Abstract:
The method of treating endotoxin shock with 2,2&#39;-[(4,8-bis(diethylamino)-pyrimido[5,4-d]pyrimidine-2,6-diyl)di-(2-methoxyethyl)imino]diethanol or a non-toxic, pharmacologically acceptable acid addition salt thereof.

Description:
This invention relates to a novel method of treating endotoxin shock with a known pyrimido-pyrimidine derivative. 
     More particularly, the present invention relates to the method of treating endotoxin shock with 2,2&#39;-[(4,8-bis(diethylamino)-pyrimido[5,4-d]pyrimidine-2,6-diyl)di-(2-methoxyethyl)imino]diethanol (hereinafter &#34;RA 642&#34;) of the formula ##STR1## or a non-toxic, pharmacologically acceptable acid addition salt thereof. 
     Examples of non-toxic, pharmacologically acceptable acid addition salts of RA 642 are those formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, maleic acid or the like. 
     The preparation of RA 642 and its non-toxic, pharmacologically acceptable acid addition salts is described in Philippine Pat. No. 9640, issued on Jan. 26, 1976. 
     BACKGROUND OF THE INVENTION 
     It has been proposed that central adrenergic neurons influence peripheral sympathetic nerve activity and thus cardiovascular regulation. The stimulation of alpha adrenergic receptors in vasomotor centers mediates a decrease in blood pressure, heart rate and in peripheral sympathetic activity [Kobinger et al., Eur. J. Pharmacol. 2, 155-162 (1967); and Hausler, Naunyn-Schmiedeberg&#39;s Arch. Pharmacol. 286, 97-111 (1974)]. We have previously indicated that E. coli endotoxin may exert its hypotensive effect by activating the central autonomic blood pressure regularoty circuits, by stimulation of central alpha adrenergic receptors leading to inhibition of brain stem sympathetic pathways that participate in the baroreceptor reflex system. Furthermore, it has recently been shown that RA 642 has a central hypertensive effect acting on the medullary cardiovascular regulatory systems [Kadatz et al., Naunyn-Schmiedeberg&#39;s Arch. Pharmacol. 292, 79-103 (1976); and Kobinger et al., Naunyn-Schmiedeberg&#39;s Arch. Pharmacol. 292, 105-111 (1976)]. 
     If the central autonomic blood pressure regulatory circuits are important to the hypotensive response to endotoxin, RA 642 may improve the hemodynamic alterations and survival rate in endotoxin shock. 
     DESCRIPTION OF THE INVENTION 
     We have now discovered that RA 642 and its non-toxic, pharmacologically acceptable acid addition salts are useful for the successful treatment of entotoxin shock in warm-blooded animals such as cats. 
     The novel utility of RA 642 was ascertained by the following tests. 
     Methods 
     General Procedures 
     16 Healthy cats weighing between 2.0 and 3.5 kg were used. Anesthesia was induced with ether and maintained with alpha chloralose (40-50 mg/kg i.v.). The cats were immobilized with gallamine triethiodide (5 mg/kg i.v.) and artificially ventilated. Body temperature was maintained between 36° C. and 37° C. The left femoral artery and vein were cannulated for blood pressure recording and drug administration, respectively. 
     Recording and Integration of Nerve Discharges 
     The left preganglionic splanchnic nerve (PSN) was approached retroperitoneally and was divided into a few multifiber strands under a dissecting microscope and covered with mineral oil. PSN discharges were recorded by means of bipolar silver electrodes at the central cut ends of the nerve. The discharges were amplified with a capacitance-coupled preamplifier (Grass P15; band width 10 Hz-3 KHz) and displayed on a dual beam oscilloscope. The neurogram was integrated by use of an R-C integrator circuit with a time constant of 0.1 second. The output signals of the integrator were calibrated in microvolts. All events were displayed on a pen recording system. The area of the integrated nerve discharges was measured by means of a planimeter (Hewlett Package; calculator 9810A, digitizer 9864A) for a given period and was expressed as PSN activity per unit time. 
     Drug Administration 
     E. coli endotoxin (Difco Lab. 0111:B4), which was freshly prepared as a suspension in physiological saline, was injected intravenously in all the cats as dosage amount of 1 mg/kg in a total volume of 1 ml. Thirty minutes after endotoxin injection, 0.25 mg/kg of RA 642 in a volume of 0.1 ml was injected intravenously into 8 cats which constituted the treated group. Another 8 cats were given only 0.1 ml of physiological saline as a vehicle 30 minutes after endotoxin injection and constituted the non-treated group. 
     Survival Experiment 
     The experiments were carried out on twelve healthy cats weighing between 2.5 and 3.5 kg, and anesthetized with pentobarbital, 35 mg/kg intramuscularly. The catheter for E. coli endotoxin and drug administration was inserted in the superficial saphenous vein below the knee joint and was kept in place for 24 hours. Thirty minutes and 6 hours after endotoxin (1 mg/kg), 0.25 mg/kg of RA 642 was injected intravenously through the catheter. Six of 12 cats served as the non-treated group and were given the same amount of saline. The animals were checked for vital signs, including the condition of stools, every three hours for 48 hours after endotoxin, and their behavior was observed for one week. 
     Statistical Analysis 
     Values for mean blood pressure, pulse pressure and PSN activity are reported as the mean percent changes ±SE from the pre-endotoxin level. Results for the time course studies were first treated with analysis of variance and then treated with paired t-test. Significant differences in survival were determined with an X 2  test (Yates&#39; modification for small samples). 
     RESULTS 
     Baseline Value 
     Pre-endotoxin values for mean blood pressure (MBP), pulse pressure and amplitude of the integrated PSN discharges in non-treated group were 118±5 mmHg, 40±3 mmHg and 52.3±2.3 μV, respectively. There were no statistical differences between the baseline values in the non-treated group and in the treated group (P&gt;0.05), as shown in Table I. 
     
