Suppression of laser-induced choroidal neovascularization by subconjunctival injection of 9alpha-fluoromedroxyprogesterone acetate (FMPA), an anti-angiogenic agent, in rats

9alpha-Fluoromedroxyprogesterone acetate (FMPA) is a synthetic analog of medroxyprogesterone acetate (MPA). FMPA exhibited more potent anti-tumor and anti-angiogenic activities in some assay systems than the parent agent, MPA. Exudative age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). Anecortave acetate, an angiostatic steroid, is clinically efficacious in patients with exudative AMD. Betamethasone is an anti-angiogenic steroid. Therefore, we examined the effects of FMPA, anecortave acetate and betamethasone on laser-induced CNV in rats. Anecortave acetate and betamethasone were included as positive controls. Crypton laser was applied to the fundus in Brown Norway rats. Laser photocoagulations were performed in each eye between the major retinal vessels of the superior retina. Subconjunctival injection of FMPA, anecortave acetate or betamethasone was performed once just after the photocoagulation (on day 0). The incidence of CNV formation was evaluated by fluorescein angiography (FAG) on day 14. On the next day, examination of the retinal function was performed by electro retinogram (ERG). Subconjunctival injection of FMPA at doses of 300, 1000 and 3000 microg/eye dose-dependently inhibited the incidence of CNV formation. Significant differences were observed at doses of 1000 and 3000 microg/eye of FMPA as compared with the control group. Anecortave acetate and betamethasone significantly inhibited the incidence of CNV formation. FMPA at the doses used in this study did not affect the retinal function in rats, as determined by ERG. FMPA appeared to be effective in a rat model of CNV, so it was demonstrated that FMPA might be useful in the treatment of AMD.     

Effect of medroxyprogesterone acetate on steroid reductases in rat liver

Male and female rats were treated with medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methyl-pregn-4-ene-3,20-dione, MPA) 600 mg/kg body weight i.p. daily for seven days. The steroid metabolizing enzymes in liver microsomes and liver homogenate were measured following MPA treatment and in control rats. The specific activity of NADPH- and NADH-5 alpha-reductase in female rats and of NADPH-5 alpha-reductase in male rats decreased by the treatment. NADP+- and NAD+-3-hydroxysteroid dehydrogenase activities were lower in female MPA rats if compared to untreated animals. In male rats only NADP+- and NAD+-3 beta-hydroxysteroid dehydrogenase activities (substrate 5 beta-dihydrotestosterone) were diminished by MPA administration. There was no effect on the cytoplasmatic 5 beta-reductases. Plasma concentrations of luteinising hormone (LH), testosterone and androstenedione were lowered by MPA treatment.     

Pharmacokinetics and bioavailability of medroxyprogesterone acetate in the dog and the rat

Medroxyprogesterone acetate (MPA) has been administered to rats and dogs. Dogs received single oral doses of 2.5, 5, and 10 mg MPA and a single intravenous dose of 1 mg MPA. Rats received single oral doses of 0.2, 1, 5, and 20 mg kg^?1 MPA and multiple oral doses (14 daily doses) of 0.2, 5, and 20 mg kg^?1 MPA. Dog plasma MPA levels from the intravenous dose were characterized by a triexponential decay with disposition half-lives of 0.3, 1.8, and 21.6 h. A Loo-Riegelman analysis of the dog plasma MPA levels from oral doses indicated absorption was not a simple first-order process. The Weibull Function was used to characterize the absorption kinetics of MPA. The oral absorption of MPA in dogs appears to be dose-linear over the dosage range studied, and the absolute bioavailability was estimated at 27 per cent. Rat plasma MPA levels from single and multiple oral doses were analyzed by a non-compartmental approach. AUC and C_max values were not dose-linear over the dosage range studied; indicative of the self-induced metabolism of MPA. Exposure of similar dosages of MPA to both the rat and the dog resulted in similar plasma profiles and pharmacokinetics.     

Role of ACTH on the effect of medroxyprogesterone in brain stem serotonin.

