Endogenous and exogenous effects of PGF2α during luteolysis in mares.

An inhibitor of PGF2α biosynthesis (flunixin meglumine, FM) was used to study the role of endogenous PGF2α on the luteolytic effect of exogenous PGF2α in mares. A 2-h infusion of PGF2α at a constant rate (total dose, 0.1 mg) on Day 10 (ovulation = Day 0) was used to mimic the maximal concentrations of a spontaneous pulse of a PGF2α metabolite (PGFM). Treatment with FM (1.7 mg/kg) was done 1 h before and 5 h after the start of PGF2α infusion. In hourly blood samples beginning 1 h before the start of PGF2α infusion, progesterone decreased (P < 0.05) similarly by 5 h in each of the PGF2α and PGF2α+FM groups but not in the controls (n = 5). In a study of spontaneous luteolysis, the same FM dose was given every 6 h from Day 13 until Day 17 or earlier if CL regression was indicated by an 80% decrease in luteal blood-flow signals. Blood was sampled for progesterone assay each day and 8 h of hourly blood sampling was done each day to characterize PGFM concentrations and pulses. Progesterone (P4) was lower (P < 0.05) in controls than in an FM group (n = 7) by Day 15. Luteolysis (P4 < 1 ng/mL) ended on Days 14-19 in individual controls. In contrast, luteolysis did not end until after Day 20 in 4 of 7 FM-treated mares. In the three mares with completion of luteolysis before Day 20 in the FM group, the interval from beginning to end of luteolysis was longer (P < 0.02) (4.5 ± 0.6 days) than in the controls (3.0 ± 0.4 days). During 8-h sessions of hourly blood sampling on Day 14, concentration of PGFM was significantly lower in the FM group for the minimal, mean, and maximal per session. Pulses of PGFM were identified by a CV methodology on each day in 7 of 7 and 3 of 7 mares in the controls and FM group, respectively. The four FM-treated mares without a CV-identified pulse were the four mares in which luteolysis did not occur before Day 20. In mares with detected pulses, PGFM was lower at each nadir and at the peak (86% lower) in the FM group than in controls, but the interval between nadirs or base of a pulse was not different between groups. Hypothesis 1 that endogenous PGF plays a role in the luteolytic effect of exogenous PGF2α was not supported. Hypothesis 2 that an inhibitor of PGF2α biosynthesis prevented or minimized the prominence of PGFM pulses and increased the frequency of persistent CL was supported.

Misoprostol-induced fever and genetic polymorphisms in drug transporters SLCO1B1 and ABCC4 in women of Latin American and European ancestry.

AIM: Misoprostol, a prostaglandin analogue used for the treatment of postpartum hemorrhage and termination of pregnancy, can cause high fevers. Genetic susceptibility may play a role in misoprostol-induced fever. SUBJECTS & METHODS: Body temperature of women treated with misoprostol for termination of pregnancy in the UK (n = 107) and for postpartum hemorrhage in Ecuador (n = 50) was measured. Genotyping for 33 single nucleotide polymorphisms in 15 candidate genes was performed. Additionally, we investigated the transport of radiolabeled misoprostol acid across biological membranes in vitro. RESULTS: The ABCC4 single nucleotide polymorphism rs11568658 was associated with misoprostol-induced fever. Misoprostol acid was transported across a blood-brain barrier model by MRP4 and SLCO1B1. CONCLUSION: Genetic variability in ABCC4 may contribute to misoprostol-induced fever in pregnant women. Original submitted 21 January 2015; Revision submitted 24 April 2015.

Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines.

Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.

Retrograde transport of a traditional Chinese medicine, alpha-trichosanthin, and its selective neural toxicity.

