The efficiency of vaginal temperature measurement for detection of estrus in Japanese Black cows.

Recently, weak estrous behavior was assumed to be the cause of a decline in breeding efficiency in cattle. The present study investigated the effect of measuring the vaginal temperature on the detection of estrus in Japanese Black cows. First, the effect of hormone administration to cows with a functional corpus luteum on the vaginal temperature was evaluated by continuous measurement using a temperature data logger. After 24 h of cloprostenol (PG) treatment, the vaginal temperature was significantly lower than on day 7 after estrus, and the low values were maintained until the beginning of estrus (P < 0.05). The cows that received PG and exogenous progesterone (CIDR) did not show a temperature decrease until the CIDR was removed. This finding suggested that the vaginal temperature change reflected the progesterone concentration. The rate of detection of natural estrus was lower for a pedometer than for the vaginal temperature (P < 0.05); synchronization of estrus resulted in a high estrus detection rate regardless of the detection method. In a subsequent experiment, the effect of vaginal temperature measurement and the use of a pedometer on estrus detection was evaluated in the cool and hot seasons. The average activities during non-estrus and the activity increase ratio (estrus/non-estrus) changed according to season (P < 0.01, P < 0.05). However, the average vaginal temperatures during estrus and non-estrus were not affected by season. The estrus detection rate of the pedometer was lower in summer and lower than that obtained using the vaginal temperature. These results indicated that vaginal temperature measurement might be effective for detecting estrus regardless of estrous behavior.

Synthesis of prostaglandin analogues, latanoprost and bimatoprost, using organocatalysis via a key bicyclic enal intermediate.

Two antiglaucoma drugs, bimatoprost and latanoprost, which are analogues of the prostaglandin, PGF2α, have been synthesized in just 7 and 8 steps, respectively. The syntheses employ an organocatalytic aldol reaction that converts succinaldehyde into a key bicyclic enal intermediate, which is primed for attachment of the required lower and upper side chains. By utilizing the crystalline lactone, the drug molecules were prepared in >99% ee.

Effects of polyquaternium- and benzalkonium-chloride-preserved travoprost on ocular surfaces: an impression cytology study.

PURPOSE: To compare the toxicity effect of polyquaternium (PQ) and benzalkonium chloride (BAK) preservation of travoprost on the ocular surface. METHODS: This prospective study included 44 eyes of 44 patients with newly diagnosed glaucoma. Twenty-two patients used PQ-preserved travoprost (PQ group) and 22 patients used BAK-preserved travoprost (BAK group). To investigate the effect on the ocular surface, conjunctival impression cytology (IC) was performed at baseline and the 1- and 6-month follow-up visits. Additionally, the ocular surface disease index (OSDI) questionnaire, Schirmer I test, and tear break-up time (TBUT) measurement were administered at baseline, and at 1-, 3-, and 6-month follow-up visits. RESULTS: While both groups showed statistically significant IC grade increases at 1 and 6 months when compared with baseline measurements, IC grades were significantly higher for patients using PQ-preserved travoprost compared with patients using BAK-preserved travoprost. The Schirmer I test and TBUT scores were not statistically significant between group 1 and BAK group at baseline and at 1-, 3-, and 6-month visits (P>0.05). OSDI scores did not statistically differ at baseline and the 1-month measurements between the 2 groups (P>0.05), but the 3- and 6-month OSDI scores were significantly higher for BAK group (P=0.001). Differences in OSDI and Schirmer I test scores were statistically significant at 1, 3, and 6 months in both groups as compared with baseline values (P<0.05). Statistically significant differences in the TBUT scores were seen for both groups at 3 and 6 months, while BAK group, but not PQ group, had insignificant score differences at 1 month as compared with baseline values of PQ group (P=0.083). CONCLUSION: PQ-preserved travoprost was found to be safer and better-tolerated than BAK-preserved travoprost. PQ-preserved travoprost provided better ocular surface comfort, and therefore a better patient experience, which would likely result in higher treatment compliance.