A clinical study with 361 female rats was conducted to elucidate the mechanism whereby MPA (medroxyprogesterone acetate) lowers 5-HT/5-HIAA ratio in the brain area and the possible role of serotoniergic mechanisms. In addition, the participation of MAO (monoamino oxidase) system and the effects of some steroids were studied in order to establish a relationship between chemical structure and activity. The effects of the following steroids were studied: MPA (medroxyprogesterone acetate), melengestrol acetate, chlormadinone, pregnenolone, -methyl pregnenolone, DOCA, acetoxi-progesterone, and ACTH (synacthen). Effects of these substances on LTP (liver tryptophan pyrrolase) activity, total and free plasma and brain stem Trp (tryptophan), and the 5HT and 5HTAA content in brain stem are tabulated. Of all the substances, only MPA and melengestrol acetate significantly raised LTP activity and both also lowered 5-HT content of brain stem. The high levels of ACTH in the blood of the adrenalectomized rats, as in those under fasting conditions, antagonized MPA effects. To further test this seeming result, ACTH and ACTH-MPA were injected into another group of animals. The ACTH not only increased plasma corticosterone but also antagonized the effect of MPA on the 5-HT content of brain stem. The study did not identify a relationship between chemical structure of the steroids studied and effects observed.     

Effect of medroxyprogesterone acetate on hepatic lipid profile of female rats under various states of nutrition

The effect of medroxyprogesterone acetate (MPA) treatment on hepatic lipid profile was studied in female rats kept on protein-deficient diet, on normal restricted diet and on normal, ad libitum diet. A significant decline in total and free cholesterol levels was observed in rats kept on protein-deficient diet and on normal, restricted diet. However, protein-deficient animals exhibited a significant rise in the liver triglyceride level. In rats on normal, ad libitum diet only, MPA treatment resulted in elevated levels of triglycerides and increased esterification of cholesterol. This was mostly due to increased incorporation of acetate into esterified cholesterol and triglyceride as evident from studies using the labelled precursor. Total phospholipid content was found to be unaffected by MPA in all the groups suggesting that the drug and dietary protein level have no effect on hepatic phospholipid content.     

Effects of medroxyprogesterone acetate on serum prolactin levels and liver prolactin binding capacity in the rat

Modifications in liver prolactin (PRL) receptor levels and serum PRL concentration induced by administration of medroxyprogesterone acetate (MPA) were investigated in rats of both sexes. MPA induced a reduction both of the levels of PRL in the serum and of liver PRL receptors in the female rat. The reduction of the number of PRL receptors caused by MPA was rapid and almost complete after 10 days of treatment and appeared earlier than that of serum PRL levels. Furthermore the MPA-induced decrease in PRL receptors was specific, since insulin binding to the same liver membranes was not affected. MPA given simultaneously with oestradiol (which increases both the number of liver PRL receptors and the serum PRL levels in the male rats) was able to counteract the increase in PRL binding induced by oestradiol. On the contrary, the oestrogen-induced increase in serum PRL was not affected by MPA treatment. Similar results were obtained using tamoxifen, a well known antioestrogenic drug. In conclusion, our results show that the reduction of PRL receptor levels induced by MPA in rat liver is specific, not correlated to serum PRL concentration, and seems to depend on the antioestrogenic activity of the drug.     

Medroxyprogesterone acetate improvement of the hepatic drug-metabolizing enzyme system in rats after chemical liver injury

Medroxyprogesterone acetate (MPA) has an inducing effect on the hepatic drug-metabolizing enzyme system in the rat. The effect of MPA on the liver metabolism was further evaluated here by investigating the restoration of hepatic function after chemical liver injury in female rats. The hepatic injury was induced by pretreating the animals with CCl4 and dimethylnitrosamine for 4 weeks, after which rats treated with MPA for a week were compared with rats showing spontaneous regeneration upon treatment with the MPA vehicle only. Changes in various parameters of the drug-metabolizing enzyme system were used as indices of hepatic function together with liver protein content. The results showed that MPA therapy increased the cytochrome P-450 content and the activity of NADPH-cytochrome c reductase, the monooxygenase enzymes benzo[a]pyrene hydroxylase and aminopyrine N-demethylase, epoxide hydrolase and glutathione S-transferase. MPA increased the relative values in the rats with liver injury almost equally to, or even more than, that seen in the intact animals in comparison to the corresponding vehicle-treated rats. MPA seemed to enhance protein synthesis during liver regeneration, as indicated by changes in total liver protein and in the gel electrophoresis pattern of the microsomal proteins. The hepatic enzyme induction and enhancement of protein synthesis achieved by MPA after liver injury may be of value in the treatment of liver diseases.     