OBJECTIVE: To investigate the peripheral neuronal toxicity of a traditional Chinese medicine, alpha-trichosanthin (TCS). METHODS: TCS and rhodamine-conjugated TCS were separately injected into the rat sciatic nerve. Saline and rhodamine were used alone and separately as control solutions. The motor neurons in the spinal cord and sensory neurons in the dorsal root ganglia were separately counted. The entry of TCS molecules into neurons was observed under the fluorescence microscope. The glial reactions were studied by lectin staining and immunohistochemical method. The muscles innervated by the sciatic nerve and distal to the injection sites, and the nerves proximal to the injection sites were also collected and examined. RESULTS: TCS was taken up and transported by peripheral axons, and at a dose of 1 nmol, killed more than 90% of the motor neurons in 5 days, but only one-third of the sensory neurons of the injected nerve. The loss of neurons was permanent, while the increase of glial activities was mild and transient. CONCLUSION: TCS is retrogradely transported by axons of the injected nerve. TCS shows a selective neurotoxicity on different types of neurons. Hence TCS is useful in producing neural lesion in research, and this use may also be of applicational value in treating chronic spasticity, hyperalgesia, and pain.

[In vitro metabolic interaction between diphenytriazol and steroid hormone drugs].

AIM: To observe the metabolic interaction between diphenytriazol and steroid hormone drugs, and provide some useful information for clinical medication. METHODS: The steroid hormone drugs which may be co-administrated with diphenytriazol were selected, such as mifepriston, estradiol, medroxyprogesterone acetate, progesterone, norethisterone and so on. Diphenytriazol was incubated with each drug in rat liver microsome. The residual concentration of diphenytriazol or steroid hormone drugs in the microsomal incubates was determined by reversed-phase high-performance liquid chromatography, separately. The inhibition constants (K(i)) for each of them were calculated. RESULTS: The inhibition constant K(is) of diphenytriazol for the metabolism of mifepristone, estradiol, medroxyprogesterone acetate, progesterone and norethisterone were (201.3 +/- 1.0), (94 +/- 4), (128.7 +/- 2.2), (64 +/- 5) and (80 +/- 4) micromol x L(-1), respectively. The inhibition constants K(i) of steroid hormone drugs for the metabolism of diphenytriazol was (66.9 +/- 2.2) micromol x L(-1) for estradiol, (60.0 +/- 2.3) micromol x L(-1) for medroxyprogesterone acetate, (163 +/- 10) micromol x L(-1) for progesterone and (88 +/- 5) micromol x L(-1) for norethisterone, respectively. CONCLUSION: Diphenytriazol shows metabolism interaction with steroid hormone drugs such as estradiol, medroxyprogesterone acetate, progesterone and norethisterone.

Biosynthesis and catabolism of prostaglandin F2alpha (PGF2alpha) are controlled by progesterone in the rat uterus during pregnancy.

Myometrial quiescence is a key factor in all species to accomplish a successful gestation. PGs play a crucial role in mediating parturition events, and their synthesis and metabolism are regulated by cyclooxygenases (COXs) and NAD(+)-dependent 15-hydroxy-PG dehydrogenase (PGDH), respectively. Progesterone (P(4)) is the hormone responsible for maintaining uterine smooth muscle quiescence during pregnancy. In this work, we have studied the effect of P(4) on the activity of COXs and PGDH, the uterine enzymes involved in the biosynthesis and metabolism of prostanoids in the rat. We found that during pregnancy PGF(2alpha) production and also protein levels of COX-1 and COX-2 were decreased. The exogenous administration of P(4) significantly inhibited the uterine production of PGF(2alpha) and also the protein level of COX-2. PGF(2alpha), metabolism was assessed by PGDH activity, which resulted high during pregnancy and increased as a result of P(4) administration. These results indicate that PGs levels were negatively modulated by P(4), which could be exerting its effect by increasing PGs metabolism through stimulation on PGDH activity and an inhibition on COX and that is a major mechanism for maintain uterine quiescence in pregnancy.

In vitro metabolism and inductive or inhibitive effect of DL111 on rat cytochrome P4501A enzyme.

