An animal model for the study of arterial thrombosis.

1. Thrombus formation induced by electrical stimulation of the carotid artery was investigated in anesthetized rabbits and rats. Occlusive Grade III thrombi were produced consistently in 34 normal New Zealand rabbits and 58 untreated albino Wistar rats. Thrombus formation was monitored continuously in some of the animals with a magnetic flowmeter or a thermistor probe applied on the carotid. 2. The usefulness of the model for the screening of drugs was tested by treating the animals with warfarin, heparin, prostacyclin (PGI2), dihydroprostacyclin (DiHPGI2), prostaglandin E1 (PGE1), and prostaglandin D2 (PGD2). 3. All of the drugs except warfarin were infused continuously into the venous circulation during the entire experimental period at a rate of 0.2 ml/min. 4. Warfarin (10 mg/kg), administered by gavage 24 h before the experiment, prevented thrombus formation, as did heparin iv (greater than 34 U/kg). 5. Of the four platelet antiaggregatory prostaglandins tested, PGI2 was the most potent inhibitor of thrombus formation and DiHPGI2 the least active, as evaluated by visual inspection of stimulated arterial segments which were excised 30 min (rabbits) or 15 min (rats) after the stimulation was stopped. PGI2 was less active in rats than in rabbits (Threshold Protective Dose ratio ca. 4:1). PGE1 and PGD2 showed intermediate activity in both animal models.

Effects of prostaglandin D2 on pulmonary arterial pressure and oxygenation in newborn infants with persistent pulmonary hypertension.

We studied the effects of prostaglandin D2 (PGD2) in six newborn infants, 1 to 2 days of age, who had persistent pulmonary hypertension syndrome and a PaO2 less than 75 torr during mechanical hyperventilation with an inspired oxygen concentration of 100%. Tolazoline and dopamine were used to treat some of the patients. No patients had congenital heart disease or sepsis. Catheters were placed to measure pulmonary and systemic arterial blood pressures. PGD2 was infused intravenously at doses of 1 to 25 micrograms/kg/min. Pulmonary and systemic arterial blood pressures, heart rate, and descending aortic blood gas values were measured before each dose change. Only two of six patients had a transient increase in PaO2. All had an increase in heart rate. Two of six patients had an increase in pulmonary arterial blood pressure. No deleterious effects occurred during the infusion. Four of six patients subsequently died. Although PGD2 is a specific pulmonary vasodilator in fetal and newborn animals, it did not lower pulmonary arterial blood pressure nor improve oxygenation in newborn infants with persistent pulmonary hypertension syndrome.

Protective effect of prostaglandins D2, E1 and I2 against cerebral hypoxia/anoxia in mice.

The protective effect of prostaglandins (PGs) against cerebral hypoxia/anoxia was investigated with a variety of experimental models in relation to their CNS depressant effects in mice. Furthermore, the effect of PGs on the changes of cerebral energy metabolites and cyclic nucleotide was examined in hypoxic mice. Mice were given s.c. doses of PGs 30 min before tests. Among the PGs tested, treatment with PGD2, PGE1 and PGI2 Na showed a consistent and dose-dependent protection against cerebral anoxia induced by all models studied: histotoxic anoxia by KCN, hypobaric hypoxia, normobaric hypoxia and decapitation-induced gasping. However, PGA1, PGA2, PGB1, PGB2, PGE2, PGF1 alpha, PGF2 alpha and 6-keto-PGF1 alpha at a dose of 3 mg/kg were without effect against normobaric hypoxia and gasping duration. The three PGs, i.e. PGD2, PGE1 and PGI2 which showed anti-hypoxic effects decreased locomotor activity and potentiated hexobarbital-induced sleep. On the other hand, PGE2, PGA1, PGA2 and PGB2 also caused a decrease in locomotor activity. Similarly, PGE2 and PGA1 caused a potentiation of hexobarbital-induced sleep, but interestingly they did not cause clear-cut increase in cerebral resistance to hypoxia, in contrast with the former three PGs. Thus general depression of CNS function appears not to be responsible for the PGD2-, PGE1- and PGI2-induced increase in cerebral resistance to hypoxia. The levels of Cr-P and ATP were significantly reduced and those of ADP and AMP were markedly elevated in hypoxic brain, resulting in a decrease in a calculated energy charge potential. The lactate level and lactate/pyruvate ratio increased and the glucose level decreased markedly.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor activity of delta 7-prostaglandin A1 and delta 12-prostaglandin J2 in vitro and in vivo.

