Metabolism of two PGF2α analogues in primates: 15(S)-15-methyl-Δ4-cis-PGF1α and 16,16-dimethyl-Δ4-cis-PGF1α

The metabolism of the prostaglandin F_2α analogues, 15-methyl-Δ⁴-$\text{cis}$-PGF_1α and 16,16-dimethyl-Δ⁴-$\text{cis}$-PGF_1α, has been investigated in the cynomolgus monkey and the human female. The two analogues, tritium labelled in the 9β-position, were administered by intramuscular injections into the monkeys and by subcutaneous injections into the human. Excretion of tritium labelled products were followed in urine (in both species) and feces (in monkeys only) and several metabolites were identified by GC/MS. The analogues were found to be resistant to the 15-hydroxy dehydrogenase and furthermore the degradation by β-oxidation was delayed. About 13% of the given dose of 15-methyl-Δ⁴-$\text{cis}$-PGF_1α was excreted unchanged into urine and feces from the monkey. The corresponding figure for 16,16-dimethyl-Δ⁴-$\text{cis}$-PGF_1α was about 20%. In addition, a large part of the metabolites had the carbon skeleton intact and were only metabolized by ω-oxidation. The relative resistance to degradation of these two analogues is likely to be the basis for their prolonged pharmacological activity.     

Metabolism of delta4-cis-PGF1alpha in the monkey.

This isomer of PGF2alpha is relatively resistant to metabolic degradation in the Cynomolgus monkey. Thus, 16-20 per cent of the amount injected was excreted unchanged in the urine. Five metabolites with 20, 18, 16 and 14 carbon atoms in the skeleton were identified. The data are similar to those earlier seen in the rat and further support the idea that this analogue of PGF2alpha could have a long half-life time in the mammalian body and thus a long duration of its pharmacological actions.     

Effects of PGF2alpha and PGF2alpha, 1-15 lactone on the corpus luteum and on early pregnancy in the rhesus monkey.

The effects of prostaglandin (PG)F2alpha and PGF2alpha, 1-15 lactone were compared in luteal phase, non-pregnant and in early pregnant rhesus monkeys. Animals treated with either PG after pretreatment with human chorionic gonadotropin (hCG) had peripheral plasma progesterone concentrations that were not statistically different from those in animals treated with hCG and vehicle. However, menstrual cycle lengths in monkeys treated with PGF2alpha, 1-15 lactone were significantly (P less than 0.02) shorter than those in vehicle treated animals. In the absence of hCG pretreatment, plasma progesterone concentrations were significantly (P less than 0.008) lower by the second day after the initial treatment with either PGF2alpha or PGF2alpha, 1-15 lactone than in vehicle treated monkeys. Menstrual cycle lengths in monkeys treated with either PG were significantly (P less than 0.04) shorter than those in animals treated with vehicle. There were no changes in plasma progesterone concentrations in early pregnant monkeys treated with PGF2alpha, and pregnancy was not interrupted. In contrast, plasma progesterone declined and pregnancy was terminated in 5 of 6 early pregnant monkeys treated with PGF2alpha, 1-15 lactone. These data indicate that PGF2alpha, 1-15 lactone decreases menstrual cycle lengths in non-pregnant rhesus monkeys. More importantly, PGF2alpha, 1-15 lactone terminates early pregnancy in the monkey at a dose which is less than an ineffective dose of PGF2alpha.     

