model. LPS induced a marked hypotensive response (Table 1): at t= 20hrs,
SAPs, MAPs and DAPs had fallen by 32%, 28.5% and 25.1% respectively. Systemic pressures spontaneously returned to pre-injection values over the ensuing 24-28hrs (t= 44-48hrs). LPS also induced a modest (ca 10%) but statistically significant tachycardia.
Time after injection t=Ohr t= 20hr p value*
SAP 166.4 ±8.8 113.2 ±5.2 0.028
MAP 110.6 ±4.9 79.1 ±6.4 0.000
DAP 82.8 ±4.9 62.0 ±7.8 0.000
HR 376.6 ±9.4 411.1 ±11.7 0.017
Table 1 Systemic blood pressure (mmHg) and heart rates (HR; beats/min) recorded with the rat tail cuff apparatus immediately before and 20hrs after injecting animals (n= 13) with bacterial LPS (4mgfKg). Student’s paired t-test (p values to the nearest 3rd decimal point).
6.3.3 JM1006, JM1226 and JM6245 reverse the hyporeactivity to PE of
RTAs from LPS-treated rats. Fig. 6.3a & 6.3b show that the addition of
JM1006 (100/<M) to the internal perfusate completely abolished the diminished sensitivity (2-way ANOVA, F= 6.78, df= 9, 136e, p= <0.001) to PE of isolated, internally-perfused RTAs from LPS-treated animals (t= 24hrs; closed circles; controls, open circles; n= 7) . Statistically, there was no significant difference between JM1006- (closed squares) and L-NMMA-treated (open triangles) arteries from LPS-treated rats (2-way ANOVA, F= 0.98, df= 9, 234e, p= 0.362). Similarly, the difference in sensitivity to PE ( 2-way ANOVA, F= 7.39, df= 9, 155e, p < 0.001) between vessels from control (open circles) and LPS-treated (t= 24hrs; closed circles, fig. 6.4a; n= 9) rats was reversed by JM1226 (100//M; closed squares, fig. 6.4b; 2-way ANOVA, F= 3.61, df= 9, 151e, p < 0.001). The effect of JM1226 (and of JM6245; data not shown) on vessels from LPS-treated rats was closely comparable to that seen after blocking NO synthesis with L- NMMA (100//M; open triangles; fig. 6.4b). Statistically, there was no significant difference between JM1226- and L-NMMA-treated arteries from LPS-treated rats (2-way ANOVA, F= 0.46, df= 9, 189e, p= 0.901). Meaned results are shown in Appendix tables 6.1 & 6.2.
Fig. 6.3 a Log D.R. curves showing the vasoconstrictor effect of bolus injections of PE on arteries isolated from animals previuosly injected with LPS 24hrs prior to sacrifice (open circles) compared to vessels from animals injected with sterile saline only (controls; closed circles). Mean ±S.E.M. shown, b Log D.R. curves to PE of vessels from LPS-treated rats (t= 24hrs) before (open circles; same data as fig. 6a) and after the addition of either JM1006 (closed squares) or L-NMMA (open triangles) to the internal perfusate. The sensitivity to PE of L-NMMA- and JM1006-treated vessels were not statistically different and both were significantly greater than that for LPS-treated arteries (see text).
Fig. 6.4a Log D.R. curves showing the vasoconstrictor effect of bolus injections of PE on arteries isolated from animals previuosly injected with. LPS 24hrs prior to sacrifice (open circles) compared to vessels from animals injected with sterile saline only (controls; closed circles). Mean ±S.E.M. shown, b Log D.R. curves to PE of vessels from LPS-treated rats (t~ 24hrs) before (open circles; same data as fig. 6a) and. after the addition of either JM1226 (closed squares) or L-NMMA (open triangles) to the internal perfusate. The sensitivity to PE of L-NMMA- and JM1226-treated vessels were not statistically different and both were significantly greater than that for LPS-treated arteries (see text).
■6
-5
-4
-3
Log[PE] (M)
6.3.4 JM1226 accelerates recovery of arterial blood pressures in LPS-
treated (hypotensive) rats. Fig. 6.5 summarises the results in the form of a
histogram in which the data from experiments 1&2 are combined for time points t= 0, 20, 24hrs, but for time points t= 29 and 32hrs the data is from experiment 2 only. The are results also shown in Appendix table 6.3.
Time (hrs)
Fig. 6.5 Blood pressure measurements for rats immediately before (t= ohr; first block) and 20hrs after (second block) a single i.p. injection (4mg/Kg) of bacterial LPS (data also in Table 1). Animals in group I (stippled columns) received an injection of saline only and those in group 2 (blank columns) received JM1226 (i.p.; lOOmg/Kg) at t= 20hrs. Blood pressure recovered more rapidly in animals treated with JM1226 and was essentially complete after ca. 9hrs. See text for details.
