Prostaglandins and congeners XIII (1). The synthesis of dℓ-erythro-16-methoxyprostaglandins

$\text{d?-Erythro}$-16-methoxy-PGE₂, PGA₂, PGF_2α, 11-deoxy PGE₁, and 11-deoxy PGF_1α have been prepared via the cuprate conjugate addition procedure. These congeners are less potent than the parent prostaglandins as stimulators of isolated gerbil colon contractions and as bronchodilators in the guinea pig Konzett assay.     

Decreased smooth muscle side effects with 16, 16-dimethyl-trans-Δ2-PGE1 methyl ester in Japanese monkey (Macaca fuscata fuscata)

The smooth muscle stimulating activity of a new PGE₁ analog, 16, 16-dimethyl-trans-Δ²-PGE₁ methyl ester (ONO-802) was evaluated by simulataneous;y recording the EMG of the uterus and intestines, along with urinary bladder pressure, and blood pressure in pregnant and non-pregnant Japanese monkeys ($\text{Macaca fuscata fuscata}$). Single intravenous injections of ONO-802 in increasing dosages (0.2–5 μg/kg) were found to be 50–100 times or more effective in inducing uterine contraction than PGF₂α and PGE₁. A mild, transient gastrointestinal muscle stimulating activity was observed, but change in urinary bladder pressure and blood pressure was not evident. ONO-802 induced uterine contractions in the pregnant animals were 10 times greater than in the non-pregnant animals. These results suggest that ONO-802 may be a suitable clinical prostaglandin for use in therapeutic abortion.     

Prostaglandin endoperoxides IV. Effects on smooth muscle

The effect on smooth muscle of the endoperoxides PGG₂ and PGH₂, which are intermediates in prostaglandin biosynthesis, was studied in different systems $\text{in vitro}$ and $\text{in vivo}$. On gastrointestinal smooth muscle (gerbil colon, rat stomach) PGG₂ and PGH₂ produced contractions comparable to those of PGE₂ and PGF_2a whereas contractions elicited on vascular (rabbit aorta) and airway (guinea-pig trachea) smooth muscle were considerably greater than those of PGE₂ and PGF_2a respectively. On intravenous injection into guinea-pigs PGG₂ and PGH₂ caused a triphasic change in blood pressure and were 8–10 times more effective than PGF_2a in producing an increase in tracheal insufflation pressure. When given as aerosols the unstable endoperoxides were less effective than PGF_2a. It is concluded that the endoperoxides are potent smooth muscle stimulants and that they are more effective than their degradation products (PGD₂, PGE₂, PGF_2a) in some systems.     

Prostaglandin synthesis in rabbit renal medulla.

Prostaglandin (PG) synthesis in rabbit renal medullary homogenates was investigated by gas chromatography-mass spectrometry utilizing two internal standards. The internal standards were added immediately after homogenization to an aliquot of the fresh homogenate. Another aliquot of the homogenate was incubated and subsequently the internal standards were added. The internal standards were 3,3,4,4 tetradeutero PGE₂(d₄-PGE₂) for quantification of PGE₂, the PGA₁ for quantification of PGA₂ and 3,3,4,4 tetradeutero PGA₂, the latter representing an $\text{in}$$\text{vitro}$ dehydration product of d₄-PGE₂ generated during work up of the samples. In 6 experiments on 6 rabbits levels of PGE₂ were 4.4 ± 1.6 μg/g (mean ± SD) renal medulla increasing during incubation to 14.95 ± 6.5 μg/g (p < 0.01) PGA₂ levels were 0.03 ± 0.02 μg/g in the non-incubated samples and did not increase during incubation. When PGA₂ levels were corrected for the amount of PGA₂ formed by $\text{in virto}$ dehydration from PGE₂ as measured by the amount of d₄-PGE₂ dehydrated to d₄-PGA₂ during workup, PGA₂ levels were indistinguishable from zero.     

Effect of castration, testosterone treatment and hereditary sterility on prostaglandin concentration in the male reproductive system of mice

Prostaglandin congeners wherein the 15-hydroxy group is moved to the C₁₆, C₁₇, or C₂₀ position or is replaced by a hydroxymethyl group were prepared $\text{via}$ the 1,4-addition of a lithium trialkyl-$\text{trans}$-alkenyl alanate to an appropriate cyclopentenone. Several of the 16-hydroxy derivatives showed significant activity as constrictors of the isolated gerbil colon and in bronchodilator and anti-secretory assays.     

