Albert Enz

In vivo detection of amyloid-β deposits by near-infrared imaging using an oxazine-derivative probe

As Alzheimers disease pathogenesis is associated with the formation of insoluble aggregates of amyloid β-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices. Quantitative analysis revealed increasing fluorescence signal intensity with increasing plaque load of the animals, and significant binding of AOI987 was observed for APP23 transgenic mice aged 9 months and older. Thus, AOI987 is an attractive probe to noninvasively monitor disease progression in animal models of Alzheimer disease and to evaluate effects of potential Alzheimer disease drugs on the plaque load.

The selective nicotinic acetylcholine receptor α7 agonist JN403 is active in animal models of cognition, sensory gating, epilepsy and pain

Several lines of evidence suggest that the nicotinic acetylcholine receptor alpha7 (nAChR alpha7) is involved in central nervous system disorders like schizophrenia and Alzheimers disease as well as in inflammatory disorders like sepsis and pancreatitis. The present article describes the in vivo effects of JN403, a compound recently characterized to be a potent and selective partial nAChR alpha7 agonist. JN403 rapidly penetrates into the brain after i.v. and after p.o. administration in mice and rats. In the social recognition test in mice JN403 facilitates learning/memory performance over a broad dose range. JN403 shows anxiolytic-like properties in the social exploration model in rats and the effects are retained after a 6h pre-treatment period and after subchronic administration. The effect on sensory inhibition was investigated in DBA/2 mice, a strain with reduced sensory inhibition under standard experimental conditions. Systemic administration of JN403 restores sensory gating in DBA/2 mice, both in anaesthetized and awake animals. Furthermore, JN403 shows anticonvulsant potential in the audiogenic seizure paradigm in DBA/2 mice. In the two models of permanent pain tested, JN403 produces a significant reversal of mechanical hyperalgesia. The onset was fast and the duration lasted for about 6h. Altogether, the present set of data suggests that nAChR alpha7 agonists, like JN403 may be beneficial for improving learning/memory performance, restoring sensory gating deficits, and alleviating pain, epileptic seizures and conditions of anxiety.

The pharmacological assessment of RS 86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion-hydrobromide). A potent, specific muscarinic acetylcholine receptor agonist

The effects of the compound RS 86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion hydrobromide) in a number of in vitro and in vivo test systems for muscarinic cholinergic activity were analyzed and compared to those of classical muscarinic receptor agonists. In radioligand binding assays RS 86 presented high nanomolar apparent affinity only for sites labeled by 3H-muscarinic receptor agonists while its apparent affinity for sites labeled by 3H-muscarinic receptor antagonists including [3H]QNB, [3H]NMS and [3H]pirenzepine was in the micromolar range. RS 86 had no or only low affinity (IC50 greater than 10 microM) for other neurotransmitter or drug receptor sites. The compound induced scopolamine-sensitive contractions of the isolated guinea-pig ileum showing a pD2 of 6 in this model. In the isolated rat superior cervical ganglion RS 86 was also an agonist with a pD2 of 6.7. When given to mice or rats by different routes RS 86 induced central and peripheral effects typical of a muscarinic receptor agonist, such as hypothermia, tremor, mydriasis, salivation, lacrimation, diarrhoea and modification of behavior as observed in an open field. In several of these tests RS 86 was about 10 times less potent than oxotremorine but more potent than arecoline, pilocarpine, aceclidine or the compound (cis) AF-30. The ED50 values for some central effects, including the induction of hypothermia and alert non-mobile behavior were lower than those for tremor and peripheral effects. Some of the effects lasted for up to 6 h, depending on the dose. Finally, RS 86 administration resulted in modifications of brain acetylcholine turnover and high affinity choline uptake typical of a central muscarinic receptor agonist. Taken together these results demonstrate clearly that RS 86 is a potent, centrally acting, selective muscarinic receptor agonist. RS 86 appears to be an adequate tool for the clinical examination of the cholinergic hypothesis of Alzheimers disease.

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Absence of receptor reserve at striatal dopamine receptors regulating cholinergic neuronal activity

N-propylnorapomorphine (NPA) dose dependently increased rat striatal ACh levels (ED50 = 18 micrograms/kg). After irreversible dopamine (DA) receptor inactivation with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), neither the ED50 nor the slope of the dose-response curve for NPA was altered, whereas the maximal response was reduced to 56% of control. Analysis of the results yielded a linear relationship between receptor occupancy and response (i.e. no receptor reserve). In contrast, DA autoreceptors display a large receptor reserve for NPA. The results support the hypothesis that a differential receptor reserve at pre- vs. postsynaptic DA receptors in rat striatum accounts for the autoreceptor selectivity of DA agonists.

Macrocyclic BACE-1 inhibitors acutely reduce Aβ in brain after po application

A series of macrocyclic peptidic BACE-1 inhibitors was designed. While potency on BACE-1 was rather high, the first set of compounds showed poor brain permeation and high efflux in the MDRI-MDCK assay. The replacement of the secondary benzylamino group with a phenylcyclopropylamino group maintained potency on BACE-1, while P-glycoprotein-mediated efflux was significantly reduced and brain permeation improved. Several compounds from this series demonstrated acute reduction of Abeta in human APP-wildtype transgenic (APP51/16) mice after oral administration.

SDZ ENS 163, a selective muscarinic M1 receptor agonist, facilitates the induction of long-term potentiation in rat hippocampal slices.