Metabolism and pharmacokinetics of vinyl acetate

The hydrolysis of vinyl acetate (formation of acetic acid) has been studied in vitro with rat liver and lung microsomes, rat and human plasma and purified esterases (such as acetylcholine esterase, butyrylcholine esterase, carboxyl esterase). Characterization of the kinetic parameters revealed that rat liver microsomes and purified carboxyl esterase (from porcine liver) displayed the highest activity.In order to establish the rate of metabolism of vinyl acetate in vivo, rats were exposed in closed desiccator jar chambers, and gas uptake kinetics were studied. The decay of vinyl acetate was dose-dependent, indicating possible saturation of metabolic pathway(s). The maximal clearance (at lower concentrations) of vinyl acetate from the system (30 000 ml/h per kg body weight) was similar to the maximal ventilation rate in this species. This indicated that under conditions when metabolic enzymes are not saturated the metabolic rate is mainly determined by pulmonary uptake.The exposure of rats to vinyl acetate resulted in a transient exhalation of significant amounts of acetaldehyde into the closed exposure system. This indicates the presence of this metabolic intermediate of vinyl acetate in the organism in vivo.     

Time course of hepatic changes produced by medroxyprogesterone acetate in the rat

The time course of the effects of medroxyprogesterone acetate (MPA) treatment (100 mg/kg, i.p.) on hepatic drug metabolism was investigated in female rats. The findings demonstrate that the maximum effects of MPA on the microsomal enzyme systems, liver weight and protein content were attained within 3-7 days depending on the measured parameter. The blood concentration of MPA reached a constant level of about 120 ng/ml in 3 days. After cessation of MPA administration the induced values decreased to the control level within about 11 days.     

Plasma concentrations of megestrol acetate and medroxyprogesterone acetate after single oral administration to healthy subjects

Plasma concentrations of megestrol acetate (MA) were measured by radioimmunoassay (RIA) after a single oral dose of 60 mg either in the form of one tablet, or four 15 mg tablets, to 10 women 21-40 years old using a cross-over design. No statistically significant difference between the two preparations was observed with respect to plasma concentrations, the area under the curve from 0 to 24 h or the maximum concentration (c(max)). For comparison, data are presented on the plasma level of medroxyprogesterone acetate (MPA) following a single oral dose of 100 mg given using a cross-over design in two different tablet forms to 10 healthy men, when no significant difference was observed for these parameters. The mean c(max) for MA after 2.6 h was 43.9 ng/ml (range 21.7-87.7 ng/ml), whereas that for MPA at 3.1 h was 13.1 ng/ml (range 4.4-29.5 ng/ml) despite the higher dose. After 24 h immunoreactive MA and MPA ranged from 9.6 to 29.0 ng/ml and from 0.2 to 4.0 ng/ml respectively. Moreover, it was found that petroleum ether extraction gives the most specific result by RIA, although considerable amounts of metabolites are still co-estimated. By comparison with selected ion monitoring using GC-MS, metabolite interference in RIA increases with time after administration of the steroids and is considerably greater for MPA than for MA. It is concluded that after oral administration the relative bioavailability of MA is significantly better than that of MPA.     