Bronchodilator activity of a PGE2 analog in animals and in man

A C-11 substituted PGE₂ analog, DHET-PGE₂ [$\text{?}$-11-deoxy-11α-(2-hydroxyethylthio)-PGE₂ methyl ester], was demonstrated to exert potent bronchodilator activity in three $\text{in vivo}$ models of augmented airway resistance: (1) acute bronchospasms, induced by 5-hydroxytryptamine, histamine and acetylcholine in the anesthetized guinea pig, (2) acute bronchospasm, induced by pilocarpine, in the anesthetized dog, and (3) chronic bronchospasm, induced by SO₂ exposure, in the unanesthetized dog. In acute and 30-day toxicological studies in the dog, no cardiovascular, respiratory or gastrointestinal side effects were observed at aerosol doses at least 1,000 times those required for efficacy. $\text{In vitro}$, DHET-PGE₂ effectively relaxed isolated preparations of dog bronchus that had been contracted with carbachol. In clinical studies, human asthmatics and bronchitics responded consistently to β-agonist bronchodilators but variably to DHET-PGE₂. Overall, increases in pulmonary resistance or decreases in FEV₁ were observed with DHET-PGE₂. Subsequent evaluation in isolated carbachol-contracted human bronchus revealed that, in contrast to the bronchodilator activity of PGE₁ and β-agonists, DHET-PGE₂ and PGE₂ induced contraction. Considered along with results from previous clinical studies on other PGs, these data underscore the difficulties in making extrapolations on this class of compounds from animal models to humans and suggest that human bronchial tissue may provide the only appropriate preclinical test system for predicting the clinical efficacy of PG bronchodilators.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

Concentrative accumulation of 3H-prostaglandins by some rabbit tissues in vitro: The chemical nature of the accumulated 3H-labelled substances

Following incubation with [³H]-PGF_2α, 73–91% of the ³H activity accumulated by rabbit uterus, choroid plexus or anterior uvea was shown to remain associated with PGF_2α on two different chromatographic systems. The tissue to medium ratios, calculated on the basis of chromatographically identified [³H]-PGF_2α, were greater than unity (2.3–10.4) for all three tissues and the extracted ³H activity could be effectively accumulated by these tissues for a second time. Under conditions when 85% of authentic [³H]-PGF_2α and only 8% of [³H]-15-keto-PGF_2α was adsorbed on rabbit anti-PGF_2α serum, 60–75% of the extracted ³H was adsorbed onto the antiserum. Following incubation with a mixture of 5,6-[³H]-PGE₁ and 2-[¹⁴C]-PGE₁, the anterior uvea and the uterus showed similar $\text{T}\text{M}$ ratios for ³H and ¹⁴C and the ${}^3\text{H}{}^{14}\text{C}$ ratios were essentially constant in their respective homogenates, extracts and chromatographic fractions, indicating insignificant β-oxidation of the accumulated PGE₁. In the case of the kidney cortex, a substantial fraction of the accumulated ¹⁴C did not extract as a PG presumably as a result of β-oxidation. It is concluded that metabolic alteration of the accumulated PG molecule does occur in some tissues, but such chemical alterations are not an integral part of the PG accumulative process. These results are consistent with the concept that some vertebrate tissues can accumulate PGs against a concentration gradient by an active transport mechanism.     

Stimulatory effect of prostaglandin E1 on thyroliberin-induced α-melanotropin release from perifused neuro-intermediate lobes of frog pituitary gland

A possible direct effect of prostaglandins on α-melanotropin (α-MSH) release at the level of the intermediate lobe of the frog pituitary was investigated $\text{in vitro}$ using a perifusion system technique. The effect of prostaglandins was studied on both spontaneous and TRH-stimulated α-MSH secretion. No significant effect of PGE₁, PGE₂, PGF_1α or PGF_2α on basal release of α-MSH could be detected. Indomethacin did not alter the α-MSH release induced by TRH. Conversely a significant increase in TRH-induced α-MSH secretion was observed in the presence of 1 x 10^?6M PGE₁. This magnifying effect was directly related to the concentration of TRH for doses ranging from 1 x 10^?8M to 1 x 10^?6M.     

