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P Leff

Publications and source records attributed to P Leff.

At least 37 records · Page 2Linked to original sources
PubMed1995

Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase.

1. FPL 67156 (6-N,N-diethyl-beta, gamma-dibromomethylene-D-ATP), is a newly synthesized analogue of ATP. 2. In a rabbit isolated tracheal epithelium preparation, measuring P2U-purinoceptor-dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP-gamma-S. UTP agonist-concentration effect (E/[A]) curves were shifted to the left by 5-fold in the presence of 100 microM FPL 67156. The differential effect of FPL 67156 on UTP and ATP-gamma-S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process. 3. FPL 67156 was tested as an ecto-ATPase inhibitor in a human blood cell assay, measuring [gamma 32P]-ATP dephosphorylation. The compound inhibited [gamma 32P]-ATP degradation with a pIC50 of 4.6. 4. FPL 67156 was then tested for its effects on ATP and alpha, beta-methylene-ATP responses at P2X-purinoceptors in the rabbit isolated ear artery. In the concentration range 30 microM-1 mM, the compound potentiated the contractile effects of ATP but not those of alpha, beta-methylene-ATP. At 1 mM, FPL 67156 produced a 34-fold leftward shift of ATP E/[A] curves. 5. The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analyzed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pKi for FPL 67156 as an ecto-ATPase inhibitor of 5.2. 6. Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X- and P2T-purinoceptors (pA2 ~ 3.3 and 3.5 respectively), and weak agonist effects at P2u-purinoceptors(p[A 50]~ 3.5).7. The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto-ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.8.This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.

Adenosine Triphosphatases↗
PubMed1995

Pharmacological profile of the novel P2T-purinoceptor antagonist, FPL 67085 in vitro and in the anaesthetized rat in vivo.

1. The role of endogenous ADP in platelet aggregation in vivo remains unclear due to the lack of suitable P2T-antagonist probes. This paper describes the potency, selectivity and specificity of the novel P 2T-purinoceptor antagonist, FPL 67085 (2-propylthio-D-beta,gamma-dichloromethylene ATP) both in vitro and in the anaesthetized rat in vivo. 2. FPL 67085 (3-30 nM) produced concentration-dependent rightward displacement of the concentration-effect (E/[A]) curve for ADP-induced aggregation of human washed platelets with no effect on ADP-independent aggregation at < or = 10 microM. 3. Logistic fitting of ADP E/[A] data indicated that the antagonist effect of FPL 67085 did not consistently accord with simple competition: in some preparations depression of the asymptote was seen. Schild analysis of data combined from all preparations, regardless of the antagonist profile observed, gave an apparent pKB of 8.9 (slope parameter 0.90). 4. The potency of FPL 67085 was unaffected by the P1-purinoceptor antagonist, 8-sulphophenyltheophylline, was similar (IC50 0.6-3.8 nM) in human and rat washed platelets or whole blood and, in rat blood, did not change following 2-30 min incubation at 37 degrees C. 5. FPL 67085 was a weak (pA50 approximately 4.2) partial agonist in tissues containing P2X- or P2Y-purinoceptors, indicating some 30,000 fold selectivity for the P2T-subtype. 6. In anaesthetized rats, intravenous infusion of FPL 67085 produced rapidly-reversible, dose-related inhibition of ADP-induced platelet aggregation measured ex vivo (ID50 1.3 micrograms kg-1 min-1) with no significant effect on haemodynamics or circulating cell counts. 7. Thus, FPL 67085 is a potent, specific and selective inhibitor of ADP-induced platelet aggregation both in vitro and in vivo. As such, it represents a novel pharmacological tool to define the role of endogenous ADP in thrombosis and the potential of P2T-purinoceptor antagonists as a novel class of infusible anti-thrombotic agents for acute use in man.

Adenosine Diphosphate↗
PubMed1994

Agonist-antagonist interactions at angiotensin receptors: application of a two-state receptor model.

Interactions between agonists and antagonists at angiotensin receptors are characterized by a number of features: variation of antagonist dynamics between apparent simple competition, insurmountable antagonism and, occasionally, augmentation; the tendency for insurmountable antagonism to be saturable; slow recovery of agonist responses following agonist-induced tachyphylaxis; and the ability of competitive antagonists to accelerate recovery from the latter intervention. Some of these phenomena have also been observed in studies of 5-HT2 receptors where they were attributed to the operation of a two-state model with an allosteric site. In this article, Mark Robertson and colleagues propose that the properties of angiotensin AT1 receptors may be explained by a similar model, but without the need to evoke an allosteric site.

Angiotensin II↗
PubMed1994

FPL 66096: a novel, highly potent and selective antagonist at human platelet P2T-purinoceptors.

