IntroductionIntact cholesterol biosynthesis is crucial for CNS homeostasis. The brain contains approximately 20-25% of all cholesterol in the human body and relies on cholesterol biogenesis that is independent of the periphery (1Korade Z. Heffer M. Mirnics K. Medication effects on developmental sterol biosynthesis.). Genetic disruptions of the sterol synthesis pathway lead to several developmental disabilities, including, but not limited to Smith-Lemli-Opitz syndrome, lathosterolosis and desmosterolosis (2Smith–Lemli–Opitz syndrome: pathogenesis, diagnosis and management., 3Allen L.B. Genaro-Mattos T.C. Porter N.A. Mirnics K. Korade Z. Desmosterolosis and desmosterol homeostasis in the developing mouse brain., 4Xie C. Turley S.D. Dietschy J.M. Cholesterol accumulation in tissues of the Niemann-pick type C mouse is determined by the rate of lipoprotein-cholesterol uptake through the coated-pit pathway in each organ.). Impaired sterol biosynthesis has been also associated with many CNS disorders including major depression, schizophrenia, Huntington’s, and Alzheimer’s diseases (5Müller C.P. Reichel M. Mühle C. Rhein C. Gulbins E. Kornhuber J. Brain membrane lipids in major depression and anxiety disorders., 6A Structure-Function Mechanism for Schizophrenia., 7Valenza M. Leoni V. Tarditi A. Mariotti C. Bjorkhem I. Di Donato S. Cattaneo E. Progressive dysfunction of the cholesterol biosynthesis pathway in the R6/2 mouse model of Huntington's disease., 8Cholesterol and the biology of Alzheimer's disease.). Unfortunately, many commonly prescribed psychotropic medications also have sterol biosynthesis inhibiting effects (1Korade Z. Heffer M. Mirnics K. Medication effects on developmental sterol biosynthesis., 9Korade Z. Kim H.-Y.H. Tallman K.A. Liu W. Koczok K. Balogh I. Xu L. Mirnics K. Porter N.A. The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and Human Fibroblasts.). Previous in vivo mouse experiments and patient biobank assessments revealed that aripiprazole, cariprazine, haloperidol, trazodone and amiodarone are strong inhibitors of post-lanosterol biosynthesis (1Korade Z. Heffer M. Mirnics K. Medication effects on developmental sterol biosynthesis., 10Tallman K.A. Allen L.B. Klingelsmith K.B. Anderson A. Genaro-Mattos T.C. Mirnics K. Porter N.A. Korade Z. Prescription Medications Alter Neuronal and Glial Cholesterol Synthesis.).ARI is an atypical antipsychotic, with >6.6M prescriptions in the US in 2019 (11

Kane, S. P. 2021. ClinCalc DrugStats Database. In. ClinCalc LLC.

). ARI is primarily utilized for treatment of schizophrenia and bipolar disorder and its beneficial effects are well documented (12A review of aripiprazole in the treatment of patients with schizophrenia or bipolar I disorder., 13The Treatment of Adult Bipolar Disorder with Aripiprazole: A Systematic Review.). TRZ is an antidepressant of the serotonin receptor antagonists and reuptake inhibitors family and it was prescribed approximately 24M times in the US in 2019 (11

Kane, S. P. 2021. ClinCalc DrugStats Database. In. ClinCalc LLC.

