TEER was accompanied by statistically significant increases in fluxes of all the agents in the mucosal to serosal direction except for FD-70. TEM of test samples exhibited tight junction dilatation, in addition to intracellular vacuolisation. The iontophoresis of FD-20 was visualised with confocal laser scanning microscopy and was localised in paracellular spaces of the monolayer. CONCLUSIONS: The fluxes of mannitol, TRH, dexamethasone, FD-4, FD-10 and FD-20 across the Caco-2 monolayer were significantly enhanced when electric field was applied. The iontophoretic effect appeared to be directly upon tight junctions