Preclinical efficacy of travoprost, a potent and selective FP prostaglandin receptor agonist.

Travoprost is the isopropyl ester prodrug of a high affinity, selective FP prostaglandin full receptor agonist. In contrast to travoprost acid's high affinity and efficacy at the FP receptor, there is only sub-micromolar affinity for the DP, EP1, EP3, EP4, IP, and TP receptors. Travoprost produced a lower incidence of ocular irritation than PGF20 isopropyl ester at a dose of 1 microg in the New Zealand albino (NZA) rabbit. Topical ocular application of travoprost produced a marked miotic effect in cats following doses of 0.01, 0.03 and 0.1 microg. In the ocular hypertensive monkey, b.i.d. application of 0.1 and 0.3 microg of travoprost afforded peak reduction in intraocular pressure (IOP) of 22.7% and 28.6%, respectively. Topical application of travoprost was well tolerated in rabbits, cats and monkeys, causing no ocular irritation or discomfort at doses up to 1 microg. Travoprost is a promising ocular hypotensive prostaglandin FP derivative that has the ocular hypotensive efficacy of PGF2alpha isopropyl ester but with less severe ocular side effects.

Effects of topically applied falintolol: a new beta-adrenergic antagonist for treatment of glaucoma.

Changes in intraocular pressure (IOP) following topical administration of falintolol (0.5%-0.25%), a new beta-blocking agent, were studied in conscious albino rabbits with alpha-chymotrypsin-induced ocular hypertension. The drug produced a reduction in IOP equal to that of timolol. A longer duration of activity was noted with falintolol. The rate of transport of topically applied falintolol through the isolated bovine cornea under conditions simulating normal physiology was linear up to three hours and twice as fast as timolol from 3 to 6 hours. Since topical ocular application of beta-adrenergic antagonists useful in glaucoma therapy can also cause a number of troublesome systemic side effects, several conclusive preclinical investigations were carried out with falintolol. Of major concern was the effect falintolol might have on the pupil, cornea, and heart rate when administered topically. The results show that falintolol does not produce any noteworthy side effects and is capable of being an effective beta-blocking agent in open-angle glaucoma therapy.

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone.

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.

Permeation of long-chain fatty acids and alcohols in rat intestine.

Uptake of long-chain fatty acids and alcohols from micellar bile salt solutions has been studied with everted sacs of rat jejunum. These unidirectional uptake rates are linearly related to lipid concentration if bile salt concentration is constant, but are essentially independent of lipid concentration over a 2.5-fold concentration range if lipid and bile salt concentrations are maintained at a constant ratio. Uptake of lipid from various micellar bile salt solutions was directly related to the experimentally varied monomer activity of solute, thus allowing conversion of uptake rates to permeation coefficients. Natural logarithm of permeation coefficients (ln P) increased linearly for saturated fatty acids of 12--18 carbons, equivalent to an incremental free energy of -695 cal.mol-1 per --CH2--. Alcohols of 10- to 14-carbon chain lengths had a similar relationship on ln P to number of carbons. Previously determined permeation coefficients for fatty acids of 2--10 carbons are now seen to be a nonlinear portion of the curve for ln P versus chain length for all saturated fatty acids.

Fatty acid and alcohol partitioning with intestinal brush border and erythrocyte membranes.

Relative partition coefficients of fatty acids and alcohols between aqueous buffers and biological membranes have been determined from the linear relationship between isotope content of sedimented membranes and aqueous concentration. This technique allows study of highly lipid soluble compounds such as long-chain saturated fatty acids. Rat intestinal brush border membranes and erythrocyte ghost membranes were studied by using homologous series of saturated fatty acids, mono-unsaturated fatty acids and 10, 12, and 14 carbon normal alcohols. The influence of chain length on partitioning was similar in the three series with an incremental free energy of -820 cal/mole per methylene group in brush borders for the saturated fatty acids. Incremental enthalpy and entropy were -1331 cal/mole and -1.64 cal/mole, degrees K respectively. Decrease in the partition coefficient due to the double bond (monounsaturated relative to saturated) had an incremental free energy of +1178 cal/mole, incremental enthalpy of -3453 cal/mole, and incremental entropy of -7.34 cal/mole, degrees K, while substitution of the hydroxyl for the ionized carboxyl group (pH 7.4) increased the partition coefficient by 72-fold. From these data it must be concluded that the lipid phase of the membrane bilayer is extremely hydrophobic, similar to heptane or polyethylene in polarity.

Determinants of fatty acid and alcohol monomer activities in mixed micellar solutions.

The determinants of monomer activities of lipids dissolved in micellar bile salt solutions have been studied using polyethylene discs as the organic phase of a partitioning system. The studies show that fatty acids and alcohols interact with micelles as a partitioning system so that the monomer activity is determined by micelle volume and the lipid's partition coefficient as well as mass of lipid in the solution. Influence of the partition coefficient is seen in the dependence of monomer activity on chain length, unsaturation and carboxyl or alcohol polar groups. Dependence on chain length is equivalent to an incremental free energy of approximately -700 cal. mol(-1) per methylene group. Substitution of an alcohol group for the carboxyl group at pH 7.4 decreases monomer activity by a factor of 900. Expansion of taurodeoxycholate micelles with 5mM monooleoylglycerol slightly decreases monomer activity whereas solutions of lipids in taurocholate have relatively greater monomer activities, demonstrating the influence of volume of the micelle organic phase. With constant micelle structure, monomer activity was linearly dependent on lipid mass in the system as predicted by partitioning theory. Addition of low concentration of lecithin, lysolecithin, or monoacylglycerol to the solutions had only small effects on the monomer activities consistent with the small change in total micelle organic phase. Data provided allow calculation of monomer activities of fatty acids and alcohols in many complex micellar solutions. Such data are important for evaluating such processes as intestinal absorption and gallstone formation and dissolution.