Macromolecular binding of (5,6-3H) prostaglandin E1 in liver cytosol in vitro

[³H] Prostaglandin E₁ (PGE₁) is bound extensively to macromolecules in liver cytosol $\text{in vitro}$. A principal binding protein accounts for 80% of the binding. This macromolecule is saturated at about 10^?10 M PGE₁. The partially purified protein has a molecular weight of 50,000 by gel filtration and a pI of about 3.5 by isoelectrofocusing. Binding is primarily noncovalent and the dissociated ligand behaves similarly to the parental [³H] PGE₁ on thin layer chromatography. Possible significance of this interaction is discussed.     

Role of endogenous and exogenous prostaglandins on the contractile functioning of isolated sow (Sus scrofa) oviducts

The output prostaglandins (PHs) E₁, E₂ and F₂ α, from ampullary and isthmic portions of sow oviducts isolated during proestrus, estrus and metestrus, was explored. Moreover, $\text{in vitro}$ cumulative dose-response curves for the contractile effect of these three PGs, on identical oviductal segments, were constructed. Isthmic preparations form proestrous and metestrous animals released more PGE₁ and PGF₂ α than PGE₂ “like material”. During estrus, the outputs of PGE₁, PGE₂ and PGF₂ α were similar, whereas, oviducts from proestrous and metestrous sows released less PGE₁ and PGF₂ α than during estrus. Although the output of PGE₂ “like material” from isthmic and ampullary segments did not differ significantly during the three stages of the sex cycle, ampullary metestrous preparations released more PGE₁ and PGF₂ α, than estrous or proestrous ones. The addition of PGE₁, PGE₂ α, consistently stimulatedthe amplitude of contractions of isthmic oviductal segments isolated from proestrous and metestrous sows. Within the concentration-range explored, dose-response curves for PGE₂ and PGE₁ were to the left of those for PGF₂ α in the isthmus obtained before ovulation (proestrus) but not in segments isolated at later times (2–3 days) of the cycle (metestrus). The stimulatory dose-response curves for PGE₁, or PGE₂, in isthmic segments of metestrous preparations incubated with phentolamine (10^?6M) were shifted to the right of controls not exposed to the adrenoreceptor blocker, whereas, the curve for PGF₂ α without phentolamine, was identical to that obtained in its presence. PGE₁ and PGE₂ did not evoke significant contractile effects on oviductal ampullary protions from proestrous sows, wherea, PGF₂ α was clearly stimulatory at concentrations of 10^?9M and higher. In ampullary segments isolated after ovulation (metestrus) the threshold for contractile enhancement following PGF₂ α was greater than during proestrus, whereas, PGE₁ elicited a significant inhibition of contractions. The spontaneous contractile pattern exhibited by isthmic and ampullary oviductal regions, prior to and after ovulation, is discussed in terms of tissue PG generation and output and is compared with results regarding tubal motility following the exposure to exogenous PGs.     

Synthesis of d1-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins including one which inhibits platelet aggregation

The synthesis of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxaprostaglandins oxaprostaglandins of the E₁ and F₁α series⁷ from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both $\text{in vitro}$ and $\text{in vivo}$ of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxa-PGE₁, methyl ester (VIII) are presented.     

Contractions of rabbit testes in vitro: Permissive role of prostaglandins for the actions of calcium and some smooth-muscle stimulating agents

Rinsing actively contracting rabbit testes $\text{in vitro}$ with fresh Tyrode's solution abolished capsular contractions and the response of this preparation to either Ca⁺⁺, serotonin, or acetylcholine. Adding exogenous prostaglandin E₂ (PGE₂) to the medium restored contractility and the responses of the preparation to each of the above three agents.⁴ A reciprocal dependency was observed between Ca⁺⁺ and PGE₂ in stimulating contractions. PGE₂ potentiated, but was not required for the stimulatory action of either epinephrine or histamine. The stimulation of contractility by epinephrine, but not prostaglandin was inhibited by the α-blocking agent, ergotamine tartrate.⁴ This action of epinephrine did not involve prostaglandin release, nor was it inhibited by indomethacin pretreatment.⁴ Isoproterenol inhibited testicular contractions evoked by PGE₂.     