1. ADP-dependent platelet aggregation is mediated by the P2T-purinoceptor and is specifically inhibited by ATP, which is a competitive P2T-purinoceptor antagonist. However, ATP functions as an agonist at other P2-purinoceptor subtypes in other tissues and is, therefore, non-selective. This paper describes the effects of the novel ATP analogue, FPL 66096 (2-propylthio-D-beta,gamma-difluoromethylene ATP), on ADP-induced and ADP-independent aggregation of human washed platelets and in standard preparations containing P2X- (rabbit ear artery) and P2Y-purinoceptors (guinea-pig aorta). 2. In suspensions of human washed platelets, FPL 66096 (1-100 nM) produced concentration-dependent rightward displacement of concentration-effect (E/[A]) curves obtained for ADP-induced platelet aggregation. Logistic fitting of E/[A] data indicated that the effect of FPL 66096 was consistent with simple competition with a pKB value of 8.66. FPL 66096 (10-1000 nM) had no effect on aggregation produced by the thromboxane A2-mimetic, U46619 (0.1-10 microM) when the response to this agent was rendered ADP-independent by inclusion of the non-selective P2-purinoceptor antagonist, suramin (100 microM). 3. The anti-aggregatory potency of FPL 66096 was not influenced by increasing the incubation time from 2 to 15 min nor by inclusion of the P1-purinoceptor antagonist 8-sulphophenyltheophylline at a concentration (300 microM) that produced a 68 fold rightward displacement of the anti-aggregatory E/[A] curve for the P1-purinoceptor agonist, 5'-N-ethylcarboxamidoadenosine (0.1-1000 microM). 4. FLP 66096 behaved as a weak (pA" 3.68) but full P2x-purinoceptor agonist in preparations of the rabbit isolated ear artery and as a weak, competitive antagonist (apparent pKB 4.71) at P2Y purinoceptors in the guinea-pig isolated aorta, indicating a selectivity of at least 9000 fold for the P2t-subtype. In the latter preparation, non-specific relaxations were produced by concentrations of FPL 66096 >10M gM.5. These results indicate that FPL 66096 is a P2-purinoceptor antagonist of unprecedented potency and selectivity and that its effects are consistent with simple competition at the P2-purinoceptor. Therefore,FPL 66096 represents a novel pharmacological tool in the classification of P2-purinoceptors and in the elucidation of the mechanisms involved in activation of platelets by ADP.

Adenosine Diphosphate↗
PubMed1994

Pharmacological analysis of ecto-ATPase inhibition: evidence for combined enzyme inhibition and receptor antagonism in P2X-purinoceptor ligands.

1. Previous studies have shown that suramin and FPL 66301 are competitive antagonists at the P2X-purinoceptor in the rabbit ear artery. Those studies employed alpha,beta-methylene ATP, a poorly hydrolysable ATP analogue, as the agonist. In this study these compounds have been tested using ATP as the agonist. 2. Suramin, in the concentration range 30-1000 microM, potentiated the contractile effects of ATP, producing a 3-fold leftward shift of the ATP E/[A] curves. FPL 66301, in the concentration range 100-1000 microM, produced a significant but small (approximately 3-fold) rightward shift of the ATP curves. These results are in marked contrast with previous studies using alpha,beta-methylene ATP in which 30-fold rightward shifts were achieved using the same concentration ranges of suramin and FPL 66301. 3. Suramin and FPL 66301 were tested as ecto-ATPase inhibitors in a human blood cell assay. Suramin inhibited the enzyme with a pIC50 of 4.3, FPL 66301 with a pIC50 of 3.3. 4. The pharmacological data were analysed using a theoretical model describing the action of a compound with dual enzyme inhibitory and receptor antagonistic properties on the effects of an agonist susceptible to enzymatic degradation. The model was found to fit the data well using the known pKB estimates for suramin and FPL 66301 and similar relative (but not absolute) pK1 estimates to those obtained for the compounds in the enzyme assay. 5. From this analysis it was concluded that the limited shifts of ATP E/[A] curves produced by suramin and FPL 66301 were the result of 'self-cancellation' of the potentiating (enzyme inhibitory) and rightward-shifting (receptor antagonistic) properties.6. The analysis also indicated that the presence of ecto-ATPase activity in the rabbit ear artery preparation has a marked effect on the apparent potency of ATP. The experimental p[A50] was 3.4,whereas the 'true' value, that is the value which would be obtained in the absence of ecto-ATPase activity, was 6.0, some 400-fold higher.7 Two conclusions are drawn from this study. Firstly, caution must be exercised in the use of suramin and FPL 66301 as tools for receptor classification. Absence of overt antagonism by these compounds when metabolically unstable agonists are used could lead to erroneous claims for receptor subtypes.Secondly, the agonist potency order currently used to designate P2X- purinoceptors may require modification.