Equilibrium binding interactions between lotrafilcon a soft contact lenses and the two prostaglandin antiglaucoma drugs bimatoprost and tafluprost.

OBJECTIVES: To determine the equilibrium binding constant (EB) values of bimatoprost and tafluprost drug product formulations in contact with lotrafilcon A soft contact lenses and to characterize the importance of drug molecule hydrophobicity in controlling the binding interactions. METHODS: Bimatoprost Ophthalmic Solution and Tafluprost Ophthalmic Solution (Saflutan) were incubated with lotrafilcon A lens material for timed intervals at 25°C and 37°C. Aliquots were withdrawn, filtered, and tested using reverse-phase ultrahigh-performance liquid chromatography with respect to [bimatoprost] or [tafluprost] remaining in the solution. A series of homologous dialkyl phthalate esters and a series of homologous p-hydroxybenzoic acid alkyl esters were also tested as reference compounds. RESULTS: Bimatoprost and tafluprost were rapidly (within 15 min) absorbed from the solution by lotrafilcon A lenses, reaching an equilibrium within 60 min. At any lens:solution (w/v) ratio, the extent of drug binding to lens material was greater for tafluprost than for bimatoprost. The log(EB) values correlated with solute octanol:water partition coefficient (logP) values, indicating that hydrophobic interactions are important in controlling solute partitioning into the lens material. CONCLUSIONS: This study established the quantitative relationships between tafluprost and bimatoprost binding to lotrafilcon A lenses. The fraction of bimatoprost or tafluprost that binds to lotrafilcon A increases with increasing lens:solution (w/v) ratio. For a 60 µL dose volume applied to a single contact lens, 16% of initially present bimatoprost remains in the solution, whereas only 6% of initially present tafluprost remains in the solution. These calculations clearly demonstrate that both drugs partition extensively into lotrafilcon A contact lens material. Although the clinical implications of such binding can only be surmised, it would seem prudent to caution contact lens wearers to remove the lenses before administering either prostaglandin drug.

A novel convergent synthesis of the antiglaucoma PGF2α analogue bimatoprost.

The 17-phenyl PGF(2α) analogue bimatoprost (10a) is the most efficacious ocular hypotensive agent currently available for the treatment of glaucoma or ocular hypertension. A novel convergent synthesis of 13,14-en-15-ol prostamideF(2α) analogues was developed employing Julia-Lythgoe olefination of the structurally advanced phenylsulfone (+)-(5Z)-15 with an enantiomerically pure aldehyde ω-chain synthon (-)-(S)-16a. Subsequent hydrolysis of protecting groups and final amidation of the diol 26a yielded bimatoprost (10a). The main advantage of the current strategy is the preparation of high-purity bimatoprost (10a). The novel convergent strategy allows the synthesis of a whole series of 13,14-en-15-ol prostamideF(2α) analogues with the desired C-15 asymmetric center configuration from a common and structurally advanced prostaglandin intermediate (+)-(5Z)-15. The preparation and identification of two synthetic impurities, 15-epi isomer (10b) of bimatoprost and a new prostaglandin related amide (+)-(5Z)-18, are also described.

Observational study results in glaucoma patients undergoing a regimen replacement to fixed combination travoprost 0.004%/timolol 0.5% in Germany.

With the launch of the fixed combination of travoprost 0.004%/timolol 0.5% (trav/tim) in Germany in May 2006, a noninterventional observational study designed as an open-label, multicenter, 6-week trial was initiated in order to evaluate the efficacy and tolerability of this new drug combination in glaucoma patients. Participants were grouped into categories according to previous drug regimens: those on timolol monotherapy; those on prostaglandin analog (PGA) monotherapy; those on concomitant therapy with a PGA and timolol; and those on fixed combinations. Trav/tim was well accepted by the patients, with 87.9% judging the tolerability of the therapy as good, very good, or excellent. Analysis of intraocular pressure (IOP) measurements showed statistically significant IOP decreases in all four categories examined in our study after regimen substitution with fixed-combination trav/tim. Fixed-combination prostaglandin analog/beta-blocker formulations are an attractive therapeutic option due to their strong IOP-lowering efficacy with once-daily dosing. In this study, glaucoma patients who underwent a regimen modification to fixed-combination trav/tim showed further reductions in IOP, irrespective of which selected monoor multiple therapies had been used previously.