Apparent monomer activity of saturated fatty acids im micellar bile salt solutions measured by a polyethylene partitioning system.

Partitioning of saturated fatty acids between discs of polyethylene film and aqueous buffer has been characterized and subsequently used to measure monomer activities of fatty acids in micellar solutions of bile salt. Partitioning of fatty acids between polyethylene and buffer achieved equilibrium in about 24-48 hr. Partition coefficients for fatty acids 10:0 and 16:0 were essentially independent of concentration, as expected for true partitioning. Experiments with various pH buffers showed that only the protonated form of fatty acids 12:0 and 16:0 participated in partitioning, and the midpoints of the partition coefficients vs. pH curves were 4.5-5.0 and 6.5-7.0, respectively. Experimentally determined partition coefficients at pH 7.4 ranged from 2.03 +/- 0.09 for 9:0 to 56,100 +/- 13,850 for 17:0. The addition of each methylene group increased the partition coefficient by a factor of about 3.75, corresponding to an incremental free energy change for each methylene group of -3433 J.mole(-1) (-820 cal.mole(-1)). Monomer activities of solutions of 14:0 and 16:0 dissolved in 20 mM taurodeoxycholate were linearly dependent on the total fatty acid concentration. 1 mM 14:0 and 16:0 in 20 mM taurodeoxycholate had monomer activities of 1.3 x 10(-5) M and 5.6 x 10(-7) M, respectively. Solutions prepared with a constant concentration ratio of fatty acid to taurodeoxycholate had essentially constant monomer activities between 8 and 20 mM taurodeoxycholate. These studies support the hypothesis that fatty acid interaction with bile acid micelles is similar to a phase distribution system, so that some measurable monomer activity of fatty acid exists in equilibrium with the mass of fatty acid contained in the micelle.

Determinants of intestinal mucosal uptake of short- and medium-chain fatty acids and alcohols.

Uptake rates across the jejunal brush border have been measured for water-soluble fatty acids and alcohols and analyzed to determine the relative roles of the unstirred water layer and the lipid cell membrane as determinants of the intestinal absorptive process. Initial studies involving measurement of time courses of electrical transients developed across the intestine exposed to poorly permeant solute molecules showed no anomalous discrimination of probe molecules of different size or charge. This finding suggests that the diffusion barrier in the intestine can be considered as an unstirred water layer. Next, uptake rates of fatty acid were found to be linear with respect to concentration of the test solute, demonstrated no competitive inhibition or contralateral stimulation, had low temperature dependency, and were insensitive to metabolic inhibition, indicating that uptake proceeds by passive diffusion. Passive permeability coefficients, *P, varied from 22 +/- 1.4 to 395 +/- 9.2 nmoles.min(-1).100 mg(-1).mm(-1) for the saturated fatty acids 2:0 through 12:0 and from 119 +/- 3.3 to 581 +/- 45.2 for the saturated alcohols 6:0 through 10:0. Vigorous stirring of the bulk buffer solution enhanced *P values in direct proportion to chain length while the presence of bile acid micelles depressed apparent permeability coefficients in proportion to fatty acid chain length. These results demonstrate that uptake of short-chain fatty acid monomers is rate limited by the lipid cell membrane but diffusion through the unstirred water layer becomes increasingly rate limiting as the chain length increases. It is also possible to conclude from these data that diffusion through the unstirred water layer becomes totally rate limiting for uptake of long-chain fatty acid monomers of physiological importance.

Determination of unidirectional uptake rates for lipids across the intestinal brush border.

An in vitro method is presented which measures valid, unidirectional uptake rates for lipids across the intestinal brush border. This method combines analysis by a newly devised, double isotope counting system for solubilized tissue with the use of a nonabsorbable marker to correct gross uptake determinations for contamination by adherent mucosal fluid. Of seven markers, only [(3)H]inulin measured adherent mucosal fluid volumes as much as 20% greater than the other markers. Diffusion of the nonabsorbable marker, as well as of the compound being studied, into the unstirred layer made the time course of uptake critically important. The time lag for diffusion of marker invalidates the use of 1-min incubation periods; however, a linear time course of uptake that intercepts essentially at zero was found for taurocholate and octanoate for periods of from 2 to 5 min. Working within this critical time period with jejunum, it was shown that tissue dry weight was an appropriate measure of the amount of tissue and that uptake rates for taurocholic, octanoic, and lauric acids were linear with respect to concentration. Tissue binding of compounds was not significant. The results demonstrate that careful use of the described method yields accurate measurement of unidirectional uptake rates of lipids across the brush border that are of critical importance in defining the characteristics of membrane penetration and the rate-limiting steps in fat and sterol absorption.

Unstirred water layers in intestine: rate determinant of fatty acid absorption from micellar solutions.

Bile acid and fatty acid uptake from micellar solutions by intestinal cells fails to reflect the incremental free energy changes expected for permeation that is rate limited by cell membranes. However, altering the size of the diffulsing particle or the thickness of tle unstirred water layer does change uptake. These observations show that the unstirred water layer is rate limiting for intestinal absorption of lipids from micellar solutions.