). The primary use of TRZ is the treatment of depression (14Fagiolini A. Comandini A. Catena Dell'Osso M. Kasper S. Rediscovering trazodone for the treatment of major depressive disorder.). However, TRZ has been also extensively utilized for off-label treatment of insomnia (15Jaffer K.Y. Chang T. Vanle B. Dang J. Steiner A.J. Loera N. Abdelmesseh M. Danovitch I. Ishak W.W. Trazodone for Insomnia: A Systematic Review., 16La A.L. Walsh C.M. Neylan T.C. Vossel K.A. Yaffe K. Krystal A.D. Miller B.L. Karageorgiou E. Long-Term Trazodone Use and Cognition: A Potential Therapeutic Role for Slow-Wave Sleep Enhancers.), opioid withdrawal symptoms (17Kurtz S.P. Buttram M.E. Margolin Z.R. Wogenstahl K. The diversion of nonscheduled psychoactive prescription medications in the United States, 2002 to 2017.), alcohol withdrawal (18Borras L. de Timary P. Constant E.L. Huguelet P. Eytan A. Successful treatment of alcohol withdrawal with trazodone., 19Le Bon O. Murphy J.R. Staner L. Hoffmann G. Kormoss N. Kentos M. Dupont P. Lion K. Pelc I. Verbanck P. Double-blind, placebo-controlled study of the efficacy of trazodone in alcohol post-withdrawal syndrome: polysomnographic and clinical evaluations., 20Roccatagliata G. Albano C. Maffini M. Farelli S. Alcohol withdrawal syndrome: treatment with trazodone.), dementia (16La A.L. Walsh C.M. Neylan T.C. Vossel K.A. Yaffe K. Krystal A.D. Miller B.L. Karageorgiou E. Long-Term Trazodone Use and Cognition: A Potential Therapeutic Role for Slow-Wave Sleep Enhancers.), fibromyalgia (21Morillas-Arques P. Rodriguez-Lopez C.M. Molina-Barea R. Rico-Villademoros F. Calandre E.P. Trazodone for the treatment of fibromyalgia: an open-label, 12-week study.) and other conditions (22Fink H.A. MacDonald R. Rutks I.R. Wilt T.J. Trazodone for erectile dysfunction: a systematic review and meta-analysis., 23Cenik B. Palka J.M. Thompson B.M. McDonald J.G. Tamminga C.A. Cenik C. Brown E.S. Desmosterol and 7-dehydrocholesterol concentrations in post mortem brains of depressed people: The role of trazodone.).Unfortunately, the side effects of ARI and TRZ have also been extensively documented (12A review of aripiprazole in the treatment of patients with schizophrenia or bipolar I disorder., 24Aripiprazole: profile on efficacy and safety., 25A review of the evidence for the efficacy and safety of trazodone in insomnia., 26Citrome L. Kalsekar I. Baker R.A. Hebden T. A review of real-world data on the effects of aripiprazole on weight and metabolic outcomes in adults., 27Hall D.A. Agarwal P. Griffith A. Segro V. Seeberger L.C. Movement disorders associated with aripiprazole use: a case series., 28Bernagie C. Danckaerts M. Wampers M. De Hert M. Aripiprazole and Acute Extrapyramidal Symptoms in Children and Adolescents: A Meta-Analysis., 29Etminan M. Procyshyn R.M. Samii A. Carleton B.C. Risk of Extrapyramidal Adverse Events With Aripiprazole., 30Pena M.S. Yaltho T.C. Jankovic J. Tardive dyskinesia and other movement disorders secondary to aripiprazole., 31Rojo L.E. Gaspar P.A. Silva H. Risco L. Arena P. Cubillos-Robles K. Jara B. Metabolic syndrome and obesity among users of second generation antipsychotics: A global challenge for modern psychopharmacology.), including their unwanted action on sterol biosynthesis: ARI and TRZ treatments lead to a strong elevation of 7-dehydrocholesterol (7-DHC) and decreased desmosterol (DES) levels in in vitro and in vivo animal models, as well as in human biomaterials (fibroblasts and plasma) (9Korade Z. Kim H.