Comparative study of the stability of bimatoprost 0.03% and latanoprost 0.005%: a patient-use study.

BACKGROUND: The stability of ophthalmic preparations in multidose containers is influenced by the preservative as well as the stability of the active ingredient. Unstable drugs may require refrigeration to preserve their active ingredient level and they are more likely to degrade over time, therefore becoming more susceptible to degradation based on patient mishandling. The purpose of this study was to determine the degree of molecular degradation that occurs in bimatoprost and latanoprost in a patient-use setting. METHODS: This was an open-label, laboratory evaluation of the relative stability of bimatoprost and latanoprost. Patients presently using bimatoprost (n = 31) or latanoprost (n = 34) were identified at 2 clinical sites in Brazil. Patients were instructed to use and store their drops as usual and return all used medication bottles between day 28 and day 34 after opening. RESULTS: Bimatoprost demonstrated no degradation, but latanoprost degraded at various levels. The mean age of bimatoprost was 43.0 +/- 3.4 days and the mean age of latanoprost was 43.9 +/- 2.8 days (P = .072). The mean percentage of labeled concentration was 103.7% in the bimatoprost bottles and 88.1% in the latanoprost bottles (P < 001). CONCLUSION: This study showed that bimatoprost maintained > or =100% concentration throughout the study period while latanoprost did not.

NMR studies of the inclusion complex of cloprostenol sodium salt with beta-cyclodextrin in aqueous solution.

PURPOSE: Cloprostenol sodium salt (referred as cloprostenol) may be used for the synchronization of estrous cycles in farm animal species. Cyclodextrins (CDs) have potential as drug delivery systems through the formation of inclusion complexes between CDs and drugs. This is the first study of the inclusion complex of cloprostenol with beta-cyclodextrin (beta-CD) in aqueous solution using NMR and 3D molecular dynamics simulations. METHODS: 1D proton NMR spectra of beta-CD, a complex of cloprostenol with beta-CD, and cloprostenol in D(2)O were assigned and confirmed. The cross relaxation interactions from ROESY were used as constraints for 3D molecular modeling studies. RESULTS: In the 2D ROESY of the complex, cross-peaks were observed between the aromatic protons of cloprostenol and protons of the beta-CD as well as between aliphatic protons and protons of the beta-CD. The stoichiometry of the complex was found that beta-CD forms a 1:1 inclusion complex with cloprostenol. The association constant K was 968 +/- 120 M(-1) at 298 K. CONCLUSIONS: Aromatic side and/or aliphatic side chains of the cloprostenol is included in the beta-CD while aliphatic side and/or aromatic side chains wraps around beta-CD, respectively. The molecular modeling also confirms that beta-CD forms a 1:1 inclusion complex with cloprostenol.

HPLC method for enantioselective analysis of cloprostenol.

A new HPLC method for the separation and quantification of cloprostenol enantiomers was developed. The optimized separation system consisted of Chiralcel OD-RH column and acetonitrile-sodium dihydrogenphosphate (pH 3.0; 20mM) (33:67, v/v) as the mobile phase. Baseline resolution of (+/-)-cloprostenol (R=2.16) was achieved and the analysis time did not exceed 10 min. Limits of detection and quantification were units of micromol/l at 274 nm. The respective values decreased an order of magnitude at 210 nm. The R.S.D. values obtained for the retention factor, peak area and peak height of each enantiomer were less than 2%. Conditions for semipreparative separation of the enantiomers can be achieved easily just by a small adaptation of the mobile phase composition.