Adenosine Triphosphatases↗
PubMed1993

Further concerns over Cheng-Prusoff analysis.

In his article in last month's issue of TiPS (14, 89-91), Douglas Craig discussed the application of the Cheng-Prusoff relationship to the analysis of antagonists in functional experiments. Craig described how the translation of this method from biochemistry to pharmacology can be accompanied by errors due to theoretical misunderstanding. He also emphasized the lower level of rigour associated with this means of estimating antagonist affinity constants compared with Schild analysis. In this article, Paul Leff and Iain Dougall extend Craig's arguments by describing some additional ways in which the application of the Cheng-Prusoff method to pharmacological experiments are likely to result in misinterpretation and erroneous estimates of antagonist affinity.

Animals↗
PubMed1993

Quantitative analysis of the agonist and antagonist actions of some ATP analogues at P2X-purinoceptors in the rabbit ear artery.

1. The agonist and antagonist effects of a series of beta, gamma-methylene dihalo- and 2-methylthio-substituted analogues of ATP at P2x-purinoceptors have been analysed on the rabbit isolated ear artery preparation. Cumulative and sequential dosing experimental protocols were employed in the construction of agonist concentration-effect curves in order to address the possible influence of acute receptor desensitization on subsequent analyses. 2. Using the cumulative curve design the following results were obtained: D-AMP-PCBr2P, 2-methylthio-D-AMP-PCCl2P, L-AMP-PCF2P, L-AMP-PCCl2P and LAMP-PCBr2P each behaved as partial agonists. D-AMP-CPP was used as a reference full agonist and these analogues were analysed by the comparative method of Barlow et al. (1967), to provide estimates of affinity and efficacy. 2-Methylthio-L-AMP-PCBr2P was virtually silent as an agonist and was analysed as a competitive antagonist by Schild analysis. 3. Two agonists, L-AMP-PCCl2P and L-AMP-PCBr2P, were analysed by the sequential curve design, and the antagonist effects of one of the agonists, L-AMP-PCBr2P were also analysed using this protocol. The resulting estimates of affinity and efficacy, while similar to those obtained with the cumulative design, indicated that acute desensitization may affect curve definition and estimation of these quantities. 4. The following structure-activity trends emerged: D-analogues tended to have higher efficacy but lower affinity than L-analogues; efficacy varied markedly and inversely with the atomic weight of the halogen while affinity was only minimally affected; 2-methylthio- substitution also reduced efficacy with minimal effect on affinity. 5. The results of this analysis are discussed in terms of the utility of affinity and efficacy information in the classification of purinoceptors and the design of chemical probes for them.

Adenosine Triphosphate↗
PubMed1993

Estimation of partial agonist affinity by interaction with a full agonist: a direct operational model-fitting approach.

1. The operational model of agonism (Black & Leff, 1983) has been extended to describe the interaction between a partial agonist and a full agonist at the same receptor. The derived equation explicitly describes the interaction and allows the affinity (and efficacy) of the partial agonist to be estimated by direct fitting of raw experimental agonist concentration-effect (E/[A]) curve data. 2. The model was used to analyse experimental E/[A] curve data generated for the interaction between pilocarpine (partial agonist) and carbachol (full agonist) at the M3-muscarinic receptor mediating contraction of the guinea-pig isolated trachea. Pilocarpine affinity estimates obtained by operational model-fitting were compared with those obtained by use of the null method (Stephenson, 1956). These analyses demonstrated that the two methods gave comparable results (mean pKB estimates were 5.79 and 5.86 for the operational model and null method respectively). 3. When multiple concentrations of partial agonist are used, simultaneous operational model-fitting of all the E/[A] curve data allows the competitive nature of the interaction to be studied. 4. We conclude that operational model-fitting is a valid and analytically simple alternative to the conventional null method of analysing full/partial agonist interactions.

Animals↗
PubMed1992

Classification of platelet and vascular prostaglandin D2 (DP) receptors: estimation of affinities and relative efficacies for a series of novel bicyclic ligands. With an appendix on goodness-of-fit analyses.

1. The DP receptors located on platelets and vasculature were examined in a human washed platelet preparation and in isolated rings of rabbit external jugular vein. 2. A series of eight novel bicyclic compounds were studied for their effects in the two assays. Seven produced agonism, inhibition of aggregation or vascular relaxation, and one compound was 'silent' in both assays. 3. The operational model of agonism (Black & Leff, 1983) was fitted simultaneously to concentration-effect curve data for the seven agonist compounds. The affinity and efficacy estimates so obtained were tested for similarity between the two tissues by analysis of variance, showing that the model could be fitted to both sets of data by assuming the same relative affinity and efficacy values. However, absolute affinity estimates were consistently lower in the vascular preparation. 4. Analysis of two of the seven agonists as antagonists was also possible. This provided pKB estimates which supported the agonist affinity estimates. The eighth compound was also analysed as an antagonist. It, like the other seven, demonstrated a difference in affinity between the two tissues. 5. The results of this study support the view that platelet and vascular DP receptors are similar, assuming that the systematic difference in affinity estimates for the series of compounds between the two tissues is the consequence of receptor micro-environment and/or accessory binding site differences.