Metabolism of toremifene in the rat

Toremifene was labelled to a specific activity of about 20 microCi/mmol with tritium at positions 3 and 5 in the para-substituted phenyl ring. At these positions tritium is not eliminated within the metabolic pathways. A mixture of unlabelled and labelled toremifene (5 or 10 mg/kg, 5 microCi/mg) was given i.v. or p.o. to Sprague-Dawley rats. The elimination of radioactivity was followed up by collecting urine and feces daily for 13 days. The elimination of toremifene which was similar after p.o. and i.v. administration took place mainly in the feces. About 70% of the total radioactivity was eliminated within 13 days, of this amount more than 90% in the feces. All applied radioactivity could be detected in three separate fractions according to the oxidative state of the side chain when counted by Berthold TLC Linear Analyzer. Each fraction was further separated into single metabolites by TLC or HPLC. Altogether 9 metabolites were identified and almost all methanol-extractable components were identified. The main metabolic pathways in the rat were 4-hydroxylation and N-demethylation. The side chain was further oxidized to alcohols and carboxylic acids. Small amounts of unchanged toremifene were found in the feces both after p.o. and i.v. administration indicating biliary secretion.     

Luteolytic action of 15-keto prostaglandin F2alpha in the rhesus monkey.

The naturally-occurring metabolite of prostaglandin F2alpha, 15-keto prostaglandin F2alpha (15-keto PGF2alpha), elicited rapid and sustained declines in serum progesterone concentrations when administered to rhesus monkeys beginning on day 22 of normal menstrual cycles. Evidence for luteolysis of a more convincing nature was obtained in studies where a single dose of 15-keto PGF2alpha was given on day 20 of ovulatory menstrual cycles in which intramuscular injections of hCG were also given on days 18-20; serum progesterone concentrations fell precipitously in monkeys within 24 hours following intramuscular administration of 15-keto PGF2alpha. However, corpus luteum function was impaired in only 4 of 11 early pregnant monkeys when 15-keto PGF2alpha was administered on days 30 and 31 from the last menses, a time when the ovary is essential for the maintenance of pregnancy. Gestation failed in 2 additional monkeys 32 and 60 days after treatment with 15-keto PGF2alpha, but progressed in an apparently normal manner in the remaining 5 animals. Two pregnant monkeys treated with 15-keto PGF2alpha on day 42 from the last menstrual period, a time when the ovary is no longer required for gestation, continued their pregnancies uneventfully. Corpus luteum function was not impaired in 9 control monkeys which received injections of vehicle or hCG at appropriate times during the menstrual cycle or pregnancy.     

Effects of analogues of prostaglandin E2 and F2 alpha on the decidual cell reaction in the rat

The identity of the prostaglandins (PGs) involved in the decidual cell reaction is uncertain. In the present study we investigated the ability of analogues of PGE2 and PGF2 alpha, 16,16-dimethyl-prostaglandin E2, methyl ester (16,16Me2PGE2) and 15(S)-15-methyl-prostaglandin F2 alpha (15MePGF2 alpha) respectively, to bring about decidualization when infused into the uterine lumen of rats sensitized for the decidual cell reaction. As indicated by uterine weights 5 days after the commencement of the infusions into rats in which endogenous PG production had been inhibited by treatment with indomethacin, 16,16Me2PGE2 produced decidualization which was equivalent to that produced by PGE2. By contrast, the infusion of 15MePGF2 alpha inhibited decidualization, even when PGE2 was infused concomitantly. As indicated by uterine radioactivity concentrations after i.v. administration of 125I-labeled bovine serum albumin, the PGF2 alpha analogue also inhibited the endometrial vascular permeability increase which precedes decidualization. Compared to PGE2, 16,16Me2PGE2 was slightly less effective at displacing 3H-PGE2 from an endometrial membrane preparation; by contrast 15MePGF2 alpha was considerably less effective. These data suggest that PGE2 mediates the decidual cell reaction, and that the decidualization obtained in response to PGF2 alpha may involve its conversion within the uterus to PGE2.     