Cyclopentenone prostaglandins (PGs) such as prostaglandin A1 (PGA1) and prostaglandin J2 (PGJ2) significantly increased the life span of Ehrlich ascites tumor-bearing mice. In order to obtain PG derivatives which have more potent antitumor activity than PGA1 and PGJ2, we synthesized a number of alkylidenecyclopentenone PGs and studied the antitumor activity of these compounds in vitro and in vivo. delta 7-PGA1, 12-epi-delta 7-PGA1, and delta 12,14-PGJ2 showed 50% inhibitory concentrations of 0.3 microgram/ml against the growth of L1210 culture cells. These compounds are several times more cytotoxic than parent compounds. From a structure-activity relationship analysis, we concluded that, as for PGA derivatives: (a) a double bond in C7-8 potentiates the cytotoxicity; (b) a 15-hydroxy group is not essential for cytotoxicity; (c) the stereochemistry of R2 chain is not essential, while 12-epi derivatives also have full activity; (d) a double bond in C12-13 seems to be essential for full activity, and for PGJ derivatives a double bond in C12-13 and C14-15 potentiates the cytotoxicity. These compounds showed marked antitumor activity against Ehrlich ascites tumor in vivo. At doses of 20-30 mg/kg/day three or five consecutive i.p. treatments with these compounds resulted in a 66 to 111% increase in life span with long-term survivors. A single i.p. injection with 12-epi-delta 7-PGA1 (100 mg/kg) resulted in 73% increase in life span with 33% of long-term survivors, indicating an activity comparable to that of cyclophosphamide (200 mg/kg). However, delta 7-PGA1 and 12-epi-delta 7-PGA1 were marginally effective against P388 leukemia. Treatment with delta 7-PGA1 (10 mg/kg/day i.p.) and 12-epi-delta 7-PGA1 (20 mg/kg/day i.p.) on Days 1-9 resulted in 25.8 and 29.6% increases in life span, respectively. delta 7-PGA1 and delta 12-PGJ2 derivatives may be a potential new class of antitumor agents.

Inhibition of human ovarian cancer cell growth in vitro and in nude mice by prostaglandin D2.

In vitro and in vivo effects of prostaglandin D2 on human ovarian tumor growth were examined by using a cell line, designated HR, derived from ascites of a patient with serous cystadenocarcinoma of the ovary. The HR cell proliferation in vitro was dose dependently inhibited between concentrations of 0.1 and 4.0 micrograms of prostaglandin D2 per ml. From results of 51Cr release assays and trypan blue dye exclusion tests the inhibitory effect seemed to result from a direct cytotoxic effect by prostaglandin D2. All DNA, RNA, and protein syntheses by the HR cells were also inhibited in a dose-dependent manner with exposure time of 48 h to prostaglandin D2. When 5 X 10(5) HR cells were inoculated to nude mice, the 50% survival time of them in untreated groups was 52 days after inoculation. Although 4 mg of prostaglandin D2 per kg caused inhibition of the tumor growth, a significant prolongation of the survival time was not observed. On the other hand, the 50% survival time of nude mice treated with 12 mg of prostaglandin D2 per kg was significantly (P less than 0.05) prolonged to 67 days, in addition to a significant inhibition of the tumor growth.

Inhibition of human melanoma growth by prostaglandin A, D, and J analogues.

