Platelet and cardiovascular activity of the hydantoin BW245C, a potent prostaglandin analogue

The platelet anti-aggregating, cardiovascular and gastro-intestinal actions of a hydantoin prostaglandin analogue, BW245C have been compared with prostaglandin and PGD₂ several species. In human plasma , BW245C was 0.2 times as active as prostacyclin and 8 times as active as PGD₂ in inhibiting platelet aggregation. In rat and rabbit plasma, BW245C was only weakly active but was more potent in sheep and horse plasma. Since the acivity of PGD₂ varied in a parallel fashion, BW245C may interactive with PGD₂ binding sites on platelets. The potency of BW245C as a vasodepressor also varied between species, being 0.5, 0.1, 0.06 and < 0.02 times as active as prostacyclin in the aneasthetised dog, monkey, rat and rabbit respectively.The relative activity of BW245C as an inhibitor of platelet aggregation was more comparable, being 0.08, 0.04 and 0.05 times as active as prostacyclin following intravenous infusion in the rabbit dog and monkey respectively. In the rabbit, BW245C was a highly selective platelet inhibitor with minimal cardiovascular actions, whereas in the dog and monkey, BW245C lowered BP in anti-aggregating doses. The potent platlet anti-aggregating actions of BW245C following parenteral or oral administration makes this hydantoin a potentially-useful anti-thrombotic prostaglandin analogue.     

Effect of a hydantoin prostaglandin analogue, BW245C, during oral dosing in man

Following an open pilot study, the effects of repeated oral doses of BW245C, a hydantoin prostaglandin analogue, were studied in man. Six healthy volunteers received 150 micrograms BW245C or placebo 6-hourly for 5 days according to a double blind randomised balanced design with 7 days interval between treatments. Measurements of headache, facial flushing, heart rate, blood pressure, systolic time intervals, ECG, platelet aggregation responses to ADP and of subjective effects were made before and 1 and 3 h after the first dose of BW245C/placebo on days 1, 3 and 5 of dosing. BW245C produced significantly (p less than 0.05) higher headache scores than placebo on days 3 and 5; facial flushing, nasal stuffiness and abdominal discomfort were more frequent on BW245C than placebo. Heart rate, derived from the ECG, was significantly (p less than 0.05) higher and pre-ejection period significantly (p less than 0.05) shorter on BW245C at 1 h after dosing on each day. Left ventricular ejection time index, QS2 index, PR interval, QRS duration and T wave height were unchanged. Heart rate, counted at the radial pulse, and systolic and diastolic blood pressure, all measured lying and standing, were similar for BW245C and placebo. Platelet aggregation responses were not significantly different between the two treatments. The results indicate that repeated oral doses of BW245C, sufficient to cause moderately uncomfortable subjective effects, do not inhibit platelet aggregation.     

Pharmacological and cardiovascular properties of a hydantoin derivative, BW 245 C, with high affinity and selectivity for PGD2 receptors

Initial experiments demonstrated that the hydantoin prostaglandin derivative, BW 245 C, has potent anti-aggregatory activity on human platelets which may result from its structural similarity with one of the natural prostaglandins. The aim of the present study was to extend this preliminary pharmacological characterization and to determine which, if any, prostaglandin receptor-type is responsible for mediating the biological activity of BW 245 C. A marked species variation was observed in the anti-aggregatory potency of BW 245 C such that in the human (0.36 X PGE1) it was about one hundred times more effective than in the rat (0.003 X PGE1). The relative potencies of PGI2 (ca. 10 X PGE1) and PGE1 were, however, similar in both species. An intravenous bolus injection of 250 micrograms/kg BW 245 C lowered systolic (-23%) and diastolic (-34%) blood pressure in spontaneously hypertensive rats. In radioligand binding studies it showed a high affinity and selectivity for PGD2 platelet receptors, binding to PGI2 or PGE2 receptors was not detectable. Therefore it is concluded that the platelet and cardiovascular actions of BW 245 C are mediated by PGD2 receptors and this accounts for the observed species variation which is a characteristic of this prostaglandin.     