Genetic polymorphism of alpha 1-antitrypsin in green monkeys studied by isoelectric focusing and family analysis

24 variants of alpha 1-antitrypsin (alpha 1-AT) were recognized in sera of 120 wild and capture-born African green monkeys by isoelectrofocusing in Ampholine PAG-plates (pH 4-6.5) and western blotting with antihuman alpha 1-AT serum. All variants had much more cathodal position than human alpha 1-AT and revealed very high microheterogeneity which was slightly different from the observed in human alpha 1-AT. The alpha 1-AT banding pattern allowed to postulate existence of 10 codominant alleles in the Pi locus of African green monkeys. The reality of 8 alleles was proved by family analysis which included 45 monkey birth cases. Two other alleles were absent in the parents available. Thus, alpha 1-AT is the most polymorphic among the known serum proteins of African green monkeys. The latter can be useful for molecular systematics of these primates.     

Retrovirus restriction by TRIM5alpha variants from Old World and New World primates

The TRIM5alpha proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5alpha orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5alpha proteins functionally resembled human TRIM5alpha, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5alpha proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5alpha, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5alpha B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5alpha proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5alpha proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.     

Excretion and measurement of estradiol and progesterone metabolites in the feces and urine of female squirrel monkeys (Saimiri sciureus)

The first objective of the present study was to determine the metabolic form and rate of excretion of ovarian hormone metabolites in the urine and feces of female squirrel monkeys injected with radiolabeled progesterone (Po) and estradiol. The major portion of the urinary metabolites of both hormones was excreted within 16-24 hr post-injection. Estrogen and Po isotopes in feces exhibited an excretion peak at 16 hr post-injection. The majority of recovered radiolabel of both hormones was excreted in feces. Chromatographic separation of fecal extractions indicated that the major estrogen metabolites in feces are in the free as opposed to the conjugated form. The radioactivity and immunoreactivity for estrone and estradiol (E(1) and E(2), respectively) in eluates of fecal samples subjected to celite co-chromatography indicated that both free E(1) and E(2) exist as excretion products in the feces of female squirrel monkeys. The major radioactive peaks for Po metabolites showed peaks in the elution profile at or very near the Po standard, and corresponded with the celite co-chromatography elution profile of Po standard when subjected to enzyme immunoassay (EIA). The second objective was to validate the application of EIA systems to measure fecal metabolites. Reproductive events of one female squirrel monkey across one annual reproductive cycle are described using the endocrine profile generated from fecal steroid assays. Examination of this profile confirmed that longitudinal fecal sampling and steroid hormone metabolite measurement in feces was not only feasible and practical, but accurately detected known reproductive events as well.     

Immunological analysis of alpha 1-microglobulin in different mammalian and chicken serum. alpha 1-Microglobulin is 5-8 kilodaltons larger in primates

Heterologous radioimmunoassays for a semiquantitative analysis of alpha 1-microglobulin were developed, exploiting the binding between polyclonal rabbit or goat antisera against human, guinea pig, or rat alpha 1-microglobulin and 125I-labeled human, guinea pig, or rat alpha 1-microglobulin. Homologues of this protein were detected in human, guinea pig, Rhesus monkey, rat, mouse, rabbit, goat, horse, and cow serum by inhibition of a set of heterologous radioimmunoassays. Serum proteins were separated by gel chromatography, and fractions were pooled, concentrated, and radiolabeled with 125I. By immunoprecipitation of the radioiodinated serum pools with heterologous anti-alpha 1-microglobulin-sera, and separating the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analogues of alpha 1-microglobulin were isolated from serum of man, guinea pig, Rhesus monkey, rat, mouse, horse, and chicken. The apparent molecular weight of alpha 1-microglobulin was 31,000-32,000 in human and monkey serum and 24,000-26,000 in guinea pig, rat, mouse, horse, and chicken serum. The possibility of an addition of a 5,000-8,000-Da peptide in primate alpha 1-microglobulin is discussed.     

