Effect of mopidamol on survival in carcinoma of the lung and colon: final report of Veterans Administration Cooperative Study No. 188.

Mopidamol (RA-233), a derivative of dipyridamole, is a phosphodiesterase inhibitor that has been shown previously to limit progression of malignancy in certain experimental animal models and in a pilot study in humans. RA-233 plus chemotherapy was compared with chemotherapy alone in a 5-year double-blind trial involving 719 patients with advanced carcinomas of the lung and of the colon. RA-233 treatment was associated with a statistically significant prolongation of survival in patients with non-small cell lung cancer (N-SCLC) limited to one hemithorax and with reduction in mean plasma fibrogen concentration. RA-233 was not toxic. The favorable effects on survival could not be explained by any factor other than the RA-233 treatment. In other tumor categories tested, no differences in survival were observed. These results suggest that RA-233 is useful in the treatment of N-SCLC of limited extent. They also suggest that therapeutic intervention aimed at modified intracellular pathways might constitute a novel investigative approach to the treatment of cancer.

Drug treatments for metastasis of the Lewis lung carcinoma: lack of correlation between inhibition of lung metastasis and survival.

The abilities of the Eli Lilly compounds LY150310, LY189332, and LY135305 to inhibit spontaneous metastasis and to increase animal survival were evaluated. These compounds represent widely varied structures and were evaluated because they have been found to inhibit thromboxane synthetase, cyclooxygenase, and thrombin activation, respectively. These biochemical processes have been proposed in the literature as targets for antimetastatic drugs. The purpose of this investigation was twofold: (a) to compare the antimetastatic activities of the Eli Lilly compounds to those of the reference antimetastatic compounds nafazatrom and RA233, and (b) to examine the correlation between inhibition of spontaneous lung metastasis and survival. Spontaneous metastasis of the Lewis lung carcinoma was used to evaluate the antimetastatic activity of the compounds. In this model 5 x 10(5) tumor cells were implanted into the gastrocnemius muscle, the primary tumor was resected on Day 14, and metastatic lung lesions were counted on Day 25. Compounds were administered every 12 h on Days 5 through 19. Nafazatrom, LY150310, LY189332, and LY135305 were found to inhibit spontaneous lung metastasis in a dose-dependent manner. The ED50 values for the respective inhibitions with these compounds were 50, 0.5, 2, and 0.35 mg/kg/day; the respective therapeutic indexes (LD50/ED50) were 7, 180, 255, and 511. To evaluate the effect of nafazatrom, LY150310, LY189332, and LY135305 on animal survival, the compounds were given at maximally antimetastatic doses of 200, 60, 20, and 6 mg/kg/day, respectively. Two dosing schedules were used: (a) on Days 5 through 19 and (b) on Day 5 until death. Neither the median survival times nor the numbers of long-term survivors were significantly changed with any of the compounds at any dosing schedule. RA233, given to a maximally tolerated dose of 200 mg/kg/day on Day 5 until death, did not inhibit lung metastasis and did not increase median survival time. Postmortem examination of animals dosed with nafazatrom, LY150310, LY189332, and LY135305 showed complete inhibition in lung lesions and the appearance of lesions in the liver, kidney, spleen, and brain. The results of this investigation show that the effect a compound has on the number of metastatic lesions in a target organ may not be predictive of its effect on survival. To successfully translate laboratory data into the clinic, survival should be considered as a predictor of a compound's potential clinical utility.

Polymeric inhibitors of platelet aggregation. II. Copolymers of dipyridamole and related drugs with N-vinylpyrrolidone.

A study has been made of the behaviour of the drugs dipyridamole and mopidamol (RA 233) attached to poly(N-vinylpyrrolidone). The inhibitory activities towards platelet aggregation induced by ADP or platelet activation factor (PAF) in sheep plasma have been examined and found to exceed the activities of the parent drugs, by factors up to 20. At the same time the abilities of the polymer-bound drugs in potentiating prostaglandin-type anti-aggregatory activities are much lower than those of the unattached drugs. The observations are explained in terms of polymer adsorption on to the platelet membranes, producing a high surface concentration of the drugs with consequent high inhibitory action. Intracellular action, such as the inhibition of phosphodiesterase, is reduced by the difficulty experienced by the polymeric drug in passing through the membrane, so that potentiation of prostaglandin activity, particularly against PAF, becomes small. A terpolymer containing units of dipyridamole and the prostaglandin analogue 5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin (245C) shows a degree of 'self-potentiation'.

Antiplatelet pyrimido-pyrimidines and metastasis.