In vitro metabolism and the inductive or inhibitive effect of DL111, a non-hormonal early pregnancy-terminating agent, toward cytochrome P450 (CYP) enzymes in rat liver microsomes were studied. In vitro metabolism of DL111 was performed in different rat liver microsomes (pretreated with phenobarbital (PB), dexamethasone (Dex), beta-naphthoflavone (BNF), DL111, respectively) and the catalytic abilities of these microsomes for DL111 were compared with control group. DL111 was well metabolized in microsomes pretreated with beta-naphthoflavone and itself. The K(m) and V(max) was 41.76 +/- 3.26 microM and 15.34 +/- 1.03 nM min(-1) mg(-1) protein for beta-naphthoflavone group, 48.17 +/- 6.06 microM and 17.54 +/- 1.79 nM min(-1)mg(-1) protein for DL111 group, 77.81 +/- 4.73 microM and 3.087 +/- 0.202 nM min(-1)mg(-1) protein for control group, respectively. The rats were pretreated intraperitoneally with the same daily dose of DL111 for different days. The DL111-pretreated microsomal enzymatic activities were evaluated by measuring the metabolic abilities for specific substrates of various enzymes. The results showed that DL111 had the same inductive function as beta-naphthoflavone (the specific inducer of CYP1A) toward rat liver microsomes. The inhibitive effect of DL111 on CYP1A was investigated by coincubating DL111 with the specific substrates of CYP1A-ethoxyresorufin or phenacetin in the microsome induced by beta-naphthoflavone, and the inhibitive level was compared with fluvoxamine (Flu), the specific inhibitor of CYP1A. DL111 inhibited significantly the metabolism of phenacetin and ethoxyresorufin with the inhibition constant (K(i)) 6.836 +/- 0.10 and 1.222 +/- 0.230 microM, respectively and its inhibition potential on CYP1A was higher than fluvoxamine.

[RP-HPLC determination of diphenytriazol in rat liver microsomal incubates and its application in in vitro metabolism].

AIM: To establish a RP-HPLC method for determination of diphenytriazol (DL111-IT) in rat hepatic microsomes. METHODS: DL111-IT in rat hepatic microsomal incubates was extracted with chloroform, using diazepam as internal standard. The determination was performed on a Lichrospher ODS-C18 reversed column (25 cm x 0.46 cm ID) with mobile phase of methanol-pH 7.5 phosphate buffer (70:30) at a flow-rate of 1.0 mL.min-1. A UVVIS detector was operated at 235 nm. RESULTS: The assaywas linear from 1.01-101.0 micrograms.mL-1 for DL111-IT. The limit of detection was 0.15 microgram.mL-1 (signal-to-noise ratio 3) and the limit of quantification was 1.01 micrograms.mL-1(RSD < 10%, n = 4). The method afforded average recoveries of (100.3 +/- 1.9)% (n = 5), and intra-day and inter-day RSD were less than 5.0%(n = 5). The method allowed study of the in vitro phase I metabolism of DL111-IT in rat liver microsomal incubates. The microsomes induced by beta-naphthoflavone showed high enzymatic activity for DL111-IT phase I metabolism. CONCLUSION: The method is simple, accurate and can be used to study the metabolism of DL111-IT in rat hepatic microsomes.

Prostaglandin F2alpha release associated with an embryo transfer procedure in the mare.

The pattern of the main metabolite of prostaglandin (PG) F2alpha was recorded following a nonsurgical embryo transfer technique in 9 mares under field conditions in Estonia. Three patterns were observed. Two of them were characterised by PG release, thereas the third was not. A tendency towards a shortened cycle was seen in 3 mares. Observations were made regarding the manipulation of the uterus as being normal or difficult to perform. In general, mares where the procedure was considered difficult were also found to have a PG release.

Interaction of hCG and Lutalyse on steroidogenesis of bovine luteal cells.

This study was designed to examine the ability of in vivo administration of human chorionic gonadotropin (hCG, 4000 IU) to alter the effects of Lutalyse (PGF2 alpha, 10 mg) in the cow. hCG significantly increased plasma progesterone concentration in midcycle cows (P less than 0.01), but these elevated levels were not maintained in the presence of Lutalyse (P less than 0.05). Responsiveness of luteal cells in vitro to luteinizing hormone (LH) (100 ng/ml), prostaglandin F2 alpha (PGF2 alpha) (1 microgram/ml), dibutyryl cyclic AMP (dbcAMP) (10 mM) and PGF2 alpha (1 microgram/ml) + dbcAMP (10 mM) during a 2 h incubation was significantly reduced following in vivo treatment with Lutalyse when compared to in vivo untreated animals. In conclusion, the luteotropic effects of hCG were incapable of preventing Lutalyse-induced regression of the corpus luteum, and treatment of animals with hCG prior to Lutalyse administration could not prevent the significant decrease in responsiveness of luteal cells in vitro.