Each of the 5 grouped vertical columns in the histogram represents (from left to right) S.b.p.s, M.b.p.s, and D.b.p.s. Both groups of LPS-treated animals were markedly hypotensive by t= 20hrs (see Table 1 which shows combined data ie.
experiments 1&2, n=13 animals) and shows some recovery thereafter. However, the recovery of saline-injected animals (stipplied columns) was incomplete even after 12hrs (t= 32hrs), whereas the rate of recovery of animals that received JM1226 (open columns) was enhanced and essentially complete within ca. 9hrs (t= 29hrs).
6.4 Discussion
From these results it is clear that ruthenium-based scavengers could offer an alternative therapeutic strategy to the use of NOS inhibitors for alleviating the symptoms of septic shock, and possibly other mediated diseases too. Two chemically-related polyaminocarboxylate-ruthenium(III) complexes - JM1226 and its aqua derivative JM6245 - were found to react with either authentic NO or with an NO donor (SNAP) in aqueous solution to yield the corresponding Ru(II) mononitrosyl adduct (Fig. 2; Flicker et al., 1995 and in press). This has yet to be confirmed for JM1006. Their ability to react with NO in several biological systems of increasing complexity (from cultured cells to whole animals) was therefore explored, following induction of the iNOS-driven, ‘high-ouput’ pathway for NO synthesis, using either LPS alone (whole animals) or combined LPS + interferon-gamma treatment (cell cultures; Flicker et al., 1995). It has been shown that ruthenium complexes reduced NO2" accumulation by cultured macrophages and afforded a significant degree of protection against the tumouricidal action of these macrophages (Flicker etal., in press).
The results of in vivo experiments using the rat model of endotoxic shock are clearly more relevant when evaluating the therapeutic potential of these compounds. The effects of LPS treatment (4mg/Kg; i.p.) have been defined according to five separate parameters all of which are consistent with the principal physiological manifestations of septic shock (Glauser et al., 1991) and therefore, establish the LPS-treated rat as a valid paradigm for testing the ability of JM compounds to scavenge pathophysiological quantities of NO under both
ex vivo (isolated RTAs) and in vivo conditions.
The hyporesponsiveness to PE displayed by internally-perfused RTAs isolated from LPS-treated animals was maximal at t= 24hrs (see Chapter 3, fig. 3.7). The addition of JM1006, JM1226 and JM6245 (results not shown) to the internal
perfusate of such vessels enhanced their sensitivity to PE: indeed, the effect was indistinguishable from that seen after blockade of NO synthesis at source using the NOS inhibitor, L-NMMA.
Treatment with LPS produced a profound but transient hypotensive response in laboratory animals. As it will be seen in Chapter 8, S.b.p.s, M.b.p.s amd M.b.p.s fall to a minimum around t= 20 - 24hrs and gradually recover over the ensuing 24- 28hrs (t= 44 - 48hrs). Fig. 6.5 shows that some spontaneous recovery of blood pressures was seen in the saline-injected (control) group of LPS-treated rats but this was clearly incomplete after 12hrs (t= 32hrs). In contrast, the recovery of the LPS-treated group that received a single i.p. injection of JM1226 given at t= 20hr was accelerated and complete after a further ca. 9hrs (t= 29hrs).
These results and those of Fricker et al., (1995) show that JM1226, 6245 and 1006 are effective scavengers of NO in a variety of biological systems and they establish these complexes as potentially useful alternatives to NOS inhibitors for alleviating some of the symptoms of endotoxic shock. It has previously been shown, and is further confirmed in this study (see Chapter 7, Section 7.4.1), that tumour-supply vessels are remarkably unresponsive to vasoactive agents (Hirst & Wood, 1989; Kennovin et al., 1993) and in this respect they strongly resemble RTAs from LPS-treated rats. It is also shown in Chapter 7 (Section 7.4.2) that the sensitivity to PE of isolated, internally perfused epigastric arteries which previously supplied an inguinal tumour implant, can be fully restored using the non-selective NOS inhibitors L-NMMA or L-NAME. Moreover, chronic oral administration of L-NAME, via the drinking water, significantly retarded solid tumour growth (Chapter 8, Section 8.3.5). Based on the data presented here it is likely that JM1226, 6245 and/or 1006, could have similarly benefical effects to L-NAME.
Further studies will have to be conducted detailing the toxicological and pharmacokinetic properties of these JM compounds. However, preliminary
toxicity tests in vivo have shown that Wistar rats can tolerate maximum doses of JM1226 some 2-4 times greater than those used here (200 - 400mg/Kg; i.p.) and pilot pharmacokinetic experiments indicate rapid plasma clearance times (Dr. Simon Fricker; personal commun.). Studies will also have to be conducted to optimize the dosing schedule of these drugs.
6.4.1 Conclusions. In conclusion, the results of this study demonstrate that by
efficiently scavenging NO, ruthenium(III) complexes JM1006, 1226 and its aqua derivative, 6245, might be usefully exploited in new therapeutic strategies aimed at combating NO mediated disease.