Analysis of metabolic profiles of prostaglandins in urine using a lipophilic anion exchanger

A method is described for the fractionation of prostaglandins and their metabolites in urine. Following acidification and extraction on Amberlite XAD-2, samples were separated by chromatography on the lipophilic anion exchanger diethyl-aminohydroxypropyl Sephadex LH-20 into fractions containing neutral compounds, monocarboxylic, dicarboxylic and polycarboxylic acids. The compounds in resulting fractions were further separated by reversed phase partition chromatography. As an application, the metabolic profiles in urine of [9β-^3H]-labeled prostaglandin F_2α¹ and prostaglandin analogs 15-methyl-PGF_2α and 16,16-dimethyl-PGF_2α were investigated in the cynomolgus monkey. It was demonstrated that the resolution of individual prostaglandin metabolites by reversed phase partition chromatography was considerably simplified by initial group separation on the anion exchanger, and several metabolites were much purified. A glucuronic acid conjugate of the main metabolite of 15-methyl-PGF_2α (dinor-15-methyl-PGF_2α) was tentatively identified using computerized gas chromatography - mass spectrometry.     

Biotransformation of cadina-4,10(15)-dien-3-one and 3alpha-hydroxycadina-4,10(15)-diene by Curvularia lunata ATCC 12017

Cadina-4,10(15)-dien-3-one (1) was metabolised by Curvularia lunata ATCC 12017 in two different growth media to give three metabolites, one of which, 12-hydroxycadina-4,10(15)-dien-3-one (4), was new. Incubation of 3alpha-hydroxycadina-4,10(15)-diene (2) with the fungus produced three new analogues, namely, (4S)-1alpha,3alpha-dihydroxycadin-10(15)-ene (5), 3alpha,14-dihydroxycadina-4,10(15)-diene (6) and 3alpha,12-dihydroxycadina-4,10(15)-diene (7).     

Precursor role of 15alpha-hydroxyestradiol and 15alpha-hydroxyandrostenedione in the formation of estetrol

Studies were designed to elucidate the origin of estetrol (15alpha-hydroxyestriol (estra-1,3,5(10)triene-3,15alpha,17beta-tetrol) or E4) during late human pregnancy. 3H-Labelled 15alpha-hydroxyestradiol (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17beta-estradiol (estra-1,3,5(10)-triene-3,17beta-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15alpha-hydroxyandrostenedion (15alpha-hydroxyandrost-4-ene-3,17-dione or 15delta4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5--6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)-triene-3,16alpha,17beta-triol or E3) containing 14C only and 15alpha-hydroxyestrone (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15delta4 and only partially via 15E2. When administered to the fetus E2 and 15delta4 contributed approximately equal amounts to urinary E4. The yield of 15alpha-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15alpha-hydroxylase. 15delta4 was a better precursor than E2 for urinary 15E2.     

Metabolism of the mutagen MeIQx in vivo: metabolite screening by liquid chromatography-thermospray mass spectrometry

2-Amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) is a potent mutagen found in cooked food. MeIQx and its isotopically labelled (13C, 15N2 and 14C) analogues were synthesised and used for metabolic studies in vivo. An equimolar mixture of MeIQx and its 13C, 15N2 stable isotope labelled analogue (containing tracer amounts of 14C-MeIQx) was given intraperitoneally to mice. Some 67% of the radioactivity was eliminated in urine and faeces within 24h. Four radiolabelled species were observed when urine was analysed by HPLC, corresponding to unchanged MeIQx and three more polar metabolites. Urine was analysed directly by HPLC-thermospray mass spectrometry. Four signals were observed containing the characteristic 1:1 isotopic doublet, corresponding to unchanged MeIQx, an MeIQx glucuronide, and two uncharacterized metabolites.     

Investigation of the heterogeneity of rhesus monkey alpha 1-antitrypsin

Rhesus monkey alpha 1-antitrypsin (n = 144) was examined for heterogeneity by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis. In contrast to other studies, no heterogeneity of Rhesus monkey alpha 1-antitrypsin could be documented using specific antisera. Rhesus monkey alpha 1-antitrypsin contained a reactive thiol. The pIs of the major isoforms of Rhesus monkey alpha 1-antitrypsin were 4.63, 4.69, 4.84 and 4.86 at 4 degrees C. No deficiency state of Rhesus monkey alpha 1-antitrypsin was detected. The six protease inhibitors in Rhesus monkey sera cross-reacted with antisera to the six human protease inhibitors.     