-Y.H. Tallman K.A. Liu W. Koczok K. Balogh I. Xu L. Mirnics K. Porter N.A. The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and Human Fibroblasts., 32Hall P. Michels V. Gavrilov D. Matern D. Oglesbee D. Raymond K. Rinaldo P. Tortorelli S. Aripiprazole and trazodone cause elevations of 7-dehydrocholesterol in the absence of Smith-Lemli-Opitz Syndrome., 33Korade Ž. Liu W. Warren E.B. Armstrong K. Porter N.A. Konradi C. Effect of psychotropic drug treatment on sterol metabolism., 34Korade Z. Allen L.B. Anderson A. Tallman K.A. Genaro-Mattos T.C. Porter N.A. Mirnics K. Trazodone effects on developing brain., 35Genaro-Mattos T.C. Allen L.B. Anderson A. Tallman K.A. Porter N.A. Korade Z. Mirnics K. Maternal aripiprazole exposure interacts with 7-dehydrocholesterol reductase mutations and alters embryonic neurodevelopment.). Importantly, 7-DHC is the most oxidizable lipid known to date, with the propagation rate constant 2,160 M-1s-1 (this is 200 times more than cholesterol and 10 times more than arachidonic acid) (36Lamberson C.R. Muchalski H. McDuffee K.B. Tallman K.A. Xu L. Porter N.A. Propagation rate constants for the peroxidation of sterols on the biosynthetic pathway to cholesterol.). 7-DHC spontaneously oxidizes and give rise to highly reactive 7-DHC derived oxysterols (37Xu L. Korade Z. Rosado J.D.A. Liu W. Lamberson C.R. Porter N.A. An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.). 7-DHC derived oxysterols are toxic, and affect cell viability, differentiation, and growth (38Korade Z. Xu L. Shelton R. Porter N.A. Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome., 39Pfeffer B.A. Xu L. Porter N.A. Rao S.R. Fliesler S.J. Differential cytotoxic effects of 7-dehydrocholesterol-derived oxysterols on cultured retina-derived cells: Dependence on sterol structure, cell type, and density., 40Xu L. Korade Z. Porter N.A. Oxysterols from Free Radical Chain Oxidation of 7-Dehydrocholesterol: Product and Mechanistic Studies.). One of the best characterized 7-DHC-derived oxysterols, DHCEO, has a profound effect on neuronal morphology, neurite outgrowth and fasciculation, potentially through sonic hedgehog signaling (41Xu L. Mirnics K. Bowman A.B. Liu W. Da J. Porter N.A. Korade Z. DHCEO accumulation is a critical mediator of pathophysiology in a Smith-Lemli-Opitz syndrome model.). In addition to DHCEO, there are at least twenty other 7-DHC-derived oxysterols identified to date, and several of these have been found in mouse and human SLOS samples (37Xu L. Korade Z. Rosado J.D.A. Liu W. Lamberson C.R. Porter N.A. An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome., 42Tomita H. Hines K.M. Herron J.M. Li A. Baggett D.W. Xu L. 7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome.). These 7-DHC derived oxysterols are not only markers of oxidative stress (43Korade Z. Xu L. Mirnics K. Porter N.A. Lipid biomarkers of oxidative stress in a genetic mouse model of Smith-Lemli-Opitz syndrome.) but are also biologically potent compounds capable of affecting sonic hedgehog signaling and interfering with immune response (44Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells.).Polypharmacy is a nationwide and worldwide challenge (45A dataset quantifying polypharmacy in the United States., 46Siwek M. Woron J. Gorostowicz A. Wordliczek J. Adverse effects of interactions between antipsychotics and medications used in the treatment of cardiovascular disorders., 47Miller C.H. Fleischhacker W.W. Managing antipsychotic-induced acute and chronic akathisia., 48

