Assessment of translational risk in drug research: Role of biomarker classification and mechanism-based PKPD concepts.

In 2005, Danhof and coauthors proposed a new biomarker classification in the context of the application of mechanism-based PKPD modeling. They defined the term 'biomarker' as a measure that characterizes a drug-induced response, which is on the causal path between drug administration and clinical outcome. The biomarker classification identified seven categories that provide different insights into the kinetics of drug action, such as target site distribution, target engagement, or into the impact of the drug on physiology or disease. The original biomarker classification has been further modified into a translational biomarker scheme that is used as a communication tool for drug hunting teams to guide designing translational and early clinical development plans as part of an integrated model-informed drug discovery and development strategy. It promotes a dedicated discussion on the topic of the translational relevance of biomarkers and enables efficient identification of translational gaps and opportunities. Based on the elucidated PKPD characteristics exhibited by a novel drug and the kinetics of the investigated biomarker, prospective predictions can be made for the drug response under new conditions; translating from the preclinical arena to the clinical setting, from the healthy volunteer to the patient, or from an adult to an elderly or a child. These drug response predictions provide support to decisions on appropriate next steps in the development of the drug, while keeping clear line of sight on the potential to address unmet medical need. Moreover, this framework enables a transparent translational risk assessment for drug hunting projects, and as such can underpin decisions at program and portfolio level.

Correct assessment of new compounds using in vivo screening models can reduce false positives.

During early drug discovery the initial in vivo efficacy testing is often performed in rodent models optimized to screen and select lead compounds rapidly, before progressing them to in vivo models that reflect the human form of the disease more closely. The way such models are frequently run can risk overestimating the efficacy of new compounds when using pre- and co-administration, as shown in three examples from different central nervous system research areas. This is undesirable for reasons ranging from good decision-making, cost efficiency and time management to the ethics of animal use. Abandoning the use of pre-treatment, monitoring crucial physiological parameters in (satellite) animals and systematically applying simple pharmacokinetic-pharmacodynamic analysis could reduce the number of false positive results.

Block of neuronal nicotinic acetylcholine receptors by organophosphate insecticides.

Chronic and acute exposure to organophosphate (OP) pesticides may lead to persistent neurological and neurobehavioral effects, which cannot be explained by acetylcholinesterase (AChE) inhibition alone. It is suggested that other brain proteins are involved. Effects of commonly used organophosphate pesticides on rat neuronal alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. Several OP pesticides, e.g., parathion-ethyl, chlorpyrifos and disulfoton, inhibited the ACh-induced ion current with potencies in the micromolar range. The potency of inhibition increased with increasing concentrations of the agonist ACh. Comparison of the potency of nAChR inhibition with the potency of AChE inhibition demonstrated that some OPs inhibit nAChRs more potently than AChE. Binding experiments on alpha4beta2 nAChRs showed that the OPs noncompetitively interact with nAChRs. The inhibitory effects on nAChRs are adequately described and explained by a sequential two-step mechanism, in which rapidly reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It is concluded that OPs interact directly with neuronal alpha4beta2 nAChRs to inhibit the agonist-induced response. This implicates that neuronal alpha4beta2 nAChRs are additional targets for some OP pesticides.

Cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) decisive for its therapeutic efficacy in sarin poisoning.

Mortality and occurrence of cholinergic symptoms upon sarin intoxication (144 micro g/kg s.c., approximately 2 x LD50) in rats is completely prevented by treatment with the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 2 mg/kg i.m.). Previously, we have shown that CPA treatment altered the distribution of sarin into the brain, presumably through its cardiovascular side effects. Therefore, the objective of the present study was to evaluate the contribution of the cardiodepressant effects of CPA to its therapeutic efficacy against sarin intoxication. Intramuscular treatment of rats with 0.5 and 2.0 mg/kg CPA 1 min after sarin poisoning attenuated most cholinergic symptoms and prevented mortality, which seemed to be directly associated with an immediate strong and long-lasting bradycardia and hypotension caused by CPA. Treatment with lower doses of CPA (0.1 and 0.05 mg/kg i.m.) caused similar levels of bradycardia and hypotension, albeit a few minutes later than at the higher doses of CPA. Upon sarin intoxication, this was correlated with increased incidence of cholinergic symptoms and decreased survival rates. Pretreatment with the peripheral adenosine A1 receptor antagonist 8- p-sulphophenyltheophylline (8-PST, 20 mg/kg i.p.) counteracted the cardiodepressant effects of 0.05 mg/kg CPA almost completely, thereby nearly abolishing its therapeutic efficacy against sarin poisoning. In conclusion, the present results strongly indicate that bradycardia and hypotension induced by the peripheral adenosine A1 receptor play a prominent role in the therapeutic efficacy of CPA in cases of sarin poisoning.

