OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Comparative metabolic conversion of aflatoxin B1 to M1 and Q1 by monkey, rat and chicken liver

We studied the $\text{in vitro}$ metabolish of flatoxin B₁ by liver microsomal preparations from monkey, rat and chicken. With all these species, both the previously recognized metabolite aflatoxin M₁ as well as the newly identified aflatoxin Q₁ were produced from the aflatoxin B₁ substrate. Aflatoxin Q₁ is an isomer of aflatoxin M₁ (with the hydroxyl on the carbon β to the carbonyl of the cyclopentenone ring) which we discovered recently in rat and monkey liver incubations with aflatoxin B₁. In our incubations we did not detect aflatoxin P₁ which has been reported as a major metabolite of aflatoxin B₁$\text{in vivo}$ in the monkey.In general the conversion to aflatoxin M₁ was comparable among the different species (1–3% of the substrate) except in the chicken in which it was lower (0.1–0.3%). Also the conversion to Q₁ was comparable to or slightly higher than the conversion to M₁ with rat and chicken liver but the conversion to Q₁ with the monkey liver was outstandingly high, accounting for 19–52% of the substrate in three species of monkeys tested.     

Metabolism of prostacyclin in cynomolgus monkey

The metabolism of prostacyclin (PGI₂) $\text{in vivo}$ was investigated in Cynomolgus monkey. Following intravenous infusion of 11-[³H]-PGI₂ for three days, pooled urine was extracted with Amberlite XAD-2, then chromatographed and purified by Sephadex LH-20, and reverse phase column chromatography. Radioactive fractions were converted to appropriate derivatives for identification by gas chromatography mass spectrometry. Twelve metabolites were characterized, the major of which was 6-keto-PGF_1α, accounting for 13% of the urinary radioactivity. The metabolic pathways are similar to those observed earlier in the rat. The excretion of substantial amounts of unchanged 6-keto-PGF_1α indicated that the monkey was not able to metabolize PGI₂ as avidly as the rat.     

Identification of the major urinary metabolite of thromboxane B2 in the monkey.

Thromboxane B2 (TxB2) was biosynthesized from prostaglandin endoperoxides (PGG2, PGH2) using guinea pig lung microsomes and infused into an unanesthetized monkey. Urine was collected and TxB2 metabolites were isolated by reversed phase partition chromatography and high performance liquid chromatography. A major metabolite (TxB2-M) was found to be excreted in greater than two-fold abundance relative to other metabolites. Its structure was determined by gas chromatography-mass spectrometry to be dinor-thromboxane B2. In vitro incubation of TxB2 with rat liver mitochondria yielded a C18 derivative with a mass spectrum identical to that of TxB2-M, substantiating that the major urinary metabolite of TxB2 in the monkey is a product of a single step of beta-oxidation.     

Rodent macrophages metabolize 25-hydroxyvitamin D3in,vitro

Rodent macrophages metabolized 25-hydroxyvitamin D₃ to an unidentified metabolite during $\text{in}\text{,}\text{vitro}$ incubations. The production of this macrophage-derived metabolite of 25-hydroxyvitamin D₃ increased as the substrate concentration was raised. A two step high pressure liquid chromatography system revealed a unique elution position of this macrophage-derived metabolite that did not match the elution positions of any of the vitamin D₃ metabolites available in this laboratory. This unique metabolite was formed $\text{in}\text{,}\text{vitro}$ within one minute by incubated macrophages although its formation increased gradually up to 60 minutes of incubation.     

Aureobasidium pullulans metabolism of prostaglandins of the A-type

$\text{Aureobasidium pullulans}$, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA₂ (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that $\text{A. pullulans}$ can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ^10,11 and Δ^13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ^10,11 → Δ^11,12). $\text{A. pullulans}$ metabolizes 15-epimeric PGA₂ equally readily with the production of similar products. PGA₁ affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. $\text{A. pullulans}$ displays some enantioselectivity, PGA₂ and 15-epi-PGA₂ are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Urinary levels of 2,3-dinor-6-oxo-PGF1α: A reliable index of the prodcution of PGI2 in the spontaneously hypertensive rat