Sangivamycin, a nucleoside analogue, is a potent inhibitor of protein kinase C

Protein kinase C functions prominently in cell regulation via its pleiotropic role in signal transduction processes. Certain oncogene products resemble elements involved in transmembrane signaling, elevate cellular sn-1,2-diacylglycerol second messenger levels, and activate protein kinase C. Sangivamycin was unique among the nucleoside compounds tested in its ability to potently inhibit protein kinase C activity. Inhibition was competitive with respect to ATP for both protein kinase C and the catalytic fragment of protein kinase C prepared by trypsin digestion. Sangivamycin was a noncompetitive inhibitor with respect to histone and lipid cofactors (phosphatidylserine and diacylglycerol). Sangivamycin inhibited native protein kinase C and the catalytic fragment identically, with apparent Ki values of 11 and 15 microM, respectively. Sangivamycin was an effective an inhibitor of protein kinase C as H-7, an isoquinolinsulfonamide. Sangivamycin did not inhibit [3H]phorbol-12,13-dibutyrate binding to protein kinase C. Sangivamycin did not exert its action through the lipid binding/regulatory domain; inhibition was not affected by the presence of lipid or detergent. Unlike H-7, sangivamycin selectively inhibited protein kinase C compared to cAMP-dependent protein kinase. The discovery that protein kinase C is inhibited by sangivamycin and other antitumor agents suggests that protein kinase C may be a target for rational design of antitumor compounds.     

New analogue of arenastatin A, a potent cytotoxic spongean depsipeptide, with anti-tumor activity

Two analogues possessing steric hindered substituents on C-15 of arenastatin A (1), a potent cytotoxic spongean depsipeptide, were synthesized and shown to enhance stability in mouse serum. Notably, 15-tert-butylanalogue (6) with higher cytotoxicity exhibited in vivo anti-tumor activity through iv administration different from 1. Additionally, conformation analysis among the two analogues and arenastatin A (1) indicated that the torsion angle from C-14 to C-20 is a conclusive factor for the potent cytotoxicity of 1.     

The effect of carbacyclin, a prostaglandin analogue, on adenylate cyclase activity in platelet membranes

The effect of carbacyclin, a chemically stable analogue of prostacyclin, on the activity of adenylate cyclase in platelet membranes was measured, and compared with the effect of PGE1. When GTP was added in concentrations up to 10 microM the activation of adenylate cyclase by carbacyclin was increased, whereas higher concentrations of GTP were inhibitory. The addition of a non-hydrolysable analogue of GDP, guanosine 5'-[beta-thio]diphosphate (GDP[beta S] ) resulted in a dose-dependent inhibition of adenylate cyclase activation by carbacyclin; this inhibition was relieved by adding increased amounts of GTP.     

Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Hypotensive activity of novokinin, a potent analogue of ovokinin(2-7), is mediated by angiotensin AT(2) receptor and prostaglandin IP receptor

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp) is a potent hypotensive peptide previously designed based on the structure of ovokinin(2-7) (Arg-Ala-Asp-His-Pro-Phe), a vasorelaxing and hypotensive peptide derived from ovalbumin. Novokinin exhibited an affinity for the angiotensin AT(2) receptor (Ki=7.35 microM). Novokinin significantly lowered systolic blood pressure at a dose of 0.03 and 0.1 mg/kg after intravenous and oral administration, respectively, in spontaneously hypertensive rats (SHRs), and the hypotensive activity was blocked by PD123319, an antagonist of the AT(2) receptor. Novokinin lowered blood pressure in C57BL/6J mice after oral administration at a dose of 50 mg/kg. However, in AT(2) receptor-deficient mice, novokinin did not reduce blood pressure. These results demonstrate that the hypotensive activity of novokinin is mediated by the AT(2) receptor. The hypotensive activity of novokinin in SHRs was completely blocked by indomethacin and CAY10441, an inhibitor of cyclooxygenase and an antagonist of the prostaglandin IP receptor, respectively. These suggest that the hypotensive activity is mediated by prostacyclin and the IP receptor downstream of the AT(2) receptor.     

The rational design of a novel potent analogue of the 5'-AMP-activated protein kinase inhibitor compound C with improved selectivity and cellular activity

We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5'-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK.     

Mutasynthesis of a potent anticancer sibiromycin analogue

Pursuit of the actinomycete pyrrolobenzodiazepine natural product sibiromycin as a chemotherapeutic agent has been limited by its cardiotoxicity. Among pyrrolobenzodiazepines, cardiotoxicity is associated with hydroxylation at position 9. Deletion of the methyltransferase gene sibL abolishes the production of sibiromycin. Supplementation of growth media with 4-methylanthranilic acid can substitute for its native 3-hydroxy congener. Cultures grown in this fashion yielded 9-deoxysibiromycin. In this study, we characterize the structure and biological activity of sibiromycin and 9-deoxysibiromycin methyl carbinolamines. Preliminary in vitro evidence suggests that 9-deoxysibiromycin exhibits reduced cardiotoxicity while gaining antitumor activity. These results strongly support further exploration of the production and evaluation of monomeric and dimeric glycosylated pyrrolobenzodiazepine analogues of sibiromycin.     