The effect of SDZ ENS 163; (+)-(3S,cis)-3-ethyldihydro-4-[(1-methyl-1H-imidazol-5-yl)methyl-2 (3H)- thiphenonedihydrogenphosphate], a selective muscarinic M1 agonist, on long-term potentiation (LTP) was studied in a rat hippocampal slice preparation. LTP was induced by theta-burst stimulation (TBS) delivered to the Schaffer/commissural fibers. In untreated slices delivery of 8 or 10 trains at 100 Hz induced a 27 +/- 8.3 and 54 +/- 7.2% potentiation of the amplitude of the excitatory postsynaptic potential (epsp), respectively (calculated as percentage of the pre-LTP amplitude). In slices pretreated with SDZ ENS 163 (2 x 10(-6) M, -30 min) delivery of 8 or 10 trains at 100 Hz induced a 62 +/- 8.4 and 54 +/- 7.1% potentiation of the epsp amplitude, respectively. In addition, treatment with SDZ ENS 163 (2 x 10(-6) M) increased the N-methyl-D-aspartate receptor-induced component of the epsp response to TBS from 21 +/- 3 (control) to 33 +/- 2%. Pretreatment with the muscarinic antagonist, scopolamine (6 x 10(-8) M), or with the M1 selective muscarinic receptor antagonist, pirenzepine (6 x 10(-8) M), did not affect LTP in untreated slices but inhibited the enhancement of LTP by SDZ ENS 163 (2 x 10(-6) M) completely. AF-DX 116 (10(-6) M, -60 min), a selective muscarinic M2 receptor antagonist did not affect LTP in control slices nor in slices treated with SDZ ENS 163 (2 x 10(-6) M). These data suggest that activation of muscarinic M1 receptors by SDZ ENS 163 facilitates the induction of LTP.

Co-administration of memantine has no effect on the in vitro or ex vivo determined acetylcholinesterase inhibition of rivastigmine in the rat brain

Rivastigmine, a cholinesterase inhibitor, is successfully used for the symptomatic therapy of Alzheimers disease (AD) in the clinic. The drug has a very low potential for drug-drug interactions, as has been demonstrated within large clinical trials. Memantine, recently approved by the FDA for the treatment of moderate to severe AD, acts as a low affinity, non-competitive NMDA-antagonist, on a completely different neurotransmitter system, the glutamatergic system. Given the different sites of action, the possibility to combine a cholinergic with a glutamatergic intervention as potentially superior AD therapy has recently been proposed. In vitro studies have demonstrated that memantine, when added to reversible AChE inhibitors, such as tacrine, donepezil or galantamine, did not interfere with the inhibitory action of any of these drugs. The results from the present study provide evidence that rivastigmine as a pseudo-irreversible (or slow-reversible) AChE inhibitor shares this property described for reversible inhibitors, since memantine (1-100 microM), irrespective of whether given prior to or after rivastigmine did not influence rivastigmines AChE inhibition in vitro. A similar observation was also made under in vivo conditions (ex vivo measurements): following a 21 day chronic, oral administration of 6 micromol/kg rivastigmine alone or of a combination of rivastigmine plus memantine (6 micromol/kg p.o. of either of the two compounds), an identical degree of AChE inhibition was observed. The concentrations of rivastigmine, its metabolite NAP 226-90 and memantine were measured in the brain of the same animals. Following an equimolar oral dose (6 micromol/kg) of both compounds, the brain level of memantine exceeded that of rivastigmine + metabolite, by a factor of around 30, when measured 2 h after the final dosing, irrespective of the duration of treatment (acute, for 3 or 21 days). This indicates that neither of the two drugs showed accumulation but also, and more importantly, that memantine does not modulate the prime therapeutic action of rivastigmine (AChE inhibition) in vitro or in vivo. Clinical trials using a combination of both drugs will provide a final proof of whether a combination therapy would lead to an increased efficacy in AD patients.

AFQ056/mavoglurant, a novel clinically effective mGluR5 antagonist: identification, SAR and pharmacological characterization.

Here we describe the identification, structure-activity relationship and the initial pharmacological characterization of AFQ056/mavoglurant, a structurally novel, non-competitive mGlu5 receptor antagonist. AFQ056/mavoglurant was identified by chemical derivatization of a lead compound discovered in a HTS campaign. In vitro, AFQ056/mavoglurant had an IC50 of 30 nM in a functional assay with human mGluR5 and was selective over the other mGluR subtypes, iGluRs and a panel of 238 CNS relevant receptors, transporter or enzymes. In vivo, AFQ056/mavoglurant showed an improved pharmacokinetic profile in rat and efficacy in the stress-induced hyperthermia test in mice as compared to the prototypic mGluR5 antagonist MPEP. The efficacy of AFQ056/mavoglurant in humans has been assessed in L-dopa induced dyskinesia in Parkinsons disease and Fragile X syndrome in proof of principle clinical studies.

7-Oxa[2.2.1]bicycloheptane-2,3-dicarboxylic acid derivatives as phosphatase inhibitors

Abstract Cantharidin (1) as well as norcantharidin or endothall (2) have been described to be inhibitors of protein phosphatase 2A, but were reported to be inactive as inhibitors of calcineurin. Using an HPLC-assay, we have discovered that 1 and 2 are inhibitors of calcineurin-dependent (appKi = 10.8 and 3.26 μM, respectively) dephosphorylation of a phosphopeptide derived from the RII subunit of c-AMP dependent kinase, H-DLDVPIPGRFDRRVS(PO4)VAAE-OH (7), which is commonly used in calcineurin assays. The phosphatase inhibitor 6 also inhibits the production of IL-2 in Jurkat cells, a calcineurin dependent process, with IC50 = 4.7 μM, indicating that some of the biological activity of this substance class may be due to calcineurin inhibition, in addition to the established PP2A inhibition.