Effects of medroxyprogesterone acetate on hepatic glucose metabolism and microsomal enzyme activity in rats with normal and altered liver

Effects of medroxyprogesterone acetate (MPA) on hepatic glucose handling and drug metabolism were investigated in female rats with intact and damaged liver. Hepatic glucose-6-phosphatase activity, glycogen content and fasting blood glucose were assessed as indices of glucose metabolism. Cytochrome P-450 content and NADPH-cytochrome P-450 reductase activity were assayed to reflect liver drug metabolism. Liver injury was induced by dimethylnitrosamine and carbon tetrachloride. The results demonstrate that hepatic glucose handling and drug metabolism were changed in a parallel fashion in intact, damaged and induced liver. The MPA-induced changes in glucose metabolism were slight in intact animals, whereas the compound has an increasing effect on glucose and drug metabolism in rats with damaged liver. The findings demonstrate the MPA enhances the normallization of hepatic glucose and drug metabolism in damaged liver.     

Medroxyprogesterone acetate pharmacokinetics following oral high-dose administration in humans: a bioavailability evaluation of a new MPA tablet formulation

The pharmacokinetics of medroxyprogesterone acetate (MPA) in healthy female volunteers have been investigated following oral administration of single doses of six different high-dose MPA tablet formulations. Blood samples were obtained over 96 hrs following administration. The plasma was separated and analyzed in duplicate for MPA by radioimmunoassay (RIA) after extraction with petroleum ether. A two compartment open model with first order absorption was computer-fitted to the plasma concentration of MPA. Following oral administration MPA is rapidly transferred from the gastrointestinal tract to the blood circulation with a half-life of the absorption process of 15-30 min. The peak plasma concentration is reached 1-3 hrs after administration, and the biological half-life of MPA is 40-60 hrs. Following administration of 1000 mg MPA the areas under the plasma concentration-time curves (AUC 0-infinity) were calculated to (mean and S.E.): 3357 (438) nmol/l and 2403 (245) nmol/l for Leo formulation A and Farlutal, respectively (P less than 0.02). Following administration of 500 mg the areas were: 2325 (389) nmol/l, 1793 (312) nmol/l, 1778 (239) nmol/l, 1178 (209) nmol/l, and 556 (89) nmol/l for Gestapuran, Leo formulation A (P = n.s.), Leo formulation B (P = n.s.), Provera (P less than 0.001), and Lutopolar (P less than 0.001), respectively. The in vitro dissolution rates of MPA from the tablet formulations were determined and compared with the results of the bioavailability studies, indicating that a rapid dissolution rate as well as the particle size of MPA are two important factors to ensure optimal absorption of MPA from the gastrointestinal tract.     

Effects of poloxamer 407‐induced hyperlipidemia on hepatic multidrug resistance protein 2 (Mrp2/Abcc2) and the pharmacokinetics of mycophenolic acid in rats

Hepatic multidrug resistance‐associated protein 2 (Mrp2) is responsible for the majority of the biliary elimination of endogenous and exogenous substances, therefore it is important to evaluate possible functional changes in Mrp2 activity under conditions of hyperlipidemia (HL). Thus, the present study assessed the protein expression and transporting activity of hepatic Mrp2 based on the in vivo biliary excretion of phenolsulfonphthalein (PSP) as a model anionic substrate for Mrp2 in poloxamer 407‐induced hyperlipidemic rats (HL rats) and compared these values with those for control rats. The pharmacokinetics of mycophenolic acid (MPA) and mycophenolic acid‐7‐O‐glucuronide (MPAG) were evaluated after the intravenous (5 mg/kg) and oral (10 mg/kg) administration of MPA to control and HL rats. In HL rats, the protein expression of hepatic Mrp2 and its biliary transporting activity exhibited significant reductions (by 24.3% and 24.6%, respectively) in the absence of a change in bile flow rate. Unexpectedly, HL and control rats showed comparable biliary excretion rates of MPAG due to the counter effects of the reduced expression and activity of Mrp2 and a 484% increase in the free fraction of MPAG in HL rats. The estimated biliary clearance value of free MPAG in HL rats was considerably slower (by 77.1%) than that in control rats. Although significant pharmacokinetic changes in total MPA and MPAG levels were not observed in HL rats, there was a marked increase in free MPA and MPAG levels. Clinically relevant pharmacokinetic changes in subjects with HL that are related to MRP2 could not be ruled out. Copyright © 2016 John Wiley & Sons, Ltd.     