The urinary levels of 2,3-dinor-6-oxo-PGF_1α (PGI₂-M), a major metabolite of PGI₂, are determined by the balance between the amount of PGI₂ synthesized and the extent of its further metabolic oxidation. The purpose of the present study was to determine if the urinary excretion of PGI₂-M can be used as a reliable index of the $\text{in vivo}$ production of PGI₂ in both normal Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). This involved the exclusion of differences in metabolism between these two strains of rats. In order to do so, we monitored the urinary excretion of PGI₂-M during paired intravenous infusions of 6-oxo-PGF_1α (the stable product of the spontaneous hydrolysis of PGI₂) in conscious, unrestrained SHR and WKY rats aged 12–15 weeks, in doses ranging from 250 to 700 ng. In one experiment, PGI₂ was infused instead of 6-oxo-PGF_1α.The results of these experiments indicate that SHR and WKY rats are equal with regard to the transformation of 6-oxo-PGF_1α and PGI₂ into PGI₂-M. For both groups, there is a good correlation between the amount of 6-oxo-PGF_1α infused and the amount of PGI₂-M excreted in urine. These observations confirm the validity of using the urinary levels of 2,3-dinor-6-oxo-PGF_1α as an index of PGI₂ production in both WKY and SHR. In addition, they support the conclusions drawn from our previous studies, namely that SHR do not produce more PGI₂ than WKY rats $\text{in vivo}$, contrary to the situation prevailing $\text{in vitro}$.     

In vivo regulation of adipose tissue protein kinase by adenosine 3',5'-monophosphate mediated glucagon stimulation.

The cytochrome P-450 content of primary hepatocyte cultures was maintained at levels close to those found $\text{in vivo}$ by using a defined medium containing testosterone, thyroxine, hydrocortisone, estradiol, glucagon, insulin, linoleic acid and oleic acid. Using these cultures, [¹⁴C]aflatoxin B₁, a potent liver carcinogen, was metabolized primarily to water-soluble metabolites. In agreement with $\text{in vivo}$ results, aflatoxin M₁ was the only nonpolar metabolite detected. In addition, a significant portion of radioactivity was covalently bound to cell constituents. These results suggest that primary hepatocyte cultures may be a good model of the liver for studying the metabolism and mechanism of action of toxic chemicals.     

Metabolism of prostaglandin A. I. The kidney cortex as a major site of PGA2 degradation

The antihypertensive and natriuretic prostaglandin A₂ (medullin) has been isolated and identified in rabbit renal papilla. Since PGA₂, unlike prostaglandin F₂ (PGE₂) and prostaglandin F_2α (PGF_2α), is not metabolized by the lung, studies were undertaken to determine if the site of PGA₂ metabolism is in the renal cortex where its primary vasodilatory and natriuretic effects occur. In $\text{in vitro}$ experiments, homogenates of renal cortex and outer medulla were incubated with ³H-PGA₂ (0.2 μc, 20 μg) at 37°C for 30 minutes. A metabolite(s) less polar than ³H-PGA₂ was observed following silicic acid chromatography of acidic lipid extracts of cortical, but not outer medullary homogenates. In $\text{in vivo}$ studies, ³H-PGA₂ (2 μc, 50 μg) was injected into the renal artery of the rabbit and blood withdrawn from the ipsilateral renal vein. At least three less polar major metabolites of PGA₂ were observed in the plasma within 15 seconds following injection. No appreciable ³H-PGA₂ was observed in venous plasma 30 seconds following injection of ³H-PGA₂. In contrast to plasma, the major urinary metabolites were more polar than PGA₂. The present study reveals that PGA₂ is almost completely metabolized in a single passage through the rabbit kidney suggesting this organ is a major site of PGA₂ metabolism.     

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.     

