Haloperidol

Clozapine was provided as a generous gift to J.H.P. from Novartis (Hanover, NJ, USA). Olanzapine was provided as a generous gift to J.H.P. from Eli Lilly (Indianapolis, IN, USA). Clozapine, olanzapine, and risperidone were supplied to D.W. by the National Institute of Mental Health’s Chemical Synthesis and Drug Supply Program. Haloperidol, prazosin, propranolol, and ritanserin were purchased from Sigma-Aldrich (St. Louis, MO, USA). J.H.P. and M.S.F. obtained CNO from the Rapid Access to Investigative Drug Program funded by the National Institute of Neurological Disorders and Stroke. D.W. obtained CNO from the National Institute on Drug Abuse Drug Supply Program.
Clozapine, olanzapine, risperidone, haloperidol, prazosin, propranolol, and ritanserin were each dissolved in distilled water with 2–3 drops of lactic acid and pH-adjusted to 6.0–7.0 with NaOH. For mouse drug discrimination studies, CNO was also dissolved in this vehicle. For rat drug discrimination studies and for mouse and rat pharmacokinetic analyses, CNO was dissolved in bacteriostatic saline containing v/v 2.5–5.0% dimethyl sulfoxide (Sigma-Aldrich) and 10% Cremophor EL (Sigma-Aldrich).
For mouse drug discrimination studies, all drugs were administered s.c. at a volume of 10 ml/kg, 30 min prior to session onset. For rat drug discrimination studies, all drugs were administered i.p. at a volume of 1 ml/kg. Clozapine was administered 60 min prior to session onset, while olanzapine, risperidone, prazosin, and propranolol were administered 30 min prior to session onset. CNO was tested at both 30 and 60 min pretreatment times. All drug doses are expressed as the salt weight.

Retrospective results from TDM analysis of antidepressants and antipsychotics were extracted from the LIMS system of the three participating Danish laboratories. These were the Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus (AUH); the Laboratory of the Danish Epilepsy Centre–Filadelfia, Dianalund (EHL); and the Department of Clinical Biochemistry, Rigshospitalet (RH), Copenhagen. Approvals were obtained from the respective hospital boards, prior to downloading the data.
Therapeutic drug monitoring at AUH is carried out by using LC-MS/MS technology and assays that have been developed in-house. The analyses are all accredited according to ISO:15189:2013, and the quality is externally monitored by proficiency testing. Results were extracted for the following drugs covering a period from 1 January 2014 to 31 December 2018: amitriptyline/nortriptyline (metabolite), aripiprazole/dehydroaripiprazole (metabolite), citalopram/escitalopram, clomipramine/desmethylclomipramine (metabolite), clozapine, duloxetine, fluoxetine/norfluoxetine (metabolite), imipramine/desipramine (metabolite), mirtazapine, olanzapine, perphenazine, quetiapine, risperidone/paliperidone (metabolite), sertraline, venlafaxine/o-desmethyl-venlafaxine (metabolite), ziprasidone, and zuclopenthixol.
All the assays performed at EHL have been developed in-house by using LC-MS/MS technology. These include the same drugs as are analysed at AUH and in addition flupentixol, haloperidol, and paroxetine. Although part of the laboratory production, fluoxetine/norfluoxetine, mianserine, and ziprasidone were excluded, owing to a low number of samples (<100). The assays (n = 26) are accredited according to ISO15189:2013, with the exception of aripiprazole/dehydroaripiprazole, and mirtazapine. The quality is externally monitored by proficiency-testing programmes, covering all analytes. For this study, data were collected from the laboratory LIMS system spanning a period from 1 January 2012 to 30 March 2022.
The analyses for therapeutic drugs at RH is performed by HPLC using UV-detection. The following drugs are included in the laboratory repertoire: amitriptyline/nortriptyline, clomipramine/desmethylclomipramine, clozapine, dosulipine/northiaden, and imipramine/desipramine, of which amitriptyline, nortriptyline, clozapine, and imipramine/desipramine are accredited according to ISO 15189:2013. The quality of the assays is monitored by external proficiency-testing schemes. For the calculations, data were collected covering the period from 9 May 2011 to 26 April 2022.

The S1R model protein was taken from PDB entry 6DJZ, S1R with haloperidol. The protein is a trimeric membrane protein containing a cupin fold [50 (link)] with each protomer bound to haloperidol and an N-terminal alpha helix that is inserted into the membrane. First, the protein was prepared for docking using the protein preparation tools of Sybyl (Tripos Corp). Missing side-chains were added in structurally reasonable positions. None of these were in the active site. The haloperidol was removed and hydrogens were added. The two carboxylates in the active site, Aps126 and Glu172, were in the deprotonated state. Minor side-chain clashes were repaired visually. Gatseiger–Huckel charges were added and the structure energy-minimized in steps: hydrogens alone, then side-chains and backbone, followed by the whole molecule. The Tripos force field was used with Simplex initial optimization, followed by Powell and terminated when the gradient reached 0.05 Kcal/mole. The molecule was then removed and used for docking experiments.
Dockings were performed with Autodock4 [106 (link)] utilizing AutodockTools. A box of approximate dimensions 30 × 30 × 30 angstroms was centered in the cupin pocket and included the majority of the pocket as well as the alpha 4 and alpha 5 helices that are juxtaposed on the membrane surface. This is including His154, Asp126, and Glu172. Both (+) pentazocine, an agonist, and haloperidol, an antagonist, were used as controls to validate that the docking procedure was correct. Haloperidol, with a proton on the nitrogen and, thus, positively charged, was found to dock in a similar way as found in the crystal structure 6DJZ with the nitrogen hydrogen-bonded to Glu172. Autodock produced the top 30 lowest energies and clustered them into groups. The docking reproduced the crystal structure in the top 10 best (lowest energy) examined. Initially, (+)-pentazocine would not dock into 6DJZ, because it interfered with Ala185 in helix 4. When helix 4 was moved slightly and energy-minimized, (+)-pentazocine docked as found in the crystal structure 6DK1, with the top 10 best dockings reproducing the interactions of the positively charged ring nitrogen interacting with Glu172. In both control compounds, Asp126 was deprotonated. The Autodock scoring is an addition of 4 intermolecular-contributing Kcal/mol energy terms as the sum of Intermolecular Energy (VdW + Hbond + desolvation + electrostatics), plus total internal energy, plus torsional energy, minus the unbound system energy. The comparison docking of (+)-pentazocine and SPH is shown in Figure 6. The positively charged nitrogen hydrogen bonded to the Glu172 as expected. In some of the less energetic dockings, the Tyr120 hydroxyl proton is involved in interactions, but Tyr103 hydroxyl is donated to the carbonyl in the interior of the binding site, stabilizing the orientation of the carboxylate on Glu172. The docking of SPH and DMS was performed under the same. The two endogenous sphingolipids, SPH and DMS, docked similarly to the control (+)-pentazocine in 6DJZ S1R. In all dockings, the protein was static and the ligand was randomly torsioned and then docked into the protein in the defined box.