Selective effects of carbamate pesticides on rat neuronal nicotinic acetylcholine receptors and rat brain acetylcholinesterase.

Effects of commonly used carbamate pesticides on rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. The potencies of these effects have been compared to the potencies of the carbamates to inhibit rat brain acetylcholinesterase. The potency order of six carbamates to inhibit alpha4beta4 nicotinic receptors is fenoxycarb > EPTC > carbaryl, bendiocarb > propoxur > aldicarb with IC50 values ranging from 3 microM for fenoxycarb to 165 microM for propoxur and >1 mM for aldicarb. Conversely, the potency order of these carbamates to inhibit rat brain acetylcholinesterase is bendiocarb > propoxur, aldicarb > carbaryl > EPTC, fenoxycarb with IC50 values ranging from 1 microM for bendiocarb to 17 microM for carbaryl and > mM for EPTC and fenoxycarb. The alpha4beta2, alpha3beta4, and alpha3beta2 nicotinic acetylcholine receptors are inhibited by fenoxycarb, EPTC, and carbaryl with potency orders similar to that for alpha4beta4 receptors. Comparing the potencies of inhibition of the distinct subtypes of nicotinic acetylcholine receptors shows that the alpha3beta2 receptor is less sensitive to inhibition by fenoxycarb and EPTC. The potency of inhibition depends on the carbamate as well as on a combination of alpha and beta subunit properties. It is concluded that carbamate pesticides affect different subtypes of neuronal nicotinic receptors independently of acetylcholinesterase inhibition. This implicates that neuronal nicotinic receptors are additional targets for some carbamate pesticides and that these receptors may contribute to carbamate pesticide toxicology, especially after long-term exposure.

Cyclopentyladenosine and some of its low-efficacy derivatives inhibit striatal synaptosomal release of acetylcholine to a similar degree.

The application of adenosine A(1) receptor agonists in regard to cerebral disorders is hampered by serious cardiovascular side effects. This problem might be circumvented by using low-efficacy agonists (partial agonists). The objective of the present study was to characterize the effects of the full agonist N(6)-cyclopentyladenosine (CPA) and its low-efficacy derivatives 3'-deoxy-CPA (3-DCPA), 8-propylamino-CPA (8-PCPA) and 8-butylamino-CPA (8-BCPA) on the 4-aminopyridine (4AP)-evoked release of [3H]-acetylcholine in a rat striatal synaptosomal system. The reason for studying these partial agonists in particular was their established low cardiovascular side effect profile. CPA reached a concentration-dependent maximal inhibition of the evoked acetylcholine release of 38+/-3%. 3-DCPA and 8-PCPA inhibited the acetylcholine release by 29+/-5% and 38+/-3%, respectively. On the other hand, 8-BCPA only diminished the acetylcholine release by 19+/-3%. This inhibitory effect was reversible upon coadministration of the nonselective adenosine antagonist theophylline, but not by the selective adenosine A(2A) receptor antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261). It is concluded that some partial adenosine A(1) receptor agonists behave as full agonists with respect to the inhibition of acetylcholine release, while lacking profound cardiovascular side effects. These preliminary results encourage further investigation of their tissue selectivity and therapeutic potential in vivo.

Characterization of the pharmacokinetics, brain distribution, and therapeutic efficacy of the adenosine A1 receptor partial agonist 2'-deoxy-N6-cyclopentyladenosine in sarin-poisoned rats.