Effects of arachidonic acid and its cyclo-oxygenase and lipoxygenase products on lymphatic vessel contracility in vitro

Lymphatic vessels exhibit rhythmical contractility $\text{in vivo}$ and $\text{in vitro}$ and this activity appears ti regulate lymph flow. A technique for measuring the cicurlar muscle contractions of isolated bovine mesenteric lymphatic vessel segments has been devised and utilized to study the pharmacological properties of these vessels. Non-contracting lympahtic vessels can be induced to contract rhythmically with a variety of mediators, the most potent being a stable PGH₂ analogue (compound U46619), and the leukotrienes B₄, C₄ and D₄ (threshold concentrations in the nanomolar range). Prostagladin F_2α, noradrenaline, serotonin and histamine also elicited rhythmical activity but much higher concentrations were required. PGE₂ and PGE₁ were potent inhibitors of spontaneous contractions or those induced with U46619. In keeping with the diverse pharmacological effects of the metabolites of arachidonic acid, the addition of arachidonate to an isolated lymphatic vessel generated both stimulatory and inhibitory activities.It is concluded that arachidonic acid products (produced in the lymphatic vessel or entering the vessel in lymph draining the tissues) regulate lymph flow through their effects on lymphatic smooth muscle.     

Effects of 19-hydroxy-prostaglandins on oviductal and uterine motility

The effects of 19-hydroxy-prostaglandins (19-OH-PGs) were tested $\text{in}$$\text{vivo}$ on the rabbit oviduct and uterus and on the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) uterus. The 19-OH-PGEs suppressed spontaneous oviductal and uterine activity in the rabbit. The qualitative effect on the rabbit oviduct of 19-OH-PGEs was similar to that of PGE₂. However, the typical response of the rabbit uterus to PGE₂ was an increase in muscle activity. With regard to the rabbit oviduct, 19(R)-OH-PGE₂ was as potent as PGE₂, but 19(S)-OH-PGE₂ was approximately $\text{1}\text{2}$ as potent as PGE₂. Based on the dose of 19-OH-PGEs usually required to cause a minimal suppression and the dose of PGE₂ required to cause a minimal stimulation of rabbit uterine activity, 19(R)-OH-PGE₂ was twice as potent as PGE₂ while 19(S)-OH-PGE₂ was $\text{1}\text{2}$ as potent as PGE₂. Stimulatory effects on the rabbit oviduct and uterus were observed following administration of 19-OH-PGEs and PGF_2α. The potency on the rabbit oviduct of 19(S)-OH-PGF_2α was about $\text{1}\text{5}$ to $\text{1}\text{10}$ that of PGF_2α; the potency of 19(R)-OH-PGF_2α was about $\text{1}\text{10}$ to $\text{1}\text{20}$ that of PGF_2α. Both 19-OH-PGFs were approximately $\text{1}\text{5}$ to $\text{1}\text{10}$ as potent as PGF_2α on the rabbit uterus. At the doses tested 19-OH-PGFs were inactive on the monkey uterus. Thus, these compounds are at least $\text{1}\text{5}$ as active as PGF_2α. In contrast, 19(R)-OH-PGE₂ had approximately the same potency as PGE₂ in stimulating monkey uterine activity; but 19(S)-OH-PGE₂ was approximately $\text{1}\text{3}$ as potent as PGE₂.     

Effects of prostaglandin PGE2 on the synthesis of placental proteins and human placental lactogen (HPL)

The biosynthesis of placental proteins and placental lactogen (HPL) was studied $\text{in vitro}$ in 10–12 week, 16–18 week and term human placenta in the presence and absence of PGE_2α. The highest ¹⁴C-leucine incorporation was detected in 10 to 12 weeks old placentas. Addition of PGE_2α to the induction medium depressed the rate of incorporation of ¹⁴C-leucine into placental proteins on a dose dependent manner. Placentas most sensitive to this action of PGE_2α were those obtained at 18 weeks gestation followed by placentas at term. $\text{In vivo}$ application of PGE_2α for tharapeutic induction of abortions resulted in the marked inhibition of placental protein synthesis $\text{in vitro}$.     

Uterine stimulant action of some 16-phenoxy analogues of (+)-11-deoxyprostaglandin E1

Some ($\text{+}$)-11-deoxy-16-phenoxyprostaglandin E₁ analogues have been evaluated as uterine stimulants in the anaesthetised pregnant rat. Gastrointestinal side effects, assessed by the antagonism of morphine-induced consitpation in the mouse, were relatively low with some of these compounds, six of which had a much more favourable relative selectivity than 16,16-dimethyl1-PGE₂ methyl ester.     