Rapid invivo metabolism of Leukotriene C3 in the monkey, Macacairus

[5,6,8,9,11,12-³H₆] Leukotriene C₃ (5 μCi) was injected through a catheter into the right atrium of an anesthetized male monkey. Blood samples were drawn from the aorta via a second catheter. The concentration of tritium in blood decreased from 100 nCi/ml after 5 sec to 1 nCi/ml 15 min after injection, suggesting that leukotriene C₃ was rapidly eliminated from the circulation. Chromatographic analyses of radioactive material in blood collected before recirculation had occurred (15 sec after injection) demonstrated that 40% of the radioactive material had been converted into two less polar metabolites. These products had the same chromatographic properties as leukotrienes D₃ and E₃, respectively. The results indicate that leukotriene C₃ is rapidly transformed by monkey lung $\text{in}\text{vivo}$. Two minutes after injection, the component corresponding to leukotriene E₃ was the predominating metabolite in blood.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

A new low-molecular-weight calcium-binding ligand in rat small intestine

Three calcium-binding ligands in the cytosol of rat small intestine have been separated using Sephadex G-75 column chromatography. The estimated molecular weights of these ligands were 85,000, 10,000, and 800, respectively. The two high-molecular-weight ligands were found only in young rats, while the low-molecular-weight calcium-binding ligand was found in all age groups and appeared to be a calcium-bound prostaglandin metabolite. Oral administration of arachidonic acid increased and PGE₂ decreased $\text{in vivo}$ calcium absorption, while PGF₂ administration showed no discernible effect on this parameter. These results suggest that the intestinal calcium transport mechanism may be modulated by prostaglandins and related metabolites.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Chemotactic activity of thromboxane B2, prostaglandins and their metabolites for polymorphonuclear leucocytes

(1) The chemotactic activities of thromboxane B₂ (TxB₂, PGE₂, PGF_2α, the 15-oxo, 15-oxo-13,14-dihydro and 13,14-dihydro metabolites of PGE₂, PGF_2α, and a metabolite of TxB₂ for polymorphonuclear leucocytes (PMN) have been investigated.(2) Thromboxane B₂ increased the directional migrationm of rat peritoneal PMN at a concentration of 2.0 μg/ml and of human peripheral neutrophils at a concentration of 0.5 μg/ml.(3) Neither PGE₂, PGF_2α nor their metabolites showed chemotactic activity for rat peritoneal PMN.(4) PGF_2α and 15-oxo-13,14-dihydro-thromboxane B₂ showed no chemotactic activity for human peripheral PMN.(5) The possible role of thromboxane B₂ in inflammation is discussed.     

Reflex ovulation in light-induced persistent estrus (LLPE) rats: Role of sensory stimuli and the adrenals

Penile intromissions have been thought to be the primary stimulus for reflex ovulation in light-induced persistent estrus (LLPE) rats, even though other stimuli also trigger reflex ovulation. To clarify the nature of these noncoital stimuli, intact (nonadrenalectomized) LLPE rats were briefly exposed to a variety of environmental stimuli, other than intromissions, and checked for ova 19–22 hr later. Summary of results (number of rats ovulating/number of rats tested): (A₁) home cage ($\text{3}\text{10}$); (B₁) home cage + vaginal taping ($\text{2}\text{9}$); (C₁) home cage + male-soiled bedding ($\text{15}\text{28}$); (D₁) novel cage ($\text{2}\text{11}$); (E₁) novel cage + vaginal taping ($\text{2}\text{11}$); (F₁) novel cage + vaginal taping + male-soiled bedding ($\text{9}\text{19}$); (G₁) novel cage + vaginal taping + male-soiled bedding + male mounts without intromissions ($\text{14}\text{26}$). The percentage of LLPE rats that ovulated in the last-mentioned test condition was related to the degree of proceptivity/receptivity of the LLPE females. Eight of eight proceptive LLPE females ovulated, but only $\text{6}\text{18}$ nonproceptive females ovulated. To account for reflex ovulation in the absence of intromission it has been suggested that adrenal progesterone (P) stimulates release of an ovulatory quota of luteinizing hormone. This study demonstrates no significant differences in percentage of LLPE females ovulating in corresponding groups of adrenalectomized (ADX) and adrenal-intact females. Summary of results: A₂ = $\text{0}\text{6}$, B₂ = $\text{5}\text{15}$, C₂ = $\text{4}\text{16}$, D₂ = $\text{2}\text{14}$, E₂ = $\text{5}\text{13}$, F₂ = $\text{7}\text{19}$, G₂ = $\text{10}\text{21}$. Conclusion: (a) Exposure to a factor in male-soiled bedding induces reflex ovulation in a significant proportion of adrenal-intact LLPE animals while exposure to a novel cage and/or vaginal taping does not, (b) penile intromissions are not the primary stimulus for reflex ovulation in intact proceptive LLPE rats, and (c) adrenal P is not required for reflex ovulation after tests with noncoital stimuli.     