Absorption and elimination of a prostaglandin F analog, fenprostalene, in lactating dairy cows.

Pharmacokinetic characteristics of the prostaglandin F2 alpha analog, fenprostalene, were studied in five lactating Holstein cows. Blood samples, milk, urine, and feces were collected for up to 7 d following a single subcutaneous injection of 1 mg of 13,14-hydrogen-3-fenprostalene in polyethylene glycol-400. The maximum concentration of tritium in plasma, observed 4 h after injection, equated to .17 ngeq/ml fenprostalene and declined with a fractional disappearance rate of .051 X h-1 to less than .04 ngeq/ml by 48 h. Likewise, milk contained .53 ngeq/ml fenprostalene at 4 h and the concentration declined with a fractional disappearance rate of .069 X h-1 to less than .03 ngeq/ml by 48 h. Milk was a very minor route of elimination of fenprostalene with only .46% of the injected dose recovered over a 7-d sampling. Recovery of tritium in urine accounted for 55% of the total dose and recovery in feces accounted for an additional 43%. Residues from fenprostalene at 7 d after injection were less than .1 ppb in all edible tissues. Differences in the molecular structure, formulation, and route of injection of fenprostalene resulted in a slower rate of absorption and elimination of this analog than previously reported for other prostaglandin products. Nonetheless, the percentage of the injected dose of fenprostalene secreted in milk was not increased appreciably, and no persistent tissue residues of fenprostalene were observed.

Absorption, excretion and tissue residue in feedlot heifers injected with the synthetic prostaglandin, fenprostalene.

Preliminary studies on use of the synthetic prostaglandin, fenprostalene, as an abortifacient had indicated that maximum effectiveness was dependent upon slow delivery. Because both route of administration and formulation control delivery rates, the influences of intramuscular (im) vs subcutaneous (sc) injections, and aqueous acetate buffer (AAB) vs polyethylene glycol-400 (PEG) vehicles on the plasma concentration and urinary excretion of fenprostalene were compared. Feedlot heifers were administered 1 mg injections of [13,14-3H]-fenprostalene. Blood samples and total urine excretion were collected during the following 96 h. The maximum concentration of tritium in plasma occurred at 2 h for AAB-im (.90 ng eq/ml), PEG-im (.75 ng eq/ml) and AAB-sc (.64 ng eq/ml), and then declined throughout 24 h with t 1/2 values of 6.1, 9.4 and 9.2 h, respectively. The peak concentration from PEG-sc was lower (.37 ng eq/ml, P less than .05), observed later (4h, P less than .05) and declined more slowly following peak concentration (t 1/2 = 15.1 h, P less than .05). Consistent with delayed absorption, a smaller fraction (P less than .05) of the total radioactivity excreted in urine was recovered during the first 24 h after injection for PEG-sc (85%) than for PEG-im (95%), AAB-sc (97%) or AAB-im (99%). In a tissue distribution study, plasma, urine and fecal samples were collected and heifers were slaughtered at various times following sc injection of 1 mg of [3H] fenprostalene in PEG. Peak concentrations of tritium in plasma occurred between 4 and 8 h and declined with a t 1/2 of 15.2 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased oral activity of a new class of non-hormonal pregnancy terminating agents.

A series of selected analogues of 2-(3-ethoxyphenyl)-5,6-dihydro-s-triazole [5, 1-a] isoquinoline (DL 204-IT) modified at the three sites of metabolism of the DL 204-IT molecule, were studied for their anti-fertility activity and absorption (in situ and in vivo) following oral administration to the hamster. All test-compounds were rather well absorbed, nevertheless, the ratios between the oral and subcutaneous pregnancy termination activity ranged between 3 and 722, suggesting a marked influence of metabolic first-pass. One of these new anti-fertility agents, 2-(1, 1'-biphenyl-4-yl)-s-triazole [5, 1-a]-isoquinoline (L 14105), showed an interesting oral activity (ED50: 0.2 mg/kg/d), 300 times greater than that of the parent compound DL 204-IT.