The relative inhibitory potency of prostaglandin A (PGA) and prostaglandin J2 (PGJ2) analogues compared to prostaglandin A1 (PGA1) was determined in a clonogenic assay system. Three human melanoma cell strains (C8146A, C8146C, and C8161), a human melanoma cell line (M1RW5) and a human neuroblastoma cell line (IMR-32) were used. Prostaglandin analogues were screened in the clonogenic assay system and the dose effect curves were analyzed by linear regression utilizing the median effect relationship. The computer-generated 50% and 95% inhibitory doses showed that 15-deoxy-16-hydroxyl-16-vinyl-prostaglandin A2 (DHV-PGA2) was from two- to three-fold more active than PGA1 in inhibiting the clonogenic growth of human melanoma cells. Based on the 50% inhibitory dose, PGJ2 and its analogues were from two to five times more potent than PGA1. The delta 12- and delta 12,14-PGJ2 were the most potent of the prostaglandins tested. However, the 95% inhibitory dose for prostaglandin D2 (PGD2), PGJ2 and its analogues against neuroblastoma did not show any enhancement in activity in comparison to PGA1, suggesting that some tumor specificity in the activity of these analogues may be signified by the neuroblastoma data. Prostaglandins which contained a fluoride substitution at position 11 were also tested for activity. As we previously observed with other analogues which did not contain an alpha, beta-unsaturated carbonyl group in the cyclopentane ring, 9 beta, 15 alpha-dihydroxy-11 beta-fluoroprosta-5-cis-13-trans-dienoic acid and 9 alpha, 15 alpha-dihydroxy-11 beta-fluoroprosta-5-cis-13-trans-dienoic acid did not inhibit the clonogenic growth of human melanoma cells. Administration s.c. to established human melanoma tumors growing in athymic nude mice caused a significant growth inhibition. The treatment schedules ranged from 1 to 8 days. Injection s.c. of PGA1 at a dose of 40 mg/kg/day resulted in a 20% suppression in tumor growth. Higher doses (100 and 200 mg/kg/day) effected an 80% reduction in tumor growth. The higher doses were associated with reversible toxicities, diarrhea and skin inflammation. Administration of DHV-PGA2 at a dose of 20 mg/kg/day resulted in 40% reduction in tumor growth. The increased in vivo potency of DHV-PGA2 corresponds to the results obtained in the clonogenic assay system.

Therapeutic opportunities in vasoocclusive disease.

There is evidence that aspirin is partially effective in the prophylaxis of various vasoocclusive disorders. This article reviews pharmacologic opportunities for improvement over and above the therapeutic effect of aspirin. It is concluded that several rational possibilities merit consideration, in particular, the use of combinations of drugs that affect the thrombotic process at different points. Such strategies will ultimately require validation by clinical trial.

The effects of PGD2 and 9-deoxy-delta 9-PGD2 on colony formation of murine osteosarcoma cells.

Effects of antineoplastic prostaglandins (PG), PGD2 and 9-deoxy-delta 9-PGD2, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD2 significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 micrograms/ml. The IC50 value was calculated to be 0.72 microgram/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-delta 9-PGD2 between 0.01 to 1 microgram/ml, the IC50 value being 0.22 microgram/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 microgram/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-delta 9-PGD2 at a concentration of 5 ug/ml. These results suggest that PGD2 and 9-deoxy-delta 9-PGD2 are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.

Prostaglandin D2 in canine endotoxic shock. Hemodynamic, hematologic, biochemical, and blood gas analyses.

This canine study was designed to evaluate the effects of the intravenous infusion of coliform endotoxin with a simultaneous infusion of prostaglandin D2 (PGD2) on hemodynamics, blood gas, blood chemistry, and some hematologic parameters. The information derived from the present study supports the view that the intravenous administration of PGD2 moderates the effects of endotoxin, with its main beneficial effect being on the renal vascular bed. Treatment with PGD2 did not change the endotoxin-induced hemoconcentration, or the reduction in the platelet and white blood cell counts. However, four of nine animals survived more than 7 days when treated with PGD2, whereas without it only one of nine animals survived the administration of the same dose of endotoxin. Although the mechanism of action is not clear, the correlation between PGD2 infusion and improved renal blood flow warrants further study in endotoxic shock.

Prostaglandin D2 inhibits the proliferation of human malignant tumor cells.

The cytotoxic effect of prostaglandin (PG) D2, PGE1 and PGF2 alpha was examined on human osteosarcoma cells (KSu cell line) in vitro, and PGD2 was most effective. DNA, RNA and protein syntheses of KSu cells were also found to be inhibited by PGD2 at a concentration of 5 micrograms/ml. Furthermore, the proliferation of various human malignant tumor cells was inhibited by PGD2 without exception so far. These results suggest that PGD2 shows an anti-neoplastic effect on a variety of human malignant tumor cells.

Cytotoxic action of prostaglandin D2 on mouse neuroblastoma cells.

Addition to the culture medium of prostaglandin (PG) D2 resulted in the degeneration in a dose- and time-dependent manner of N18TG-2 cells cloned from mouse neuroblastoma. The ED50 for PGD2-induced cytotoxicity was about 10 microM. The degenerative changes were irreversible when the cells were exposed for more than 10 h. Scanning and transmission electron microscopic examination revealed that treatment with PGD2 resulted in appearance of numerous blebs of various sizes along the cell surface and also in destruction of surface membrane and of cytoplasmic organelles. Tumor weight of N18TG-2 neuroblastoma inoculated subcutaneously on the backs of A/J mice was about 35-70% less than that of controls after 14 days of single daily i.p. or s.c. injections of 0.5-1 mg/kg of PGD2. The results indicate that PGD2 has growth-inhibitory effects on mouse neuroblastoma cells in vitro and in vivo.

