AF-DX 384

The binding of [3H]AF-DX 384 is reduced in the caudate-putamen of subjects with schizophrenia.[Pubmed:10353630]

Life Sci. 1999;64(19):1761-71.

Clinical studies of cholinergic pharmacotherapy, together with the putative role of the muscarinic receptor system in the neurophysiology of human behavior, support a possible muscarinic cholinergic involvement in schizophrenia. The present study has measured the density of [3H]AF-DX 384 labelled receptors (muscarinic M2 and M4) in the caudate-putamen, obtained at autopsy, from 19 subjects who had schizophrenia, and 20 subjects who did not have schizophrenia. [3H]AF-DX 384 binding was reduced in caudate-putamen from schizophrenic subjects (104 +/- 10.3 vs 145 +/- 901 fmol mg(-1) TE; mean +/- s.e.; p = 0.007). Preliminary analysis of patient drug data as well as rat studies suggest that the reduced [3H]AF-DX 384 binding in caudate-putamen of schizophrenic subjects is not wholly due to antipsychotic drug treatment, or anticholinergic medication for the treatment of extrapyramidal effects. These data suggest that the muscarinic cholinergic system may be involved in the pathology of schizophrenia.

Development of a new type of allosteric modulator of muscarinic receptors: hybrids of the antagonist AF-DX 384 and the hexamethonio derivative W84.[Pubmed:15163212]

J Med Chem. 2004 Jun 3;47(12):3324-7.

Various fragments of the hexamethonio-type allosteric agent W84 were linked to the secondary amino group of the muscarinic M(2) acetylcholine receptor-preferring antagonist AF-DX 384 to increase the area of attachment with the allosteric site. Addition of only the phthalimido moiety of W84 gave an allosteric enhancer of NMS binding. Thus, a new lead structure for the development of allosteric enhancers of NMS binding has been discovered.

Comparative distribution of binding of the muscarinic receptor ligands pirenzepine, AF-DX 384, (R,R)-I-QNB and (R,S)-I-QNB to human brain.[Pubmed:12297267]

J Chem Neuroanat. 2002 Sep;24(3):211-23.

Quinuclidinyl benzilate (QNB) and its derivatives are being developed to investigate muscarinic receptor changes in vivo in Alzheimer's disease and dementia with Lewy bodies. This is the first study of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB binding in vitro in human brain. We have compared the in vitro binding of the muscarinic ligands [3H]pirenzepine and [3H]AF-DX 384, which have selectivity for the M1 and M2/M4 receptor subtypes, respectively, to the binding of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB. This will provide a guide to the interpretation of in vivo SPET images generated with [123I]-(R,R)-I-QNB and [123I]-(R,S)-I-QNB. Binding was investigated in striatum, globus pallidus, thalamus and cerebellum, and cingulate, insula, temporal and occipital cortical areas, which show different proportions of muscarinic receptor subtypes, in post-mortem brain from normal individuals. M1 receptors are of high density in cortex and striatum and are relatively low in the thalamus and cerebellum, while M4 receptors are mainly expressed in the striatum, and M2 receptors are most evident in the cerebellum and thalamus. [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB density distribution patterns were consistent with binding to both M1 and M4 receptors, with [125I]-(R,R)-I-QNB additionally binding to a non-cholinergic site not displaceable by atropine. This distribution can be exploited by in vivo imaging, developing ligands for both SPET and PET, to reveal muscarinic receptor changes in Alzheimer's disease and dementia with Lewy bodies during the disease process and following cholinergic therapy.

Syntheses of R and S isomers of AF-DX 384, a selective antagonist of muscarinic M2 receptors.[Pubmed:10732976]

Bioorg Med Chem. 2000 Mar;8(3):591-600.