The metabolic fate of triamcinolone acetonide in laboratory animals

The metabolic fate of 9-fluoro-11β,16α,17,21-tetrahydroxy-l, 4-pregnadiene-3,20-dione cyclic 16,17-acetal with 2-¹⁴C-acetone, triaacinolone acetonide (TA) was studied in rabbits, dogs, monkeys and rats and found to be qualitatively similar in all species. In the dog, rat and monkey the major excretory route was the feces irrespective of the mode of administration. In the rabbit the excreted radioactivity was equally distributed between urine and feces. The metabolites were isolated by preparative thin layer chroma tography, located by autoradiography, eluted and analyzed by MS, IR, UV and NMR. The major metabolites of triamcinolone acetonide (TA) were identified as the C-21 carboxylic acids of TA and of the 6β hydroxy-TA,(6β-OH-TA) and the previously identified (1,2) 6β-OH-TA. In addition MS and UV data indicate the presence of 9-fluoro-11β,16α, 17-trihydroxy-3,20-dioxo-1,4,6-pregnatrien-21-oic acid cyclic 16,17 acetal with 2-¹⁴C-acetone.     

Additional studies on pregnancy termination and inhibition of the monkey corpus luteum with 5-oxa-17-phenyl-18,19,20-trinor-PGF1α methyl ester and structurally related prostaglandins

Oral administration of 5-oxa-17-phenyl-18,19,20-trinor-PGF_1α methyl ester (PGF-analog) resulted in a consistent and dose-dependent inhibition of corpus luteum progesterone production in nonpregnant rhesus monkeys concomitantly treated with human chorionic gonadotropin. Similarly, vaginal suppositories containing PGF-analog also inhibited the monkey corpus luteum. Side effects by the oral route of administration were minimal, whereas side effects of following vaginal treatment with PGF-analog were higher. Five prostaglandings with structural similarity to PGF-analog were studied for their ability to inhibit the monkey corpus luteum, but none showed an advantage over the parent molecule. PGF-analog did not synergize with 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ for the inhibition of the monkey corpus luteum, nor did it synergize with (15S)-15-methyl-PGF₂α methyl ester for the interruption of early pregnancy in the monkey. 9-Deoxo-9-methylene-5-oxa-17-phenyl-18,19,20-trinor-PGE₁ methyl ester did not terminate early gestation in the monkey at doses of 8 or 24 mg.     

Study on the metabolites of 1alpha,25-dihydroxyvitamin D4

Three major metabolites of 1alpha,25-dihydroxyvitamin D(4) were isolated from the bile of rat and the structures were elucidated on the basis of spectral data and the periodate oxidative cleavage of the diol structures of the metabolites. One of the metabolites was the known calcitroic acid. Another two metabolites were isomers and identified as 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-26-oic acid and 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-28-oic acid. It was found that 1alpha,25-dihydroxyvitamin D(4) is metabolized in a similar manner in vivo to that of 1alpha,25-dihydroxyvitamin D(2) but differently from 1alpha,25-dihydroxyvitamin D(3).     

Decreased rate of metabolism induced by a shift of the double bond in prostaglandin F 2alpha from the delta5 to the delta4 position.

The synthesis of an isomer of prostaglandin F 2alpha,9alpha,11alpha,15(S)-trihydroxyprosta-4-cis,13-transdienoic acid is described. The metabolism of this compound in the rat has been investigated. The rate of degradation by beta-oxidation was slowed down considerably. Thus 10-20% of the injected isomer was excreted in the urine unchanged indicating a longer half-life in the circulation than for prostaglandin F 2alpha. More over 2% was excreted as C20 metabolites, 11-18% as C18 metabolites and 8-15% as C16 metabolites. This relative resistance to degradation by beta-oxidation is of considerable biochemical and pharmacological interest.