Chlorpromazine

SKOV3 exosomes were labeled with carboxyfluoresceine diacetate succinimidyl-ester (CFSE) (Invitrogen) as previously described [12 (link)]. Briefly, exosomes (20 μg) collected after a 100,000 × g ultracentrifugation were incubated with 7.5 μM CFSE for 30 min at 37°C in a final volume of 200 μl PBS containing 0.5% BSA. Labeled exosomes (Exos-CFSE) were 65-fold diluted with DMEM supplemented with 10% vesicles-free fetal calf serum and pelleted by ultracentrifugation for 16 h at 10,0000 × g, 12°C. Exos-CFSE were resuspended in DMEM and incubated with SKOV3 cells at 37 or 4°C.
When indicated Exos-CFSE or cells were treated for 30 min with 100 μg/ml proteinase K, or for 2 h with 15 mU neuraminidase from V. cholerae or from A. urefaciens (Roche), before uptake. SKOV3 cells were also incubated, 30 min prior to and during uptake, with the inhibitors 10 μg/ml chlorpromazine, 5 μg/ml cytochalasin D, 50 μM 5-ethyl-N-isopropyl amiloride (EIPA) or 2% methyl-beta-cyclodextrin, or with 150 mM of the monosaccharides D-glucose, D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine, and the disaccharide β-lactose (Sigma).
Uptake assays were always performed in the presence of the compounds and analyzed after 2 or 4 h by immunofluorescence microscopy or flow cytometry.

Chi-square for categorical variables and Mann–Whitney U test for continuous variables due to non-normality were used to compare the baseline characteristics between patients with RLS and RLS-free controls. Cox proportional hazards regression models were applied to explore the association between RLS and the risk of dementia after adjusting for age, sex, income, residence, CCI, and history of other comorbidities. Among the Cox regression models, we used the Fine–Gray subdistribution hazard model with mortality as a competing risk given the old age of the study population. The proportional hazard assumption was satisfied in our Cox model (Schoenfeld individual test p-value > 0.05).
Sensitivity analyses were performed using four different models. In model 1, dementia was defined as the prescription of anti-dementia medications (donepezil, rivastigmine, galantamine, and memantine) at least twice and a diagnosis of the ICD-code of dementia. Although these medications were approved for only AD (rivastigmine additionally for Parkinson’s disease dementia), they can be used for cognitive symptoms in other types of dementia based on recommendations from multiple guidelines [31 (link)–33 (link)]. The previous study revealed that the definition of all-cause dementia by ICD-10 code plus anti-dementia medications had a positive predictive value of 94.7% when reviewing the medical records of 972 patients in two hospitals [34 (link)]. In model 2, medication history was added to the ICD code to define RLS. Patients with RLS ICD-code (G25.8) who had taken dopamine agonists (ropinirole or pramipexole) twice or more were regarded as patients with RLS (n = 1458). In this sensitivity model, we excluded patients with Parkinson’s disease because they could also take dopamine agonists. In model 3, patients taking antipsychotic agents were excluded because the antidopaminergic property of antipsychotic agents could lead to a misdiagnosis of RLS (n = 2482). The following antipsychotic agents approved in South Korea were used in this study: haloperidol, sulpiride, chlorpromazine, perphenazine, pimozide, risperidone, olanzapine, quetiapine, paliperidone, amisulpride, aripiprazole, ziprasidone, clozapine, blonanserin, and zotepine. In model 4, patients with RLS only diagnosed by psychiatrists or neurologists were included (n = 1154) to preclude the possible misdiagnosis by non-expert physicians.
To evaluate the effect of dopamine agonists (pramipexole and ropinirole) on the development of dementia, the risk of dementia was compared after dividing RLS patients by dopamine agonist use. Patients with RLS who were prescribed pramipexole or ropinirole at least once were considered dopamine agonist users. All missing data were addressed using listwise deletion. Data processing and statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA). Statistical significance was set at a two-tailed p-value of < 0.05.

This study used a cross-sectional design in a consecutive sample of 122 Japanese patients with schizophrenia (10 inpatients, 112 outpatients; 51 men, 71 women; mean age ± standard deviation, 43.2 ± 13.5 years) who were treated at Fujisawa Hospital, Asahinooka Hospital, or Yokohama City University Hospital in Japan from June 2016 to August 2019. All patients had received oral aripiprazole as monotherapy or AOM as monotherapy for more than 3 months at the same dosage and without adjustment in the previous 3 months. Fifty-two patients in the oral aripiprazole group and 9 patients in the AOM group were those involved in our previous study [9 (link), 23 (link)]. In addition, patients with schizophrenia receiving oral aripiprazole or AOM were newly recruited for the present study. Exclusion criteria were as follows: nonadherence to medication prescribed (evaluated using medical interviews); current or previous cardiovascular, respiratory, neurological, or endocrine illness; and current or previous substance abuse that could obscure diagnosis. Patients taking non-psychotropic medications such as antihypertensive drugs other than laxatives, which do not affect HRV, were excluded. Psychiatrists with sufficient clinical experience made the diagnosis of schizophrenia according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition [24 ]. They also evaluated patients’ positive, negative, and general signs using a Japanese translation of the Positive and Negative Syndrome Scale (PANSS; [25 (link)]) to determine symptom severity on the same day as an electrocardiogram (ECG) was recorded.
We collected participants’ clinical information from the medical records and calculated the doses of all psychotropic medications prescribed, including aripiprazole, anticholinergic, and benzodiazepine agents, using conversions to standard equivalents of chlorpromazine, biperiden, and diazepam [26 (link)].
This study was approved by respective ethics committees of Fujisawa Hospital, Asahinooka Hospital, and Yokohama City University Hospital and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants following a full explanation of the study.