Enantiomers of 5,11-dihydro-11-[2-[2-[(N,N-dipropylaminomethyl)piperidin-1- yl]ethylamino]-carbonyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (AF-DX 384) 1, have been synthesized from (S)-(+) and (R)-(-)-2-[N,N-dipropylaminomethyl]piperidine 4. The enantiomeric excess of 1 has been determined by capillary electrophoresis by using the alpha-highly sulphated cyclodextrin (alpha-HSCD) as chiral selector within the running electrolyte. (S)-(+)-(4) was prepared from (S)-(-)-pipecolic acid in a 4-step procedure (overall yield: 30%, ee: 99%) and (R)-(-)-AF-DX 384 from (R)-(+)-pipecolic acid. The (R)-(-) isomer exhibited in vitro a 23-fold higher affinity than its enantiomer (S)-(+) towards muscarinic receptors of subtype 2.

AF-DX 384 binding in rabbit cingulate cortex: two site kinetics and section autoradiography.[Pubmed:7616446]

J Pharmacol Exp Ther. 1995 Jul;274(1):562-9.

Autoradiographic studies of muscarinic receptors are limited by the lack of selective ligands. Inasmuch as AF-DX 384 has a higher affinity for m2 than m4 receptors and pirenzepine (PZ) has a reverse affinity profile, competition between these ligands was used to label m2 receptors in homogenized and sectioned tissue. Rabbit cingulate cortex was used because m2 receptors are expressed by anterior thalamic axons in posterior cingulate cortex (PCC) and this region is easily deafferented with undercut lesions to demonstrate presynaptic binding. Saturation isotherms and Scatchard analysis of [3H]AF-DX 384 binding showed one binding site with a KD of 9 +/- 2.3 nM (mean +/- SEM) and a Bmax of 1405 +/- 146 fmol/mg protein. Competition studies with [3H]AF-DX 384 (2 nM) and 10(-10)-10(-4) M PZ were performed in anterior cingulate cortex (ACC) and PCC. In both regions, the best fit was a two site model for low (BL) and high (BH) affinity binding in which Bmax values were similar (ACC: BL = 535 +/- 62 fmol/mg, BH = 676 +/- 85; PCC: BL = 552 +/- 41; BH = 675 +/- 85). Although affinities for KH were similar in each region (ACC: KH = 4.69 +/- 1.36 nM; PCC: KH = 8.53 +/- 3.69 nM), those for KL were significantly different (ACC: 181 +/- 15.4 nM; PCC: 285 +/- 42; P = .018). Binding of [3H]AF-DX 384 with PZ (150 nM) was best fit with a single site model (KD = 6 +/- 0.01 nM; Bmax = 688 +/- 31 fmol/mg), suggesting that PZ blocks the lower affinity site. (ABSTRACT TRUNCATED AT 250 WORDS)

Binding of [3H]AF-DX 384 to cloned and native muscarinic receptors.[Pubmed:1941609]

J Pharmacol Exp Ther. 1991 Nov;259(2):601-7.

The binding selectivity of [3H]AF-DX 384 [(+-)-5,11-dihydro-11- ([(2-(2-[(dipropylamino)methyl]-1- piperidinyl)ethyl)amino]carbonyl)-H-pyrido(2,3-b)(1,4)benzodiazepine-6-o ne] was evaluated with cloned human muscarinic receptors (M1-M4) in Chinese hamster ovary (CHO-K1) cell lines as well as in rat heart and brain. There were uniform classes of sites for the radioligand in the M2-rich tissues, heart (Kd = 2.3 nM) and brainstem (Kd = 2.4 nM). However, [3H]AF-DX 384 bound to all four cloned receptor subtypes. Using kinetic methods, the calculated Kd values were M2 (1 nm) greater than M4 (2.2 nM) greater than M3 (15 nM) greater than M1 (55 nM). Scatchard analysis with the CHO cells confirmed the high affinity of this radioligand for the M2 (1.8 nM) and M4 (2.5 nM) receptors. To evaluate the potential for selectively binding to M2 and M4 receptors in cortex and striatum, low concentrations (0.5-0.8 nM) of the radioligand were used and a two-site competition model was used to derive the binding constants for pirenzepine and AF-DX 116 [(+-)-11-2((-((diethylamino) methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)-benzodiazepine-6-one] and to compare them with values obtained with cloned M2 and M4 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)