Persistent pulmonary hypertension of the neonate (persistent fetal circulation syndrome).

This 15-year-old disease has been clearly described anatomically. Some understanding of possible in utero predisposing conditions has emerged from clinical and animal studies. However, we have very little understanding of the cellular processes that trigger and/or prolong the abnormal medial smooth muscle hypertrophy underlying the condition. Empiric observation has resulted in the development of hyperventilation as a fairly successful treatment modality, although the underlying mechanism of this salubrious effect is unknown. Drugs, a major focus of clinical and laboratory investigations, sometimes are marginally successful (and sometimes are utter failures). Translated into the neonatal intensive care unit, the disease is more frequently accurately diagnosed than in the past, but it remains frustratingly difficult to manage, and thus far is impossible to prevent. The research foundations laid in the past decade provide impetus for accelerated search into the fundamental cellular and biochemical derangements that cause persistent pulmonary hypertension. It is to be hoped that the next decade will yield major advances in both mechanistic understanding and in treatment.

Prostaglandin D2 reverses induced pulmonary hypertension in the newborn lamb.

We investigated the effects of PGD2 in six near-term newborn lambs with induced pulmonary hypertension (mean pulmonary arterial pressure equal to mean systemic arterial pressure). In each lamb PGD2 decreased mean pulmonary arterial pressure, increased pulmonary blood flow, and therefore decreased pulmonary vascular resistance without changing mean systemic arterial pressure. A bolus dose of 20 micrograms PGD2 decreased pulmonary arterial pressure by 30%, increased pulmonary blood flow by 45%, and decreased pulmonary vascular resistance by 54%; systemic arterial pressure increased by 4%. At all doses, pulmonary vascular resistance fell further than systemic vascular resistance, the ratio of percent change in pulmonary vascular resistance to percent change in systemic vascular resistance was approximately 2:1. Similar changes occurred with continuous infusions of PGD2. These effects suggest a role for PGD2 in the normal regulation of pulmonary vascular resistance and blood flow at birth. In addition, because PGD2 in these circumstances increases pulmonary blood flow and reduces pulmonary arterial pressure, it may merit further trials in nonhuman primates; it may be an appropriate agent for treating newborn infants with persistent pulmonary hypertension.

Evaluation of prostaglandin D2 (PGD2) as an anticoagulative agent for haemodialysis in comparison with prostaglandin E1 (PGE1).

Prostaglandin D2 (PGD2), a potent antiplatelet agent, was evaluated as an antithrombotic agent for haemodialysis in comparison with prostaglandin E1 (PGE1) and prostaglandin I2 (PGI2). When antiplatelet action was evaluated, taking the degree of associated hypotension into consideration, PGD2 was found to be superior to PGE1 and PGI2 as the latter two had the negative effect of inducing hypotension, while PGD2 had a less hypotensive effect. The suppression of the platelet function by PGD2 was observed to have only a slight influence on platelet function in systemic blood.

Suppression of immune complex vasculitis in rats by prostaglandin.

Immune complex-induced vascular damage can be markedly suppressed by treatment of rats with either prostaglandin (PG)E1 or its stable derivative, 15-(S)-15-methyl PGE1, but not with PGF2 alpha. In addition, PGD2 and PGE2 also show suppressive effects. The PGE1 derivative is considerably more effective than PGE1 and shows potent anti-inflammatory activity even after oral administration. Suppression of the vasculitis reaction is reflected by a greatly diminished increase in vasopermeability, indicating little or no vascular damage. In suppressed animals, the infiltration of neutrophils is greatly reduced, and those leukocytes that have appeared at tissue sites fail to show phagocytic uptake of immune complexes. In suppressed animals, the skin sites nevertheless show deposits of immune complexes and C3 fixation in vascular walls. Neutrophils harvested from the blood of rats treated with PGE1 show depressed responsiveness in chemotaxis and in enzyme secretion after incubation with chemotactic peptide. These studies indicate that certain PG have potent anti-inflammatory activity, which may be related to their effects on leukocytes.