Algorithms↗
PubMed1992

Adenosine 5'-(2-fluorodiphosphate) is not a selective P2Y purinoceptor agonist in the rabbit jugular vein.

The relaxant properties of the putative selective P2Y agonist adenosine 5'-(2-fluorodiphosphate) (ADP-beta-F) and its structural analog adenosine 5'-(2-thiodiphosphate) have been investigated in the rabbit precontracted jugular vein preparation. In tissues with intact endothelium, ADP-beta-F produced a multiphasic agonist concentration/effect curve made up of two vasorelaxant components which were kinetically and pharmacologically distinct. The higher potency phase (p[A50] 5.58 +/- 0.13), characterized by slow, tonic responses, was retained after endothelial denudation and blocked by the selective P1 purinoceptor antagonist 8-sulphophenyltheophylline. The lower potency phase (p[A50] 3.98 +/- 0.07), characterized by fast, phasic responses, was abolished by endothelial denudation and is presumed to be mediated at P2Y purinoceptors. By contrast, the agonist concentration/effect curve to adenosine 5'-(2-dithiophosphate) in endothelium-intact tissues appear monophasic and was unaffected by 8-sulphophenyltheophylline (p[A50] 6.86 +/- 0.12), although endothelial denudation revealed a secondary P1-induced relaxant component (p[A50] 5.73 +/- 0.20). This study demonstrates that in the rabbit jugular vein, relaxant responses to ADP-beta-F are mediated primarily by activation of P1 purinoceptors, and it is, therefore, invalid to regard ADP-beta-F as a selective probe for P2Y purinoceptors, whereas adenosine 5'-(2-thiodiphosphate) does show some selectivity for this receptor.

Adenosine Diphosphate↗
PubMed1991

Further subclassification of ATP receptors based on agonist studies.

Recent studies using agonist analogues of ATP and other nucleotides have generated some surprising observations which may have ramifications for the classification of P2 receptors, particularly for those responses currently attributed to P2Y receptor activation. 2-MethylthioATP (2-MeSATP), the conventional P2Y receptor agonist, does not interact with ATP in the expected fashion in various models of endothelial function, suggesting that it acts by a different mechanism. Furthermore, in certain cell types where responses to ATP are mediated by phospholipase C activation, 2-MeSATP has little or no activity. Interestingly, the pyrimidine uridine triphosphate (UTP) invariably shows similar potency to ATP in systems where 2-MeSATP is inactive. In this article Steve O'Connor and colleagues discuss these data and their significance, and propose that separate receptors may be responsible: one sensitive to 2-MeSATP and the other, a 'nucleotide' receptor, sensitive to UTP.

Animals↗
PubMed1991

Estimation of the efficacy and affinity of the beta 2-adrenoceptor agonist salmeterol in guinea-pig trachea.

1. We have attempted to estimate the affinity and efficacy of the new beta 2-adrenoceptor agonist salmeterol in the guinea-pig isolated trachea and to compare these estimates with those obtained for salbutamol. 2. The kinetics of the relaxation to salmeterol were considerably slower than those to salbutamol and the responses were poorly defined. Nevertheless, in experiments employing a cumulative curve design both salmeterol and salbutamol exhibited partial agonist behaviour. Operational model-fitting of these data indicated that the intrinsic efficacies of the two agonists were indistinguishable but that salmeterol had an affinity (pKA = 7.4) some 30 fold greater than salbutamol (pKA = 5.9). 3. Single dose experiments, using maximally effective concentrations of salmeterol or salbutamol were carried out in an attempt to quantify more reliably the relative efficacies of the two agonists. In these experiments salbutamol was found to have an efficacy (2.5) approximately 3 fold greater than that of salmeterol (0.8). 4. Full/partial agonist interaction studies were carried out to provide another estimate of the affinity of salmeterol. Isoprenaline and adrenaline were employed as full agonists in these experiments and the affinity (pKB) estimates obtained for salmeterol against these agonists were 7.3 and 7.4 respectively. These affinity estimates were similar to the pKA value (7.4) obtained by operational model-fitting. 5. Salmeterol has slightly lower efficacy (3 fold) but markedly greater affinity (30 fold) than salbutamol at beta 2-receptors. The slightly lower efficacy of salmeterol is unlikely to have clinical implications for this drug as a bronchodilator beta 2-adrenoceptor agonist in asthma.

Adrenergic beta-Agonists↗