title: Simultaneous in-vivo receptor occupancy assays for serotonin 1A, 2A, and dopamine 2 receptors with the use of non-radiolabelled tracers: Proposed method in screening antipsychotics.
abstract: BACKGROUND
  Conventionally, receptor occupancy assays employ radiolabelled tracer. However, recent advances with non-radiolabelled tracers brought a revolution in target engagement assays. Non-radiolabelled tracer based receptor occupancy uses LC-MS/MS based quantification. It offers simultaneous quantification of more than one tracer; thus, provides the feasibility of evaluating multiple targets in a single animal. In the present study, we demonstrated simultaneous measurement of serotonin 1A, serotonin 2A, and dopamine 2 receptor occupancy using non-radiolabelled tracers in rats.
  
  
  METHODS
  Non-radiolabelled WAY-100635 or MDL-100,907 or raclopride were used as tracers for 5-HT1A, 5-HT2A, and D2receptors, respectively. In-vivo brain distribution of these tracers was measured after administration as individual or as a mixture of tracers (cocktail tracer). Similarly, in-vitro brain free fractions were evaluated with the single and cocktail tracer in brain homogenates. The mass spectrometer was used for simultaneous quantification of tracers in both in-vivo and in-vitro samples. A ratio method was employed for calculation of receptor occupancy after single and cocktail tracer administration. Pindolol, olanzapine, and ziprasidone were used as tool compounds for demonstrating receptor occupancy at 5-HT1A, 5-HT2A, and D2receptors.
  
  
  RESULTS
  In optimization studies, regional distribution and concentration ratios (specific to non-specific) of these tracers were unaltered with individual and cocktail tracer. Non-significant variability was observed in brain free fraction of tracers' indicating the minimal binding interactions in this tracer combination. The half-maximal effective dose (ED50) for pindolol (5-HT1A1.37 & 2.42mg/kg, i.v.), olanzapine (5-HT2A1.37 & 2.12 and D21.90 & 2.72mg/kg, p.o.), and ziprasidone (5-HT1A10.92 & 9.57; 5-HT2A0.03 & 0.04 and D20.11 & 0.08mg/kg, i.v.) were comparable with individual or cocktail tracer.
  
  
  CONCLUSIONS
  The present study demonstrated the utility of non-radiolabelled tracers in simultaneous measurement of multiple target engagement. Use of this method will minimize the time, in addition to the cost in translational research.
authors.affiliation: Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad, India.. Electronic address: jagadeesh_thentu@suven.com.
authors.forename: Jagadeesh Babu
authors.initials: JB
authors.lastname: Thentu
authors.affiliation: Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad, India.. Electronic address: nvsrk@suven.com.
authors.forename: Ramakrishna
authors.initials: R
authors.lastname: Nirogi
authors.affiliation: Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad, India.. Electronic address: gopi@suven.com.
authors.forename: Gopinadh
authors.initials: G
authors.lastname: Bhyrapuneni
authors.affiliation: Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad, India.. Electronic address: devender@suven.com.
authors.forename: Devender Reddy
authors.initials: DR
authors.lastname: Ajjala
authors.affiliation: Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad, India.. Electronic address: raghupathi@suven.com.
authors.forename: Raghupathi Reddy
authors.initials: RR
authors.lastname: Aleti
authors.affiliation: Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad, India.. Electronic address: raghava_palacharla@suven.com.
authors.forename: Raghava Choudary
authors.initials: RC
authors.lastname: Palacharla
mesh_terms: D000818:Animals; D014150:Antipsychotic Agents / Q000378:metabolism* / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D018492:Dopamine Antagonists / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D008297:Male; D011485:Protein Binding / Q000502:physiology; D011849:Radioactive Tracers; D051381:Rats; D017208:Rats, Wistar; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism*; D044402:Receptor, Serotonin, 5-HT2A / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D014150:Antipsychotic Agents; D018492:Dopamine Antagonists; D011849:Radioactive Tracers; D044402:Receptor, Serotonin, 5-HT2A; D017448:Receptors, Dopamine D2; D012702:Serotonin Antagonists; D044282:Receptor, Serotonin, 5-HT1A
keywords: 5HT1A; 5HT2A; Acetic acid (PubChem CID: 176); Cocktail tracer; D2; Dimethyl sulfoxide (PubChem CID: 679); Hydroxypropyl beta-cyclodextrin (PubChem CID: 44134771); LC-MS/MS; MDL-100,907 (PubChem CID: 60858); Non-radiolabelled tracer; Occupancy; Olanzapine (PubChem CID: 4585); Pharmasolve-d3 (PubChem CID: 71750536); Pindolol (PubChem CID: 4828); Raclopride (PubChem CID: 3033769); Saline (PubChem CID: 5234); WAY-100635 (PubChem CID: 5684); Ziprasidone (PubChem CID: 60854)