Effect of dexcloprostenol on endogenous prostaglandin F2alpha release in dairy heifers.

Cloprostenol was previously believed to be unable to release endogenous prostaglandin F2alpha (PGF2alpha) when administered during early bovine diestrus. A prostaglandin release is, however, seen in late diestrus. The aim of this study is to find out whether dexcloprostoenol (containing the only biologically active isomer, d-isomer, of cloprostenol) induces endogenous PGF2alpha release during early and late diestrus. Twelve heifers of the Finnish Ayrshire breed were allocated into two equal groups. Their estrous cycles were synchronized with dexcloprostenol. A further luteolysis was induced with 0.15 mg of dexcloprostenol either on Day 7 (group D7 or early diestrus) or on Day 14 (group D14 or late diestrus) after ovulation. Blood for progesterone and the PGF2alpha metabolite 15-ketodihydro-PGF2alpha determinations was collected immediately before dexcloprostenol treatment and thereafter every second hour for 48 h. Five of the six heifers in both groups showed significantly increased blood levels of 15-ketodihydro-PGF2alpha at some time during the 48-h experimental period. The intervals from treatment to the first significant increases of the PGF2alpha metabolite were 32.8+/-2.3 h (min. 30 h, max. 36 h) and 20.0+/-4.2 h (min. 14 h, max. 24 h) in groups D7 and D14, respectively (P < 0.01). We have concluded that dexcloprostenol induced endogenous PGF2alpha release in most cases, regardless the time of its administration (early or late diestrus). This release, however, differs from that observed during spontaneous luteolysis.

Travoprost: a potent ocular hypotensive agent.

Travoprost (isopropyl (Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(1E,3R)-3-hydroxy-4-[alpha,alpha,alpha,trifluoro-m-tolyl)oxy]-1butenyl]cyclopentyl]-5-heptenoate) is an isopropyl ester prodrug and a high-affinity, selective FP prostaglandin- receptor full agonist. This prodrug is a synthetic prostaglandin analogue, which in appropriate cases is administered topically for the treatment of glaucoma and ocular hypertension. The isopropyl ester prodrug is rapidly hydrolyzed by esterases in the cornea to the biologically active, free acid. Travoprost has demonstrated preferential affinity and full agonist activity for the FP receptor in the nanomolar range, with no meaningful affinity or activity at other receptors. Like other compounds of this class, the reduction of intraocular pressure by travoprost is due at least in part to increased uveoscleral outflow. Results from phase II and phase III pivotal studies for FDA approval in the United States have demonstrated that travoprost is an effective topical agent for treatment of elevated intraocular pressure in patients with open-angle glaucoma and ocular hypertension. Travoprost is a safe drug, with local side effects including hyperemia, eyelash growth and iris color change. The dosing is once per day in the evening, and storage does not require refrigeration. Travoprost will be a helpful new drug in the medical management of glaucoma.

Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells.

Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.

Identification and characterization of the ocular hypotensive efficacy of travoprost, a potent and selective FP prostaglandin receptor agonist, and AL-6598, a DP prostaglandin receptor agonist.

The structure-activity studies that led to the identification of travoprost, a highly selective and potent FP prostaglandin analog, and AL-6598, a DP prostaglandin analog, are detailed. In both series, the 1-alcohol analogs are very effective and are thought to be acting as prodrugs for the biologically active carboxylic acids. The efficacy of amide prodrugs depends on the degree of substitution and the size of the substituents. Selected compounds are profiled in vitro and in vivo preclinically. Clinical studies show that travoprost 0.004% (isopropyl ester) provided intraocular pressure control superior to timolol 0.5% when used as monotherapy in patients with open-angle glaucoma or ocular hypertension. In clinical studies, AL-6598 0.01% provided a sustained intraocular pressure reduction with q.d. application; b.i.d. provided greater intraocular pressure control. The acute and, apparently, conjunctival hyperemia associated with topical ocular AL-6598 can be attenuated while maintaining intraocular pressure-lowering efficacy by formulating with brimonidine.