                       TABLE I______________________________________Baseline Values.  Before Endotoxin                30 min. after Endotoxin  Non-              Non-  treated Treated   treated   Treated______________________________________MBP      118 ± 5              119 ± 6                        79 ± 7*                                78 ± 9*(mmHg)Pulse     40 ± 3               38 ± 5                        33 ± 3*                                28 ± 3*Pressure(mmHg)Amplitude    52.2 ± 2.3              55.4 ± 6.4                        24.8 ± 6.2*                                24.9 ± 4.6*of PSNDischarges(μV)______________________________________ MBP and PSN discharges = mean blood pressure and preganglionic splanchnic nerve discharges, respectively. *P &lt; 0.01 in comparison with each preendotoxin level. 
    
    
    
     In the accompanying drawings: 
     FIG. 1 shows the percent changes in mean blood pressure following endotoxin and RA 642. 
     FIG. 2 shows the percent changes in pulse pressure following endotoxin and RA 642. 
     FIG. 3 shows the percent changes in PSN activity following endotoxin and RA 642. 
     FIG. 4 shows the effects of RA 642 on blood pressure and sympathetic nerve activity. From top to bottom: mean blood pressure, systemic blood pressure and integrated nerve discharges. 
    
    
     Non-treated Group 
     The data for MBP, pulse pressure and PSN activity in this group are shown by the triangles connected by the dotted lines in FIG. 1, FIG. 2, and FIG. 3, respectively. Following intravenous administration of endotoxin, MBP fell to 77±8% (P&lt;0.01) of pre-endotoxin level (pre-E1) within 5 minutes after endotoxin injection. There followed a slight temporary recovery of MBP to 82±4% (P&lt;0.05) of pre-E1.  There was a secondary decay in MBP so that after 30 minutes MBP reached to 68±7% (P&lt;0.01) of pre-E1. Following vehicle injection 30 minutes after endotoxin injection, MBP continued to decline reaching 54±8% (P&lt;0.01) of pre-E1 60 minutes after endotoxin (FIG. 1). The pulse pressure decreased to 82±11% (P&lt;0.05) of pre-E1 5 minutes after endotoxin which persisted until 30 minutes after endotoxin. Following vehicle injection the pulse pressure dropped to 78±11% (P&lt;0.01) of pre-E1 5 minutes after vehicle treatment. Finally, there was a gradual decline in the pulse pressure to 67±13% (P&lt;0.01) of pre-E1 (FIG. 2). 
     PSN activity decreased to 71±11% (P&lt;0.05 of pre-E1 within 5 minutes after endotoxin injection. After a small recovery in PSN-activity, a secondary decrease in PSN-activity reached to 47±10% (P&lt;0.01) of pre-E1 30 minutes after endotoxin injection. Following vehicle saline injection, PSN activity did not show any changes so that after 60 minutes, PSN activity was 42±8% (P&lt;0.01) of pre-E1 (FIG. 3). 
     Treated Group 
     The data for MBP, pulse pressure and PSN activity in this group are shown by the stars connected by solid lines in FIG. 1, FIG. 2 and FIG. 3, respectively. There were no significant differences in the time course of each parameter for the first 30 minutes after endotoxin injection between the two groups (P&lt;0.05). MBP, pulse pressure and PSN activity 30 minutes after endotoxin injection were 65±5% (P&lt;0.01), 73±7% (P&lt;0.01) and 46±8% (P&lt;0.01) of pre-E1, respectively. These reduced levels by endotoxin were not significantly different from the levels in the non-treated group (P&lt;0.05). 