Alkylation and carbamoylation intermediates from the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl -1-nitrosourea (CCNU)

Evidence is presented which shows that 1-(2-chloroethyl) -3-cyclohexyl-1-nitrosourea (CCNU) upon degradation provides a 2-chloroethyl alkylating intermediate, possibly 2-chloroethyl carbonium ion, and 2-chloroethanol. Thiol alkylation occurs $\text{in vivo}$ and a major urinary metabolite of CCNU is thiodiacetic acid. A rapid microsomal hydroxylation of the cyclohexyl ring occurs which yields varying ratios of at least five metabolites: cis or trans 2-hydroxy, trans- 3-hydroxy, cis-3-hydroxy, cis-4-hydroxy and trans-4- hydroxy-CCNU. $\text{In vivo}$ carbamoylation appears to not be due to cyclohexylisocyanate but to the various hydroxy-cyclohexylisocyanates which are formed from hydroxy CCNU metabolites.     

Microbial transformations of natural antitumor agents. 23. conversion of withaferin-A to 12β- and 15β-hydroxy derivatives of withaferin-A.

Microbial transformation experiments were conducted with the antitumor lactone withaferin-A. $\text{Cunninghamella elegans}$ NRRL 1393 transformed withaferin-A ($\text{1a}$) to 15β-hydroxywithaferin-A ($\text{2a}$) and 12β-hydroxywithaferin-A ($\text{3a}$). The hydroxylated metabolites were isolated by solvent extraction and were purified by column and thin-layer chromatography. Structures of the hydroxylated metabolites were determined by protonand carbon-13 NMR, IR and mass spectral analyses, and by the preparation of acylated derivatives. Compounds $\text{2a}$ and $\text{3a}$ inhibited the growth and biochemical functions of $\text{in vitro}$ grown P-388 lymphocytic leukemic cells.     

Neocarzinostatin chromophore: Presence of a cyclic carbonate subunit and its modification in the structure of other biologically active forms

On the basis of spectroscopic evidence, opening of a five-membered cyclic carbonate ring (1,3-dioxolan-2-one) in the C₁₅-subunit of the previously determined partial structure $\text{1}$ (Fig. 1) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$), is proposed to account for its base-catalyzed methanolysis to NCS-Chrom $\text{C}$. NCS-Chrom $\text{B}$, apparently an authentic natural product present as a minor component in all preparations of NCS studied, was found to be formally equivalent to the hydrolysis/decarboxylation product of the cyclic carbonate functionality in NCS-Chrom $\text{A}$. The mercaptan-dependent DNA strand-scission activity, equivalent for NCS-Chrom $\text{A}$, $\text{B}$ and $\text{C}$, is independent of the integrity of the cyclic carbonate ring system and implicates a secondary site in the C₁₅-substructure for mercaptan activation.     

Hepatic microsomal mixed function oxygenase: enzyme multiplicity for the metabolism of carcinogens to DNA-binding metabolites

Microsome-mediated metabolic activation of aflatoxin B₁ (AFB₁) and benzo[a]-pyrene (BP), as determined by the $\text{in vitro}$ formation of DNA binding metabolites, was studied, using hepatic microsomes from untreated, phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated male rats. Contrasting results were obtained for the two substrates: in the case of AFB₁, microsomes from PB-treated rats were twice as active as microsomes from untreated and MC-treated rats, whereas, in the case of BP, microsomes from MC-treated rats were several fold more active than microsomes from untreated and PB-treated rats. These data strongly suggests enzyme multiplicity of microsomal mixed function oxygenase for the activation of carcinogens, especially AFB₁ and BP whose reactive metabolites are believed to be epoxides.