Prostaglandin F2alpha-induced nest-building behaviour is associated with increased hypothalamic c-fos and c-jun mRNA expression.

Intramuscular injection of the naturally occurring prostaglandin F2alpha (PGF2alpha) to sexually mature female pigs induces luteolysis and rapidly elicits a behavioural response consistent with pre-partum nest-building. Intramuscular injection of the synthetic prostaglandin F2alpha (cloprostenol) also induces luteolysis but no nest-building behaviour is observed. The effects of PGF2alpha, but not cloprostenol, on nest-building behaviour may be mediated via peripheral PGF2alpha receptors (FP) or via direct action on central FP receptors. We have previously shown FP receptor mRNA to be localized in porcine paraventricular nucleus (PVN), supraoptic nucleus (SON) and pars dorso-medialis of the suproptic nucleus (SOD), suprachiasmatic nucleus, choroid plexus and anterior and intermediate pituitary lobes. In this experiment, we examined hypothalamic expression of the immediate early genes c-fos and c-jun mRNA after treatment with PGF2alpha or cloprostenol. Twenty-one 8-month-old nulliparous female pigs (gilts) were injected intramuscularly with a luteolytic dose of PGF2alpha (15 mg), cloprostenol (175 microg) or saline control, their behaviour was recorded and they were killed 60 min later. Coronal hypothalamic sections and control ovarian tissues were incubated with 45-mer oligonucleotide probes complementary to porcine c-fos and c-jun genes using standard in situ hybridization histochemistry techniques. Significantly higher c-fos and c-jun mRNA expression was found in PGF2alpha-treated compared to saline or cloprostenol-treated pigs in the PVN, SON and SOD. Significantly higher c-fos and c-jun mRNA expression was found in corpus lutea of PGF2alphaand cloprostenol-treated pigs compared to saline controls. Treatment with PGF2alpha induced nest-building behaviour whereas treatment with cloprostenol and saline did not. This suggests that PGF2alpha, or one of its metabolites, and not cloprostenol, crosses the blood-brain barrier and acts directly on hypothalamic receptors to mediate its effect on nest-building behaviour.

Synthesis and biological activity of a novel 11a-homo (cyclohexyl) prostaglandin.

The racemic cyclohexane-for-cyclopentane ring substitution analogue of the potent prostaglandin FP agonist cloprostenol (7) was synthesized from cyclohexenediol 11 in 21 steps and 0.07% yield. In a prostaglandin FP receptor-linked second-messenger assay, racemic analogue 7 exhibited an EC(50) value of 319 nM (72% response relative to cloprostenol); the corresponding values for PGF(2)(alpha) and cloprostenol were 23 nM (91% relative response) and 1 nM (defined as 100% response), respectively. Key features of the synthesis were the selective manipulation of four hydroxyl groups to direct independent elaboration of the alpha and omega chains and a new method for synthesis of aryloxy-terminated omega chains involving Horner-Emmons elongation of an aldehyde to a methyl enone, regioselective bromination adjacent to the carbonyl, and phenoxide displacement of bromide.

Enantioselective behaviour of drugs used in domestic animals: a review.

The chirality of drugs, with particular reference to agents used in veterinary medicine, is reviewed. Basic concepts of chirality and aspects of the methodology for the separation of enantiomers are considered. Chiral compounds are in common use in animals and their pharmacological actions and side-effects (pharmacodynamics) and absorption into and fate within the body (pharmacokinetics) are of fundamental importance; pharmacodynamic and pharmacokinetic properties of enantiomeric pairs commonly differ and this has major implications for their effective and safe therapeutic use. As examples of the particular significance of chirality in veterinary medicine, the following drug classes are reviewed; benzimidazole anthelmintics, cloprostenol, verapamil, ketamine, halogenated hydrocarbon anaesthetics and 2-arylpropionic acid anti-inflammatory drugs. The implications of chirality for drug product development and approval by registration authorities are discussed.