Induction of luteolysis in the mare with a prostaglandin analogue

Five mares were administered 0.5 to 2.0 mg of a prostaglandin analogue, RS 9390 (Syntex), during nine estrous cycles in February and March. Luteolysis as measured by peripheral plasma progesterone occurred in four cycles, transitory luteolysis following 0.5 mg RS 9390 in two cycles, while functional corpora lutea were not present in three cycles. In 8 out of 9 of these cycles the mares returned to estrus 1.5 to 5 days following treatment. It appears that RS 9390 can be used as a regulator of cycle length in mares.     

3,4,4'-Trihydroxy-trans-stilbene, an analogue of resveratrol, is a potent antioxidant and cytotoxic agent

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a naturally occurring polyphenol widely distributed in food and dietary plants. This phytochemical has been intensively studied as an efficient antioxidant and anticancer agent, and a variety of substituted stilbenes have been developed in order to improve the potency of resveratrol. In this work, we described the synthesis of 3,4,4 -trihydroxy-trans-stilbene (3,4,4'-THS), an analogue of resveratrol, and studied its antioxidant and cytotoxic activity in vitro. 3,4,4 -THS was much more efficient than resveratrol in protecting against free radical-induced lipid peroxidation, photo-sensitized DNA oxidative damage, and free radical-induced hemolysis of human red blood cells. More potent growth inhibition in cultured human leukemia cells (HL-60) was also observed for 3,4,4 -THS. The relationship between the antioxidant efficiency and cytotoxic activity was discussed, with the emphasis on inhibition of the free radical enzyme ribonucleotide reductase by antioxidants. The result that this subtle structure modification of resveratrol drastically improves its bioactivity provides important strategy to develop novel resveratrol-based molecules.     

Effects of a synthetic prostaglandin analogue, cloprostenol, on the corpus luteum of the guinea pig

The effects of a synthetic prostaglandin analogue, cloprostenol, on luteal function in a guinea pig were studied. At a dose of 250μg, cloprostenol administered I-P on day 9 of the oestrous cycle caused a reduction in the length of the oestrous cycle from 17.4±s.d. 0.9 to 14.5±1.1 days (p<0.01). Lower doses were ineffective, and post-treatment cycles were not different in length from pre-treatment cycles. Cloprostenol also caused a dose-dependent reduction in luteal weight, which fell from 3.52±0.82 to 1.82±0.4mg (<0.01) 48 h after administation of a 250μg dose on day 9. Plasma progesterone, measured by radioimmunoassay, was reduced from 4.67±0.59 to 2.69±0.66 ng ml⁻¹(p<0.01) 48 h after administration of 250μg cloprostenol on day 9. 250μg cloprostenol also reduced blood flow per corpus luteum, measured by ⁸⁵Sr-labelled 15μm microspheres, both at 3 h (20.20±10.36 to 9.40±4.2μ1 min⁻¹; p0.05) and at 48 h 18.47±8.27 to 5.23±1.90μl min⁻¹; p<0.01) after administration on day 9. No adverse side-effects were observed at any dose level of cloprostenol used. It was concluded that cloprostenol is a useful experimental luteolysin in the guinea pig.     

Efficacy of a prostaglandin analogue in reproduction in the anestrous mare

A prostaglandin F analogue was studied in anestrous mares: a dose-response study; a study in mares presumed pregnant; and a field evaluation of effective doses in breeding establishments. A dose of 2.0mg given by single subcutaneous injection to mares with initial plasma progesterone levels greater than 1.0ng/ml, caused luteolysis on the basis of decline in plasma progesterone concentrations. Follicle maturation leading to ovulation, accompanied by estrus, was observed, and fertility at mating either by natural service or artificial insemination was satisfactory. A dose of 1.0mg was generally effective for luteolysis, but pregnancy rates were lower than after 2.0mg. A proportion of mares which had less than 1.0mg of plasma progesterone at the time of injection ovulated and became pregnant.     

Treatment of pregnant gilts with a prostaglandin analogue, Cloprostenol, to control oestrus and fertility

Oestrus was induced 4-7 days after treatment with Cloprostenol (ICI 80,996) in gilts between 12 and 40 days pregnant. Fertility at this synchronized oestrus was good (85%).     