The role of antiplatelet drugs in relation to their potential antimetastatic activities has been reviewed and the effects of two pyrimido-pyrimidine derivatives (RX-RA69 and RX-RA85) with strong antiplatelet activities investigated in metastasizing tumour models. The routes of administration and drug dosages were always chosen in such a way that good antiplatelet activities were obtained. RX-RA69 (20 mg/kg/day) given in the drinking water had no effect on spontaneous metastasis of Lewis lung carcinoma. RX-RA85 (20 mg/kg/day) did not influence spontaneous metastasis of B16 melanoma. On the other hand, giving RX-RA85 (8 mg/kg) daily i.p. to Lewis lung carcinoma bearing mice significantly increased the number of lung metastases but had no significant effect on primary tumour implant growth. Pretreating mice orally with 20 mg/kg RX-RA85 1 h before i.v. injection of B16 melanoma cells had no significant effect on lung colony number or distribution of extrapulmonary tumours while injecting the same dosage of RX-RA85 i.v. 1-2 h before tumour-cell injection decreased lung colony formation, but increased extrapulmonary tumour burden. This investigation like many others does not support the importance of platelets in metastasis formation.

Role of platelets in vasogenic brain edema. II. Contribution of platelet serotonin in brain edema.

In this study of the function of platelets after CNS injury, platelets were treated with serotonin labeled with radioactive carbon (14C) in animals subjected to a freezing lesion of the cerebrum. The distribution of platelet serotonin was measured by counting the specific activity of 14C-labeled serotonin in tissue and by autoradiography. Some of the animals were treated with RA-233, which inhibits the formation of platelet plugs after endothelial damage and the release of serotonin from platelets. Platelet serotonin accumulated near the surface of the cortex at the site of injury in all animals. More cerebral edema developed in animals treated with the platelet inhibitor than in untreated animals, probably because platelet aggregates were inhibited from forming and were not available to plug leaks in the traumatized vessels. Serotonin did not appear to facilitate the spread of edema.

Role of platelets in vasogenic brain edema. I. Significance of thrombus formation in the damaged vessels.

In an investigation of the role of platelets in vasogenic edema in cats, direct observation of the cortex revealed that within several minutes after cold injury, platelet thrombi formed in small veins at the point where the veins emerged from the depths of the brain. Later, edema fluid extravasated from the veins at this same point. Pretreatment with a platelet inhibitor, RA-233, abolished the formation of platelet thrombi and remarkably enhanced the leakage of edema fluid. The microcirculation was assessed by carbon black perfusion and was found to fill better in the cats that received the platelet inhibitor. The better filling may be ascribed to a decreased number of thrombi and consequent improved blood flow in small blood vessels. We conclude that platelet aggregates play a major role in controlling the leakage of edema fluid after cold injury.

Effects of RA 233 treatment on the adhesive, invasive and metastatic properties of 13762NF rat mammary tumor cells.

The pyrimido-pyrimidine analogue RA 233 has pleiotropic and differential effects on cultured tumor cell clones isolated from the 13762NF rat mammary adenocarcinoma. A nonresponsive clone of low metastatic potential (MTC) was not modified in its cell fragility or invasive, adhesive or lung-colonizing properties by RA 233 treatment. In contrast, a drug-responsive clone of high metastatic potential (MTLn3) was rendered less invasive and its cell fragility was decreased with RA 233 treatment, although its adhesiveness to lung microvascular endothelial cells and subendothelial matrix was unaffected by RA 233. Lung colonization of intravenously injected MTLn3 cells in syngeneic rats was significantly increased by RA 233 treatment, whereas spontaneous metastasis from the mammary fat pad to lung sites was decreased, although this decrease was not statistically significant.

Antitumor effect of RA233 alone and combined with radiotherapy.

The effect of RA233 alone or in combination with radiation was investigated in vivo on the S180 sarcoma, the B16 melanoma and the Lewis lung carcinoma. The combined treatment was a significant improvement over radiation alone for the B16 and S180 tumours. RA233 alone did not influence the growth of these tumours. When the primary 3LL was irradiated, tumour size was unaffected but the number of pulmonary metastases was reduced. They were further reduced by the combination of RA233 and radiation. The number, volume and cytokinetics of the B16 cells and the 3LL cells were affected to varying degrees by RA233. The significance of these changes relative to the effects of RA233 are discussed.

The effect of 2,6-bis(diethanolamino)-4-piperidinopyrimido [5,4-d]-pyrimidine (RA233) on growth, metastases and lung colony formation of B16 melanoma.