The objective of the present study was to determine (1) the influence of sarin poisoning (144 microg/kg s.c.) on the pharmacokinetics and brain distribution of the adenosine A1 receptor partial agonist 2'-deoxy-N6-cyclopentyladenosine (2'dCPA), and (2) the effect of 2'dCPA (20 mg/kg i.v.) on the central acetylcholine (ACh) release and protection against sarin toxicity. A five-compartment model successfully described the pharmacokinetic profile of 2'dCPA in blood and brain microdialysate. A covariate analysis revealed that the volume of distribution of 2'dCPA in blood was different in sarin-poisoned rats, 177 +/- 7 versus 148 +/- 8 ml in control rats. However, the transport of 2'dCPA from blood to the brain was unaffected as reflected by the values of the intercompartmental transport clearances, 0.21 +/- 0.02 and 0.21 +/- 0.04 microl/min in control and sarin-poisoned rats, respectively. Also the area-under-curve (AUC) ratios of brain microdialysate and blood were identical with values of 0.02 +/- 0.001 and 0.02 +/- 0.002, respectively, demonstrating the restricted transport of 2'dCPA into the brain in both treatment groups. Treatment of sarin-poisoned rats by 2'dCPA did not adequately prevent the accumulation of ACh in the central nervous system. 2'dCPA delayed the emergence of concomitant symptoms compared to untreated rats, but eventually only 29% of the animals survived 24 h. In conclusion, the pharmacokinetic profile of 2'dCPA in blood was slightly changed by sarin, but not the distribution of 2'dCPA into the brain. The therapeutic efficacy of 2'dCPA against sarin was limited, presumably due to insufficient quantities of 2'dCPA reaching the brain.

Partial adenosine A(1) receptor agonists inhibit sarin-induced epileptiform activity in the hippocampal slice.

Organophosphate poisoning can result in seizures and subsequent neuropathology. One possible therapeutic approach would be to employ adenosine A(1) receptor agonists, which have already been shown to have protective effects against organophosphate poisoning. Using an in vitro model of organophosphate-induced seizures, we have investigated the ability of several adenosine A(1) receptor agonists to inhibit epileptiform activity induced by the organophosphate sarin, in the CA1 stratum pyramidale of the guinea pig hippocampal slice. Application of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) or the partial adenosine A(1) receptor agonists 2-deoxy-N(6)-cyclopentyladenosine (2-deoxy-CPA) and 8-butylamino-N(6)-cyclopentyladenosine (8-butylamino-CPA) abolished epileptiform activity in a concentration-related manner. The rank order of potency was CPA (IC(50) 4-5 nM) >2-deoxy-CPA (IC(50) 113-119 nM)=8-butylamino-CPA (IC(50) 90-115 nM). These data suggest that partial adenosine A(1) receptor agonists, which have fewer cardiovascular effects, should be further evaluated in vivo as potential treatments for organophosphate poisoning.

Low efficacy adenosine A1 agonists inhibit striatal acetylcholine release in rats improving central selectivity of action.

The objective of this study was to characterize the effects of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) and its low efficacy derivatives 2'-deoxy-CPA (2DCPA), 3'-deoxy-CPA (3DCPA), 8-ethylamino-CPA (8ECPA) and 8-butylamino-CPA (8BCPA) on the release of acetylcholine (ACh) using intrastriatal microdialysis. These low efficacy agonists exhibited lower effects on the cardiovascular system than CPA. A concentration-dependent inhibition of ACh release was observed with a maximum of 60.5+/-2.4% for CPA, 42.5+/-2.3% for 2DCPA, 45.3+/-5.8% for 3DCPA, 57.1+/-1.4% for 8ECPA and 93.1+/-10.9% for 8BCPA, respectively. This effect was counteracted by the adenosine A(1) receptor antagonist 8-cyclopentyltheophylline. These findings show that low efficacy adenosine A(1) agonists inhibit striatal ACh release equally effective as CPA, suggesting that central nervous system-selective actions can be obtained with these compounds.

Adenosine A1 receptor agonist N6-cyclopentyladenosine affects the inactivation of acetylcholinesterase in blood and brain by sarin.