Cheine, M., J. Ahonen, and K. Wahlbeck. 2000. Beta-blocker supplementation of standard drug treatment for schizophrenia. Cochrane Database Syst Rev: CD000234.

). Both ARI and TRZ are commonly prescribed, often utilized together as part of the treatment plan. Yet, the sterol biosynthesis inhibiting side-effects of these two medications (alone or in combination) in adulthood have not been systematically explored to date. As a result, our study was designed to evaluate the effects of ARI, TRZ and ARI+TRZ polypharmacy on adult brain sterol biosynthesis. Post-lanosterol lipid profiling was performed using LC-MS/MS. For our experiments we utilized in vitro primary neuronal and astroglial cultures, as well as in vivo studies on adult mice. The experimental design is presented in Figure 1.

Figure 1Experimental Design. In vitro experiments: Cortical neurons and astrocytes were cultured from C57BL/6J mice (embryonic day 18 (E18) for neurons and postnatal day 2 (P2) for astrocytes), and treated with five different concentrations of ARI, TRZ or ARI+TRZ. CHOL, DES, 7-DHC, and LAN were analyzed by LC-MS/MS after 3, 6, and 9 days of treatment using the PTAD derivatization assay. In vivo experiments: Following daily exposure to either ARI (2.5mg/kg), TRZ (10mg/kg), ARI+TRZ (2.5mg/kg+10mg/kg), or vehicle (VEH) for eight days, eight brain regions of each adult mice were dissected (n=8-9/group). Sterols, ARI, TRZ and their main metabolites were measured by LC-MS/MS.

Materials and MethodsChemicals

Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich Co (St. Louis, MO). HPLC grade solvents were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). TRZ and ARI were obtained from Selleckchem (Radnor, PA) and dissolved in sterile DMSO solution for the experiments. All sterol standards, natural and isotopically labeled, used in this study are available from Kerafast, Inc. (Boston, MA).

Aripiprazole and trazodone injections in miceAdult male C57Bl/6J stock # 000664 mice, 3 months old were purchased from Jackson Laboratories. Mice were housed under a 12 h light-dark cycle at constant temperature (25°C) and humidity with ad libitum access to food (Teklad LM-485 Mouse/Rat Sterilizable Diet 7012) and water in Comparative Medicine at the University of Nebraska Medical Center (UNMC), Omaha, NE. In humans, TRZ (Desyrel) is given at a starting dose 150 mg/day; and may be increased by 50 mg per day every 3 to 4 days to a maximum dose of 400 mg per day for outpatient use. For treatment of insomnia, TRZ is given at a starting dose of 50 mg/day. If we take a dose of 50 mg/60 kg human body weight, this translates to 0.83 mg/kg/day. Animal Equivalent dose (AED in mg/kg) is calculated as AED (mg/kg) = human dose (mg/kg) 50 mg per day) x Km ratio (12.3) = 10 mg/kg (49A simple practice guide for dose conversion between animals and human.). As a result, we chose to use a low dose of 10 mg/kg in our mouse experiments, which translates back to about 50 mg/day in humans, depending on the weight of the patient. Based on these calculations and literature data, we used ARI (Abilify) at 2.5 mg/kg in our mouse experiments (which corresponds to ARI tablet 10-15 mg/day in humans). Range of doses in humans is 2 mg – 30 mg/day (). Total number of 35 adult male mice were used in our study with 9 animals assigned to each group, except for control group that had 8 mice. We applied intraperitoneal injections as a method for drug (or vehicle) delivery, every day at 8.00 am. The treatment did not influence mouse body mass for the duration of the experiment (Supplemental Figure S1). All procedures were performed in accordance with the Guide for the Humane Use and Care of Laboratory Animals. The use of mice in this study was approved by the Institutional Animal Care and Use Committee of UNMC.Tissue collection and preparation for sterol analysis

Four to six hours after the last injections, mice were euthanized with Isoflurane overdose ((Forane® isofluranum, Abbott Laboratories LTD; Lake Bluff, IL, USA). Brains were dissected and brain regions were frozen in pre-chilled methyl-butane and stored at -80°C. Frozen samples were sonicated in ice-cold PBS containing butylated hydroxytoluene (BHT) and triphenylphosphine (PPh3). First set of aliquots (10μL) of homogenized tissue were used for sterol extraction. The second set of aliquots (20μL) of homogenized tissue were used for protein measurements. The protein was measured using BCA assay (PierceTM BCA Protein Assay Kit, ThermoFisher Scientific, Waltham, MA). Sterol levels were normalized to protein measurements and expressed as nmol/mg protein. The third set of aliquots (100μL) of homogenized tissue were used for drug measurements.