The dissemination of malignant cells from a primary tumor to distant host sites appears to be influenced by blood platelets of the hemostatic system. Under conditions that did not inhibit primary tumor growth, RA233 decreased both the incidence and frequency of spontaneous lung metastases by 66% and 69%, respectively. Although RA 233 effectively inhibited the formation of spontaneous metastases, oral administration of RA233 prior to and after the intravenous inoculation of 1 X 10(5) B16F10 murine melanoma cells failed to inhibit subsequent lung colony formation. These findings indicate that RA233 has antimetasttic activity, and that inhibition of platelet aggregation is not the sole determinant of this action.

Cytotoxic effect of RA233 on hypoxic B16 melanoma cells in vitro.

The pyrimido-pyrimidine derivative RA233 was found to selectively kill cultured mouse B16 melanoma cells after prolonged hypoxia. At the optimum cytotoxic concentration (100 microM), RA233 reduced cell clonogenicity by about 80% when administered during long-term hypoxia of 4 days. Comparable cytotoxicity was also evident when RA233 was present only during re-oxygenation following 4 days of hypoxia. RA233 treatment during both hypoxia and re-oxygenation resulted in the greatest cytotoxicity, with only about 1% of cells surviving such treatment. By contrast, the hypoxic cell sensitizer misonidazole was cytotoxic only when administered during hypoxia. RA233 appears to be a unique hypoxic cell sensitizer that kills long-term hypoxic tumor cells principally during re-oxygenation.

Role of adenosine uptake and metabolism by blood cells in the antiplatelet actions of dipyridamole, dilazep and nitrobenzylthioinosine.

Adenosine (Ado, 10 microM) did not inhibit ADP-induced human platelet aggregation in whole blood. However, if the blood was preincubated with dipyridamole (10 microM), a potent inhibitor of the erythrocytic nucleoside transport system (NTS), Ado acted as a strong inhibitor of platelet aggregation. Similarly, Ado inhibited platelet aggregation in whole blood in the presence of other potent NTS inhibitors, dilazep (1 microM) and p-nitrobenzylthioinosine (NBMPR, 1 microM). RA 233 (10 microM), an analog of dipyridamole which is a potent inhibitor of platelet cAMP phosphodiesterase (PDE), did not evoke the Ado effect in whole blood. However, in platelet-rich plasma (PRP), RA 233 potentiated strongly Ado-mediated inhibition, whereas dipyridamole, dilazep and NBMPR were without activity. 5'-Methylthioadenosine (MTA), an Ado receptor antagonist, reversed the inhibition produced by a nucleoside transport system inhibitor plus Ado in whole blood. Dipyridamole (10 microM), dilazep (1 microM) or NBMPR (1 microM) blocked [14C]Ado (10 microM) uptake by blood cells in whole blood, whereas RA 233 (10 microM) was not effective. The combination of 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of adenosine deaminase (ADA), plus 5-iodotubercidin (ITu, 10 microM), a potent inhibitor of adenosine kinase (Ado kinase), gave comparable Ado-mediated inhibition of platelet aggregation in whole blood as was obtained when the blood was pretreated with dilazep. These studies suggest that the in vivo antiplatelet actions of drugs such as dipyridamole and dilazep result from their abilities to block erythrocytic Ado uptake and subsequent metabolism, thus elevating the extracellular steady-state concentration of the physiologically occurring, antiplatelet agent, Ado.

Differential effects of the pyrimido-pyrimidine derivatives, dipyridamole and mopidamol, on platelet and vascular cyclooxygenase activity.

The chronic administration of 10 mg/kg/day of dipyridamole to rats produced 33.7% inhibition of platelet aggregation induced with ADP and a 93% increase in 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in vascular samples, versus saline-treated rats. Mopidamol, 8.3 mg/kg/day, caused 50.6% inhibition of ADP-induced platelet aggregation, 37.6% inhibition of aggregation induced with arachidonic acid, a 47.6% decrease in serum levels of thromboxane B2 and a 23.7% increase in the vascular production of 6-keto-PGF1 alpha, versus saline-treated rats. Dipyridamole showed a higher in vitro anti-aggregating effect in whole blood (IC50 6.6 microM) than in platelet-rich plasma (PRP) (IC50 210 microM), when ADP was used as inducer, and had no effect in the presence of arachidonic acid. Mopidamol exerted a similar effect in whole blood (IC50 3.7-20 microM, depending on the inducer) and PRP (IC50 11-17.3 microM), and showed a dose-dependent inhibition of platelet aggregation and thromboxane B2 synthesis induced with arachidonic acid (IC50 16.8-22.3 microM). Mopidamol also inhibited enzymatically induced lipid peroxidation) (IC50 89 +/- 5.9 mumol/L) and had no effect on free radical-induced lipid peroxidation. The dose-dependent increase in 6-keto-PGF1 alpha in vascular samples after incubation with dipyridamole showed a negative linear correlation with inhibition of lipid peroxidation (r2 = 0.77). It is concluded that the phosphodiesterase inhibitors, dipyridamole and mopidamol, interfere in a different manner with platelet function. It seems that mopidamol may also exert a selective effect on platelet thromboxane synthesis.