Preliminary investigation of the nasal delivery of liposomal leuprorelin acetate for contraception in rats

The purpose of the study was to investigate the nasal route as a non-invasive alternative for delivery of leuprorelin acetate (leuprolide acetate, LEU) and to achieve an effective concentration of leuprorelin acetate in blood after nasal administration for contraception in rats. The plain drug solution, physical mixture (plain drug along with constituents of liposomes), or drug encapsulated in either neutral or charged liposomes containing 5 microg leuprorelin acetate were administered to rats through the nasal route. The plain drug solution was administered subcutaneously (s.c.). Simultaneous evaluation was performed on the influence of a mucoadhesive agent (chitosan) on nasal absorption of the plain drug and the liposome-encapsulated drug. Blood samples were taken at regular time intervals and subjected to luteinising hormone (LH) analysis using a specific immunoassay kit. The plasma luteinising hormone concentration vs time data of nasal and subcutaneous treatments were plotted and compared with that of subcutaneous administration. Relative percentage of bioavailability (F) for nasal treatments was calculated from plasma concentration vs time plots. Sperm count and fertility performance studies were carried out for selected formulations in rats. Neutral liposomes (LLEU) and negatively-charged liposomes (LLEUn) showed higher relative percentage of bioavailability (F 27.83 and 21.30%, respectively) as compared with the plain drug and the physical mixture (F 10.89 and 10.96%, respectively) after nasal administration. Hence, work on neutral liposomes was continued. F was further improved after incorporation of chitosan i.e. 10.89 to 49.13% for plain leuprorelin acetate and 27.83 to 88.90% for liposomal leuprorelin acetate formulations. Liposomal chitosan formulation administered nasally and leuprorelin acetate solution subcutaneously achieved complete azoospermia. No implantation sites were observed after the mating of female rats with treated males. It was observed that in the treated female rats, the estrous cycles ceased with the same formulations from the first treatment cycle. The findings of these investigations demonstrated that the bioavailability of the nasally-administered liposomal leuprorelin acetate with chitosan formulation was comparable with that of the subcutaneously administered drug. Complete contraception was obtained in male and female rats that had been treated with either the nasally administered liposomal leuprorelin acetate with chitosan or the subcutaneously administered drug.     

Improved bioavailability of a new oral preparation of medroxyprogesterone acetate.

Medroxyprogesterone acetate (MPA) is widely used in the hormonal therapy of breast cancer. So far, oral formulations of MPA commercially available present a very low bioavailability, with a less than 10% extent of oral absorption. A new oral preparation of MPA has been recently developed. Based on a pilot study, an open, randomized, crossover trial has been performed on 22 breast and endometrial cancer patients to evaluate the relative bioavailability of this new oral formulation (200-mg sachet, twice daily) as compared with a standard formulation (Farlutal, 500-mg tablet, twice daily). The bioavailability evaluation was mainly based on the area under the curve measured between two administrations at steady state, after 15 days of continuous therapy. Wide interpatient variability of MPA plasma levels after oral MPA administration was confirmed. The MPA plasma levels were higher in patients treated with the new formulation than in patients treated with Farlutal. The relative bioavailability of the new preparation was 3.5 times higher than that of the standard. This new formulation represents a great improvement in the extent of oral absorption of MPA and could lead to better management of hormone-responsive tumors by hormonal therapy.     

基于HPLC法测定醋酸甲羟孕酮的平衡溶解度与油水分配系数

目的对醋酸甲羟孕酮(MPA)在不同溶剂中的平衡溶解度及其在正辛醇-缓冲液体系中的油水分配系数进行测定。方法采用HPLC法测定MPA的质量浓度,测定MPA在水、有机溶剂、缓冲溶液和加十二烷基硫酸钠的缓冲溶液中的平衡溶解度,采用摇瓶法结合HPLC法测定MPA在正辛醇-缓冲液体系中的质量浓度,从而计算油水分配系数。结果 37℃时,MPA在蒸馏水和丙酮中的平衡溶解度分别为0.52±0.04和238.70±0.47μg·mL~(-1);在体积分数为90%的乙醇溶液中的平衡溶解度最大;在加十二烷基硫酸钠的缓冲溶液中的平衡溶解度增大;在水中的表观油水分配系数为1.99(lgP=0.30),脂溶性较强;在pH值为1.2~6.8的缓冲溶液中,平衡溶解度随pH值的升高而减小,油水分配系数随pH值的升高而增大。结论该实验建立的测定方法可准确测定MPA的平衡溶解度和油水分配系数。     