The disulfide bonded ring of iso-rANP, unlike that of rANP, has potent cardiovascular activity

Iso-atrial natriuretic peptide (iso-rANP), a 45 amino acid peptide with a disulfide bond between residues 23 and 39, is a newly discovered second atrial hormone with considerable homology with rat atrial natriuretic peptide (rANP). We have reported that iso-rANP(1-45) has effects similar to rANP in anesthetized rats in causing hypotension, a decrease in heart rate, and an increase in urine flow and electrolyte excretion. In the present experiments, we found that, unlike the ring peptide of rANP (rANP(105-121)), bolus injections of the ring peptide of iso-rANP (iso-rANP(23-39)) had considerable effects on the circulation but only small effects on the kidney, compared with iso-rANP(1-45). However, the effects of iso-rANP(23-39) in causing hypotension and decreasing heart rate were transient compared with those of iso-rANP(1-45). When we substituted a glycine for arginine into the ring portion of iso-rANP at position 36, so that there were only three amino acids different from the ring of rANP, biological activity of iso-rANP(23-39) was retained. In our bioassay system, all of the circulatory effects and most of the renal effects of rANP are mediated via vagal sensory afferents. We found that the effects of the ring peptide of iso-rANP on the circulation and the kidney were unchanged following vagotomy. The results indicate that iso-rANP(23-39) mediates at least some biological effects by membrane receptor mechanisms different from those of rANP and that the ring portion of iso-rANP probably contributes to nonvagally mediated responses of iso-rANP(1-45).     

Platelet sensitivity to a prostacyclin analogue in normal and pathological pregnancy

Platelet sensitivity to a prostacyclin analogue (PSP) was investigated at different stages of normal and pathological pregnancies. During uncomplicated pregnancy PSP decreased progressively by about 30% and returned to almost normal values in the puerperium. In pre-eclamptic women PSP was reduced by 49% and in pregnant diabetics by 50% compared with normal pregnant individuals of corresponding gestational age. Reduced PSP in normal and pathological pregnancy seems to be another aspect of increased coagulability and platelet aggregation found in these patients.     

Biological activity and anti-prostaglandin effects of prostaglandin analogue, ent-11-epi-15-epi PGE2 methyl ester

The biological activity and anti-prostaglandin property of the prostaglandin analogue, ent-11-epi-15-epi PGE2 methyl ester, were studied on isolated guinea pig ileum. Ent-11-epi-15-epi PGE2 methyl ester contracted guinea pig ileum and produced a concentration-response curve parallel to that of PGE2. However, the former exhibited a lower maximal effect than PGE2. At concentrations greater than 10(-6)M, ent-11-epi-15-epi PGE2 methyl ester selectively antagonized contractile actions of PGE2 and PGE2 alpha without affecting contractions induced by acetylcholine. These observations suggest that the PG analogue acted like a competitive antagonist to PGE2 and PGF2 alpha on guinea pig ileum in vitro.     

Synthesis and biological activity of Boc [Nle28, Nle31]CCK27-33, a highly potent CCK8 analogue

A new CCK8 related peptide, Boc[Nle28,Nle31]CCK27-33 (Boc[diNle]CCK7) was synthesized and tested for cholecystokinic activity, at both the peripheral and the central level. This analogue, protected against both chemical oxidation and enzymatic degradation by aminopeptidases, was shown to be equipotent to CCK8 in releasing amylase from rat pancreas fragments. In addition, the EC50 values of Boc[diNle]CCK7 in the guinea pig gallbladder and ileum contraction assays (3.2 nM and 3.0 nM respectively) were similar to those of CCK8 (6.0 nM and 2.0 nM). Moreover both Boc[diNle]CCK7 and CCK8 elicited similar effects on the open field test over the same concentrations range. These results demonstrate the ability of Boc[diNle]CCK7 to be a suitable tool for investigating the physiological role of native CCK8.     

Efficacy of a prostaglandin analogue in reproduction in the cycling mare.

A prostaglandin F analogue caused luteolysis in normal cycling non-lactating mares, and lactating mares (treated after the foal estrus). Effective doses ranged from 1.0 to 4.0mg given as a single subcutaneous injection 8–10 days after ovulation. A dose of 0.5mg was ineffective, hence the dose-response relationship was steep, indicative of a quantal type of response. Mares usually returned to estrus within 2–4 days and ovulated by 7 days after treatment. Mares bred naturally or by artificial insemination at the induced estrus and ovulation were fertile. The compound was without side-effects, and hence should be of value in manipulating the estrous cycle of the mare.