The pyrimido-pyrimidine derivative RA-642 protects from brain injury in a combined model of permanent focal ischemia and global ischemia reperfusion.

The effects of pyrimido-pyrimidine derivatives (dipyridamole, RA-642 and mopydamole) on lipid peroxidation (inhibition of the production of malondialdehyde, MDA) in different regions of the rat brain were studied. Ferrous sulfate and ascorbic acid (FeAs) were used to induce lipid peroxidation via the formation of hydroxyl anions. The antiperoxidative effect of RA-642 (in the microM range) was 10 times more potent than that of dipyridamole. Mopydamole did not exert any inhibitory effect on MDA production. In a model of ischemia reperfusion with bilateral occlusion of the common carotid arteries for 1 h and restoration of circulation for a period of 2 h, dipyridamole inhibited FeAs-induced MDA production but did not protect from postischemic brain tissue damage (measured by mitochondrial reduction of tetraphenyl tetrazolium). RA-642 inhibited FeAs-induced MDA production and showed 50-67% protection from tissue damage as compared with untreated animals, while mopydamole did not inhibit MDA production and showed 30-48% protection. No correlation was found between inhibition of lipid peroxidation and protection from brain tissue damage.

The pyrimido-pyrimidine derivatives, dipyridamole, mopidamol and RA-642, prevent from retinal vascular defects in experimental diabetes mellitus.

We compared the effects of dipyridamole, RA-642, and mopidamol on platelet activity and thromboxane/prostacyclin balance in relation to the degree of retinal vascularization in a model of experimental streptozotocin-induced diabetes in rats. After 3 months, collagen-induced platelet aggregation in whole blood was 25% higher in diabetic animals than in nondiabetics. Dipyridamole inhibited 43% platelet aggregation, mopidamol 39%, and RA-642 36%. Platelet production of thromboxane B2 was 87% higher in untreated diabetic rats. Mopidamol and RA-642 produced a 46% and 41% inhibition of thromboxane B2. Dipyridamole did not inhibited thromboxane B2 synthesis. Aortic production of 6-keto-PGF1 alpha was 43% lower in untreated diabetic animals and showed no change after treatment with either mopidamol or RA-642. In contrast, dipyridamole caused a 90% increase in aortic production of prostacyclin. Computerized analysis of retinal vascularization showed that untreated diabetic rats had a 81% decrease in the area occupied by peroxidase-labelled vessels as compared with nondiabetics. Treatment with dipyridamole, mopidamol, and RA-642 caused 2.5-fold, 2.8-fold and four-fold increases, respectively, in the percentage of retinal surface occupied by peroxidase-labelled vessels. Differences in retinal vascularization between diabetic animals given RA-642 and nondiabetic controls were negligible.

Potentiation of the antiplatelet action of adenosine in whole blood by dipyridamole or dilazep and the cAMP phosphodiesterase inhibitor, RA 233.

Adenosine (Ado, 10-50 microM), a potent inhibitor of ADP-induced human platelet aggregation in platelet-rich plasma (PRP), does not inhibit aggregation in whole blood. However, the Ado analogs, 2-fluoroadenosine, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) which are resistant to deamination (2-fluoroadenosine) or deamination and phosphorylation (2-chloroadenosine and NECA), inhibit aggregation in whole blood with IC50 values of 12 microM, 2.3 microM and 0.26 microM, respectively. The inhibitory effect of NECA (200 nM) is potentiated by the platelet cAMP phosphodiesterase (PDE) inhibitor RA 233 (5 microM). Inhibition of the erythrocytic nucleoside transport system by dilazep (1 microM) or dipyridamole (10 microM), or blockade of Ado metabolism by 2'-deoxycoformycin (5 microM) plus 5-iodotubercidin (10 microM), evokes the antiaggregatory action of Ado in whole blood (IC50 congruent to 2 microM). RA 233 (5 microM) potentiates Ado-mediated inhibition about 10-fold when nucleoside transport or Ado metabolism is blocked. Ado (10 microM or 200 nM) is rapidly metabolized within 1 min in whole blood. When nucleoside transport is inhibited by dilazep or dipyridamole, or when Ado metabolism is blocked by 2'-deoxycoformycin and 5-iodotubercidin, 50-60% of the Ado remains in the plasma after 5 min. These results show that the failure of Ado to inhibit platelet aggregation in whole blood results from its rapid uptake and metabolism by erythrocytes. More importantly, these data emphasize the key role of nucleoside transport inhibition in the antiplatelet actions of dipyridamole and dilazep. In addition, superior therapeutic results may be obtained from the combination of blockade of nucleoside transport system with inhibition of platelet cAMP PDE.