Methylprednisolone-loaded PLGA microspheres: a new formulation for sustained release via intra-articular administration. A comparison study with methylprednisolone acetate in rats

Methylprednisolone (MP) released by poly(d,l-lactide-co-glycolide) microspheres (PLGA MS) was monitored in plasma after intra-articular (i.a.) administration into rat joint. A validated LC-ESI-MS/MS method was used to quantify the plasmatic concentrations of MP. The calculated pharmacokinetic parameters were compared to those obtained after the i.a. administration of a commercially available suspension of MP acetate (MPA). Different pharmacokinetic profiles were observed in the two formulations, and a lower peak level (C(max) = 13.7 ± 4.3 ng · mL(-1)) and AUC(0-72 h) (198 ± 45 ng · mL(-1) · h) were observed for MP-PLGA MS than MPA (C(max) = 18.4 ± 2.7 ng · mL(-1)) and AUC(0-72 h) (943 ± 249 ng · mL(-1) · h). The administration of MP-PLGA MS resulted in a rapid increase in the MP concentration at 30 min, with a t(max) at 0.8 ± 0.3 h. Instead, for the MPA suspension the t(max) was 32.0 ± 13.9 h. These differences were indirectly confirmed by the evaluation of the extra-articular effects, namely, carrageenan-induced paw edema, since MP-PLGA MS showed a lower anti-inflammatory activity than MPA.     

Microsomal effects of cyproterone acetate and flutamide in rat testis

1. Effects of two doses of cyproterone acetate (CA) and flutamide (FLU) (50 and 100 mg/kg/3 days) on the testicular steroidogenesis in male rats have been investigated, by measuring the content of cytochrome P-450, cytochrome b5 and cytochrome c reductase activity in the microsomal fraction. 2. CA provoked a significant decrease in cytochrome P-450 content while FLU induced an increase with the lowest dose but a significant decrease after administration of 100 mg/kg. 3. On the other hand, in all cases the cytochrome b5 and cytochrome c reductase activity remained unchanged. 4. The plasma levels of LH and testosterone were measured after CA and FLU injection (50 and 100 mg/kg/3 days). 5. CA administration provoked a reduction in blood levels of these hormones while FLU induced a significant increase in both. 6. These data suggested that CA and FLU modified the cytochrome P-450 in rat testes by an indirect mechanism, probably through the modification of LH plasma levels.     

Elimination of methoxyacetic acid and ethoxyacetic acid in rat.

1. The pharmacokinetics of methoxyacetic acid (MAA) and ethoxyacetic acid (EAA) have been determined in the male and female rat following bolus intravenous administration at 100 mg/kg. The plasma-concentration data of MAA fitted well to a one-compartment model, and the plasma-concentration data of EAA to a two-compartment model. 2. The elimination half-life of MAA estimated from plasma data was higher in females (18.6+/-2.0 h) than in males (13.2+/-0.4 h). There was no difference in the elimination half-lives estimated from urine data. The apparent volume of distribution was lower in the male than in the female rat estimated from plasma data only, while AUC, total and non-renal clearances and the relative amount MAA excreted unchanged in urine was similar in the male and female rat. 3. Clearance of EAA is higher than of MAA, and this appears as a result of metabolic capacity. The elimination half-lives of EAA were similar in the male and female rat, 9.4+/-3.7 and 10.5+/-2.6 h respectively. AUC was higher in the female compared with the male rat. The fraction of EAA eliminated during the distribution phase was 44.0+/-15.4 and 41.0+/-17.4% in the male and female rat respectively. The initial volume of distribution, the apparent volume of distribution, total and non-renal clearance are higher in the male compared with the female rat.