Metabolism of arachidonic acid in vitro by porcine blastocysts and endometrium

Metabolism of radiolabeled arachidonic acid (¹AA) by blastocysts and endometrial slices recovered from five gilts 16 days after detection of estrus was studies $\text{in vitro}$. Blastocysts from each gilt were divided into four 216 ± 18 mg, and each portion was placed into a separate petri dish containing 15 ml modified minimum essential medium (MEM)_. The incubates from each gilt received either 25, 50, 100 or 200 μg radioinert arachidonic acid (AA). Endometrium was dissected from each uterin horn, sliced and duplicate 509 ± 3 mg portions from each gilt were placed into petri dishes containing 15 ml MEM and 200 μm AA. All incubates received 5 νCi of ¹AA (either [¹⁴C]-arichidonic acid or [³H]-arichidonic acid). The incubates were rocked at 37°C for 24 h in an atmosphere of 50% n₂:45% O₂:5% CO₂. After incubation, tissues and MEM were separated by centrifugation. Metabolism of ¹AA was assessed in extracts of MEM and tissue homogenates by separating ¹AA and its metabolites on columns of Sephades LH-20. Blastocysts produced compounds that migrated with [³H]-13,14-dihydro-15-keto-PGF_2^α (¹PGFM), [³H]-PGE₂ (¹PGE₂) and [³H]-PGF_2^α (¹PGF_2^α). The greatest (P<.05) proportion (35.7 ± 1.8%) of the radioactivity in blastocyst MEM was recovered as PGE₂. In blastocyst homogenates, most (66.2 ± 3.3%; P<0.05) of the radioactivity was in a nonporal peak assumed to be arachidonate esters. The concentration of AA ni MEM did not alter metabolism of ¹AA by blastocysts. Endometrial slices produced ¹PGFM and ¹PGE₂ but only in small amounts, and they were capable of producing nonpolar, probably esterified, forms of ¹AA. It was concluded that porcine blastocysts produced and metabolized prostaglandins $\text{in vitro}$ and that they make a contribution to the uterine milieu during early pregnancy.     

Multiple sites of interaction between prostaglandins and non-steroidal anti-inflammatory agents

Several non-steroidal anti-inflammatory agents (NSAIA) are shown to inhibit the net production of prostaglandin (PG)- like activity from arachidonic acid by a cell-free preparation of guinea-pig lung. Moreover, these agents also antagonize PGE₁-induced contractions of the isolated gerbil colon. The anti-spasmogenic effects are reversible and specific. At high concentrations, indomethacin and mefenamic acid interfere with the binding of PGE₁ to a broken cell preparation of rat epididymal adipocytes. Taken together the data indicate that NSAIA interact with prostaglandins at multiple sites and are consistent with the suggestions reported previously that NSAIA may have multiple actions.     

Relative contracting adn relaxing potencies of a series of prostaglandins on isolated canine mesenteric artery strips

The contracting and relaxing potencies of anf interactions between a number of prostaglandins (PGs) were studied $\text{in vitro}$ on spiral strips of small canine mesenteric arteries (outside diameter < mm). PGF₂α and PGE₂, the most potent contracting PGs, were nearly equal in potency (EC₅₀ 4 × 10^?7M) and did not cause relaxation under our experimental conditions. PGI₂ and PGE₁ were equal and the most potent relaxing PGs (EC₅₀ 3 × 10^?9M). PGE₁ also caused contraction, but this effect was not consistent. PGI₂ did not cause contraction in concentrations up to 3 × 10^?6M. In higher concentrations, however, it caused abrupt and near maximal contraction. PGD₂ was weak in both respect, causing incomplete relaxation and contraction or biphasic effects. Interaction studies showed that PGE₁ and PGI₂ mutually excluded the relaxing effects of each other. PGE₁ also reversed the relaxing effect of isoproterenol. However, pre-exposure to PGD₂ did not attenuate the relaxing effect of PGE₁ or PGI₂ nor was the relaxing effect of PGD₂ changed by pre-exposure to PGE₁. Two different orders of potency of PGs suggest two PG receptors subserving contraction and relaxation, respectively. Further, it appears that several PGs can act upon both receptors which may explain unusual interactions between the PGs and some of their atypical effects. Finally, the data also suggest that there may be subtypes of the PG receptors subserving contraction and relaxation.