     Within 5 minutes after injection of RA 642, MBP rapidly recovered to 83±6% (P&lt;0.05) of pre-E1. This level of MBP was significantly higher than the level at 30 minutes after endotoxin (P&lt;0.01) and was maintained until the end of the experiment (FIG. 1). The pulse pressure did not change significantly, so that 60 minutes after endotoxin (30 minutes after treatment) pulse pressure was 70±6% (P&lt;0.01) of pre-E1. There was no significant difference in the time course of changes in pulse pressure after treatment between both groups (P&lt;0.05) (FIG. 2). 
     Following injection of RA 642, PSN activity rapidly increased to 62±10% (P&lt;0.01) of pre-E1 10 minutes after treatment. This increased PSN activity by RA 642 was significantly higher than the level at 30 minutes after endotoxin (P&lt;0.01) and was maintained until the end of the experiment. There was statistically significant difference in the time course between the non-treated group and the treated group (P&lt;0.01) (FIG. 3). 
     FIG. 4 is an example of the pattern of response to RA 642 following intravenous injection of E. coli endotoxin. 
     Survival 
     Five of the six cats in the non-treated group (83%) died within 48 hours after endotoxin injection (average 28.7±2.9 hours). Three of the six cats had bloody diarrhea. The surviving one only recovered normal appetite and behavior 4 days after endotoxin injection. In contrast, all of the 6 cats treated with RA 642 (100%) survived, as shown in Table II. 
     
                       TABLE II______________________________________Effects of RA 642 on survival and condition of stool duringendotoxin shock.                     Bloody   Mucous     Survival            Mortality                     Diarrhea Diarrhea______________________________________Non-treated group       1        5        3      2(n = 6)Treated group       .sup. 6* .sup. 0* 0      3(n = 6)______________________________________ Values are number of animals in each group (n = 6)? *P &lt; 0.05 in comparison with nontreated group. 
    
     Immediately after injection of RA 642, the animals showed deep regular breathing and an increase in muscle tonus. The corneal reflex and the reflex movements of limbs to pain stimuli were facilitated. Three of the treated cats showed a mucous diarrhea without blood during the initial period (6 hours after endotoxin). Within 48 hours after endotoxin injection, normal appetite and behavior returned in all treated animals. During the observation period, the animals treated with RA 642 did not show any signs of clonic or tonic convulsions. 
     For pharmaceutical purposes RA 642 or a non-toxic acid addition salt thereof is administered parenterally to warm-blooded animals suffering from endotoxin shock as the active ingredient in customary pharmaceutical compositions, that is, compositions consisting essentially of an inert pharmaceutical carrier and an effective amount of the active ingredient, such as injectable solutions or the like. The effective dosage range of RA 642 and its non-toxic acid addition salts is from 0.1 to 1.0 mgm/kg body weight, depending upon the severity of the shock.