Problems with solubility determined that only a small selection of the compounds prepared were suitable for testing. The femoral artery, when used with this system, was found to be sensitive to both DMSO and ethanol. Both of these caused substantial dilation of the artery and so, with the time available, we were only able to use such compounds as could be dissolved in an aqueous system at concentrations of 1 0"^ and
below. In this series of experiments we used the iV-acyl penicillamines, SNOPP, SNVP and SNOPHE, the derivatives bearing 3, 5 and 7 membered chains respectively. We were also able to use RIG 200, which was at its solubility limits at -2.5 x lO'^M. Sonication was necessary to dissolve these compounds. Absorbance studies verified that this did not cause any loss of the NO group. In each of these studies the artery response to the 5-nitrosothiol was compared to that of SNAP.
M eth od s.
A protocol similar to that of 3.2 was used. Experiments were carried out on isolated segments of femoral artery from adult male W istar rats (400 - 550 g; n=36). The perfusion system was as described for rat tail tirtery perfusion. The animals were killed by cervical dislocation and the femoral arteries exposed and canulated immediately distal to the epigastric arterial branch. Cannulated arterial segments ( 7 - 8 mm long) were dissected free and transferred to a perspex organ bath chamber ( 1 ml volume) at 37°C.
The rest of the procedure was as 3.2, except that the signal from the converter was fed to a Macintosh LCIII computer as to make data storage and analysis easier.
Endothelial denudation.
The endothelium is an essential part of the arterial machinery for the production of NO. It is of interest to note any differences in response to NO donors in the presence and absence of the endothelium. The endothelium is easily damaged during exposure and cannulation of the artery but can be surprisingly difficult to remove intentionally. In human studies it is enough to pass water through the blood vessel, whereby the osmotic pressure kills the endothelium. In animal model studies it is easier to pass air through the artery which also kills the endothelium. In order to determine the endothelial viability, the pressure after denudation is compared to that of the healthy artery in the presence of an NO scavenger such as haemoglobin. In both cases there should be no nitric oxide and so the pressure should be identical.
The apparatus allows the use of three different modes of ^-nitrosothiol delivery to the vessel:
a) Bolus injection (10 |il) through a resealable rubber septum into the perfusate immediately upstream of the vessel. The artery is exposed to the drug for -300ms.
b) Addition of the drug to the perfusate, allowing the inside of the artery to be continuously exposed to the drug.
c) Addition of the drug to the superfusate, continuously exposing the outside of the artery to the drug.
Bolus injection studies.
Bolus injections of 5-nitrosothiol (lOpl; 10'^ - lO'^M) were made sequentially into the perfusate of the precontracted arteries. The was performed on endothelium intact and endothelium denuded arteries. The responses were deemed to have recovered once pressure was maintained for more than 2 ^ 2 min. Following the highest dose (IQ-^M) the
arteries were allowed to recover for periods of between 15 min and 5 h before being
perfused with haemoglobin (10 |iM).
R esu lts.
Experiments using endothelium intact artery showed the nitrosothiol to have only a tiansient effect. Injections of all of the nitrosothiols showed dose dependent vasodilation which rapidly recovered to their original pre-injection pressures, as expected. The results for endothelium denuded vessels varied substantially between compounds and will be dealt with separately, in comparison with SNAP.
3.3.1. RIG 200
Injections of RIG 200 into endothelium intact arteries gave a dose dependent increase in dilatory response. The arteries recovered fully to their pre-injection pressures. The studies showed it to have a lesser effect than that caused by SNAP. Bolus injections of
AcO AcO CH2OAC
o = c
\ SNO RIG 200: R = CH3 RIG 300: R= CHgCHg RIG 500: R = (CHgjsCHg RIG 700: R = (CHglsCHg HN^ H3C (>=0 RRIG 200 into endothelium denuded arteries showed a full recovery following 10'^ and 10‘^M injections but thereafter failed to recover to pre-injection pressure. There appears to be no difference in the sensitivity to the NO donors between the endothelium denuded arteries and those with intact endothelia. The results are shown in figure 3. The upper trace shows the bolus injection of increasing doses of SNAP. At all but lO’^M the pressure recovers fully. The trace shows the recoveries to be rapid. At the lO'^M dose there is a rapid initial recovery which then tails off. The addition of haemoglobin, via perfusion, gives a rapid return to original pre-injection pressure.
Figure 3 SNAP
RIG200
The lower trace shows the bolus injection of RIG 200 in increasing doses. The recovery between injections is much less rapid and to a lesser degree, leading to a sustained depression of tone Typically recovering to only about 50 % of pre-injection pressure following a lO'^M bolus injection. The perfusion of haemoglobin gives a very slow rise in pressure. The difference between the recovery periods for SNAP and RIG 200 is dem onstrated by the traces in figure 4. Trace a) relates to RIG 200, a slow and incomplete recovery, trace b) relates to SNAP, a full and rapid recoveiy of tone.
Figure 4
a
25%
30 min
The comparisons between SNAP and RIG 200 are shown in figure 5 below. The gi’aph gives the maximum dilation (as a percentage of perfusion pressure) caused by increasing doses of each drug. The gi aph also shows the degree of recovery shown by the artery at each dose. From the graph we can see that there is a sti’ong dose dependency for both drugs, with an increase in peak amplitude with increasingly concentrated bolus injections. SNAP (filled circles) is shown to be more potent at all but the highest dose than RIG 200 (filled tiiangles). Following bolus injections of SNAP the artery is shown to recover fully (100 %) at all but the highest doses (empty circles). The artery shows a dose dependent decrease in recovery following doses of RIG 200 (empty triangles) and only recovers fully at the lowest doses.
Figure 5
125%-|
100%
e
>H 75%-
SNAP PEAK AMPLITUDE SNAP RECOVERY RIG200 PEAK AMPLITUDE RIG200RBOOVERY 0% ■2 -3 -4 6 -5 ■8 ■7 -9 log DOSE (M)
3.3.2. Penicillam ine derivatives.
As the penicillamines prepared are close analogues of SNAP it is reasonable to expect that their activities and stabilities should be of similar magnitudes to that of SNAP. The acyl chain, increasing the lipophilicity of the molecule, enhances prospects of entering the tissue lining the lumen. However, unlike RIG 200, they still retains some degree of hydrophilicity through the free acid group. This makes the handling of the compound somewhat more simple. It is easier to get these derivatives into solution than RIG 200 and consequently it is possible to use them in higher concentrations.
Penicillam ine derivatives
HO2C N H C H2 SNOPP: R = GH3 SNVP: R = (GH2)2CH3 SNOPHE: R = (GH2)4GH3
Figure 7