Role of plasma adenosine in the antiplatelet action of HL 725, a potent inhibitor of cAMP phosphodiesterase: species differences.

The potent inhibitor of platelet cAMP phosphodiesterase (PDE) HL 725 (9,10-Dimethoxy-2-mesitylimino-3-methyl-3, 4,6,7-tetrahydro-2H-pyrimido(6,1-A)-isoquinoline-4-one-hydrochloride), was examined for its effects on human and rat platelet aggregation. Strong inhibitory effects are seen on collagen-induced platelet aggregation both in rat platelet-rich plasma (PRP) (IC50, 54 +/- 12 nM) and whole blood (IC50, 57 +/- 25 nM). Compared to the effects on rat platelets, HL 725 is about two-fold less inhibitory in human PRP (IC50, 94 +/- 29 nM) and whole blood (IC50, 126 +/- 50 nM). The inhibitory action of HL 725 can be reversed by washing and resuspension of the platelets, suggesting that HL 725 does not bind tightly to cAMP PDE. If human or rat PRP is pretreated with adenosine deaminase, an enzyme that degrades adenosine or 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, the inhibitory effect of HL 725 is reversed. Similar blockade of the inhibitory actions of several other inhibitors of cAMP PDE such as RA 233, RX-RA 69 (analogs of dipyridamole) and oxagrelate is seen by adenosine deaminase pretreatment. The nucleoside transport inhibitors, dilazep and dipyridamole which are non-inhibitory alone to platelet aggregation, strongly potentiate (about 10-fold) the inhibitory action of HL 725 on collagen-induced platelet aggregation in human whole blood. However, if the whole blood is pretreated with adenosine deaminase, no inhibitory effect of dipyridamole plus HL 725 is seen on platelet aggregation. These studies demonstrate that plasma adenosine plays a crucial role in the antiaggregatory actions of HL 725 and several other inhibitors of cAMP PDE both in human and rat blood.

Inhibition of ferrous-induced lipid peroxidation by pyrimido-pyrimidine derivatives in human liver membranes.

The effects of pyrimido-pyrimidine derivatives (dipyridamole, RA-642, and RA-233) on lipid peroxidation, using d-alpha-tocopherol as standard, were studied in enriched membrane fractions from human and rat hepatocytes. Equimolar concentrations of ferrous sulfate and ascorbic acid were used to induce lipid peroxidation. The amount of peroxidized lipids observed in membrane fractions from human liver was smaller than in those from rat liver. In both species, however, pyrimido-pyrimidine derivatives, except for RA-233 in rat liver, inhibited lipid peroxidation dose-dependently in the following sequence: RA-642 greater than dipyridamole greater than d-alpha-tocopherol RA-233.

The pyrimido-pyrimidine derivative RA-642: a potent inhibitor of ferrous-induced lipid peroxidation in cell membranes.

The effects of three pyrimido-pyrimidine derivatives (RA-642, dipyridamole and mopidamol) on hydroxyl anion-induced lipid peroxidation in cell membranes from liver, brain, kidney, lung and heart rat tissue were studied using d-alpha-tocopherol as standard for lipid peroxidation. Ferrous sulfate and ascorbic acid (FeAs) were used to induce lipid peroxidation via the formation of hydroxyl anions. The products resulting from the reaction with thiobarbituric acid were taken to be indicators of lipid peroxidation. Thiobarbituric acid reactive substances (TBARS) were produced by different rat tissues in the following sequence: brain greater than liver greater than kidney greater than heart greater than lung. Dose-response and time-response curves were plotted for all compounds. Inhibiting concentrations, 50% (IC50), ranged from 0.3-1.4 microM for RA-642, and 2.5 and 4.6 microM for dipyridamole. In liver mitochondrial membranes, IC50s of these compounds were 0.4 +/- 0.2 and 5.8 +/- 1.2 microM, respectively. At 15 min after beginning TBARS production, dipyridamole and RA-642 did not exert any inhibitory effect.