The objective of the present study was to develop a kinetics of pharmacodynamics model to properly describe and investigate the in vivo interaction between the selective adenosine A(1) agonist N(6)-cyclopentyladenosine (CPA), acetylcholinesterase (AChE) in blood and brain, and the AChE-inhibitor sarin (isopropylmethylphosphonofluoridate). The direct interaction of CPA (2 microM) on the inhibition of AChE by sarin was studied in vitro in heparinized rat blood and in 10% (w/v) brain homogenate. CPA did not directly influence the sarin-mediated inactivation of AChE in either system. In sarin-poisoned (144 microg/kg s.c.) rats not treated with CPA, AChE was completely inactivated in blood and brain within 7 min. CPA (2 mg/kg i.m.) treatment, 1 min after sarin administration, caused a small delay in the inhibition of AChE in blood. Treatment with CPA, 2 min before sarin, protected the neuronal AChE partially from being inhibited, but not the enzyme localized in blood. With a dose-response-time model the proportion of the dose of sarin reaching the site of action was estimated to be 48 +/- 12 or 13 +/- 3% after CPA post- or pretreatment, respectively. A correlation between the residual AChE activity in the brain and the incidence of cholinergic symptoms could be established with logistic regression analysis: lower inhibition of AChE in the brain precluded the onset of critical symptoms. In conclusion, CPA affects the concentration of sarin reaching the site of action, which contributes to the protection previously observed in sarin-poisoned rats.

Therapeutic efficacy of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) against organophosphate intoxication.

The objective of the present study was to investigate whether reduction of central acetylcholine (ACh) accumulation by adenosine receptor agonists could serve as a generic treatment against organophosphate (OP) poisoning. The OPs studied were tabun ( O-ethyl- N-dimethylphosphoramidocyanidate), sarin (isopropylmethylphosphonofluoridate), VX ( O-ethyl- S-2-diisopropylaminoethylmethylphosphonothiolate) and parathion ( O, O-diethyl- O-(4-nitrophenyl)phosphorothioate). The efficacy of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) against an OP intoxication was examined on the basis of the occurrence of clinical symptoms that are directly associated with such intoxication. CPA (1-2 mg/kg) effectively attenuated the cholinergic symptoms and prevented mortality in lethally tabun- or sarin-intoxicated rats. In contrast, CPA (2 mg/kg) proved to be ineffective against VX or parathion intoxication. Intracerebral microdialysis studies revealed that survival of sarin-poisoned and CPA-treated animals coincided with a minor elevation of extracellular ACh concentrations in the brain relative to the baseline value, whereas an 11-fold increase in transmitter levels was observed in animals not treated with CPA. In VX-intoxicated rats, however, the ACh amounts increased 18-fold, irrespective of treatment with CPA. The striatal acetylcholinesterase (AChE) activity following a lethal sarin intoxication was completely abolished in the vehicle-treated animals, whereas 10% and 60% AChE activity remained in animals treated with 2 mg/kg CPA 1 min after or 2 min prior to the poisoning, respectively. In VX-intoxicated animals the AChE activity in the brain was strongly reduced (striatum 10%, hippocampus 1%) regardless of the CPA treatment. These results demonstrate that CPA is highly effective against tabun or sarin poisoning, but fails to protect against VX or parathion. Survival and attenuation of clinical signs in tabun- or sarin-poisoned animals are associated with a reduction of ACh accumulation and with protection of AChE activity in the brain.

Effects of the adenosine A1 receptor allosteric modulators PD 81,723 and LUF 5484 on the striatal acetylcholine release.

The objective of the present study was to characterize the adenosine A(1) receptor allosteric enhancing and antagonistic actions of (2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)(3,4-dichlorophenyl)methanone (LUF 5484) and (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81,723) on striatal acetylcholine release. Upon local administration in conscious rats, LUF 5484 or PD 81,723 caused a concentration-dependent increase of extracellular acetylcholine levels of approximately 40%, which was similar to that obtained by the selective adenosine A(1) receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine (8CPT) and N(6)-cyclopentyl-9-methyladenine (N0840). In interaction experiments, LUF 5484 or PD 81,723 did not change the inhibition of acetylcholine release by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA), whereas 8CPT caused an eightfold rightward shift. Acetylcholine concentrations were diminished with 62+/-3%, 48+/-11% and 56+/-9% by CPA, CPA+LUF 5484 and CPA+PD 81,723, respectively. In conclusion, the antagonistic action of LUF 5484 and PD 81,723 seems to counteract the putative allosteric actions with respect to the reduction of striatal acetylcholine release.