LC-MS/MS (SRM) analysesSterols were extracted and derivatized with PTAD as described previously (51Genaro-Mattos T.C. Tallman K.A. Allen L.B. Anderson A. Mirnics K. Korade Z. Porter N.A. Dichlorophenyl piperazines, including a recently-approved atypical antipsychotic, are potent inhibitors of DHCR7, the last enzyme in cholesterol biosynthesis.) and placed in an Acquity UPLC system equipped with ANSI-compliant well plate holder coupled to a Thermo Scientific TSQ Quantis mass spectrometer equipped with an APCI source. Then 10 μL was injected onto the column (Phenomenex Luna Omega C18, 1.6 μm, 100 Å, 2.1 mm × 100 mm) with 90% MeOH and 10% ACN (0.1% v/v acetic acid) mobile phase for 1.7 min runtime at a flow rate of 500 μL/min. Natural sterols were analyzed by selective reaction monitoring (SRM) using the following transitions: Chol 369 → 369, 7-DHC 560 → 365, desmosterol 592 → 560, lanosterol 634 → 602, with retention times of 0.7, 0.4, 0.3 and 0.3 min, respectively. SRMs for the internal standards were set to: d7-Chol 376 → 376, d7-7-DHC 567 → 372, 13C3-desmosterol 595 → 563, 13C3-lanosterol 637 → 605.ARI and TRZ measurementsMedications were extracted from 100μL aliquots for all brain regions using methyl tert-butyl ether and ammonium hydroxide as described previously (52Allen L.B. Genaro-Mattos T.C. Anderson A. Porter N.A. Mirnics K. Korade Z. Amiodarone Alters Cholesterol Biosynthesis through Tissue-Dependent Inhibition of Emopamil Binding Protein and Dehydrocholesterol Reductase 24.). ARI levels were acquired in an Acquity UPLC system coupled to a Thermo Scientific TSQ Quantis mass spectrometer using an ESI source in the positive ion mode. Ten μL of each sample was injected onto the column (Phenomenex Luna Omega C18, 1.6 μm, 100 Å, 2.1 × 50 mm2) using water (0.1% v/v acetic acid) (solvent A) and acetonitrile (0.1% v/v acetic acid) (solvent B) as mobile phase. The gradient was: 10–40% B for 0.5 min; 40–95% B for 0.4 min; 95% B for 1.5 min; 95–10% B for 0.1 min; 10% B for 0.5 min. ARI, TRZ and their metabolites were analyzed by SRM using the following transitions: ARI 448 → 285, dehydroaripiprazole 446 → 285, TRZ 372 → 176, m-CPP 197 → 153. The SRM for the internal standards (d8-ARI and d8-m-CPP) were set to 456 → 293 and 205 → 157, respectively. Final medications levels are reported as ng/mg of protein.Primary neuronal culturesPrimary cortical neuronal cultures were prepared from E18 C57BL/6J mice as previously described (41Xu L. Mirnics K. Bowman A.B. Liu W. Da J. Porter N.A. Korade Z. DHCEO accumulation is a critical mediator of pathophysiology in a Smith-Lemli-Opitz syndrome model., 53Allen L.B. Genaro-Mattos T.C. Anderson A. Porter N.A. Mirnics K. Korade Z. Amiodarone Alters Cholesterol Biosynthesis through Tissue-Dependent Inhibition of Emopamil Binding Protein and Dehydrocholesterol Reductase 24.). The brain tissue was placed in prechilled HBSS solution (without Ca2+ or Mg2+), and two cortices were dissected, cut with scissors into small chunks of similar sizes, and transferred to Trypsin/EDTA (0.25%) for 25 min at 37°C. Trypsin was removed and residual trypsin was inactivated by adding Trypsin Inhibitor (Sigma, cat. no: T6522) and DNase for 5 min. Solution was removed and small tissue chunks were resuspended in Neurobasal medium (NBM) with B-27 supplement (Gibco, cat. no: 17504-044). Samples were triturated with a fire-polished Pasteur pipet. The cells were pelleted by centrifugation for 10 min at 80g. The cell pellet was resuspended in NBM with B-27 supplement, and the cells were counted. The cells were plated on poly(d-lysine) coated 96-well plates at 70,000 cells/well. The growth medium was NBM with B-27 supplement and Glutamax. Cells were incubated at 37 °C in 5% CO2 for 3-10 days in presence and absence of different concentrations of ARI, TRZ, and ARI+TRZ.Primary astrocyte culturesPrimary astrocyte cultures were prepared from postnatal day 2 (P2) C57BL/6J mice as previously described (54Tallman K.A. Allen L.B. Klingelsmith K.B. Anderson A. Genaro-Mattos T.C. Mirnics K. Porter N.A.