Sigma-1 and dopamine D2/D3 receptor occupancy of pridopidine in healthy volunteers and patients with Huntington disease: a [18F] fluspidine and [18F] fallypride PET study

Purpose Pridopidine is an investigational drug for Huntington disease (HD). Pridopidine was originally thought to act as a dopamine stabilizer. However, pridopidine shows highest affinity to the sigma-1 receptor (S1R) and enhances neuroprotection via the S1R in preclinical studies. Using [18F] fluspidine and [18F] fallypride PET, the purpose of this study was to assess in vivo target engagement/receptor occupancy of pridopidine to the S1R and dopamine D2/D3 receptor (D2/D3R) at clinical relevant doses in healthy volunteers (HVs) and as proof-of-concept in a small number of patients with HD. Methods Using [18F] fluspidine PET (300 MBq, 0–90 min), 11 male HVs (pridopidine 0.5 to 90 mg; six dose groups) and three male patients with HD (pridopidine 90 mg) were investigated twice, without and 2 h after single dose of pridopidine. Using [18F] fallypride PET (200 MBq, 0–210 min), four male HVs were studied without and 2 h following pridopidine administration (90 mg). Receptor occupancy was analyzed by the Lassen plot. Results S1R occupancy as function of pridopidine dose (or plasma concentration) in HVs could be described by a three-parameter Hill equation with a Hill coefficient larger than one. A high degree of S1R occupancy (87% to 91%) was found throughout the brain at pridopidine doses ranging from 22.5 to 90 mg. S1R occupancy was 43% at 1 mg pridopidine. In contrast, at 90 mg pridopidine, the D2/D3R occupancy was only minimal (~ 3%). Conclusions Our PET findings indicate that at clinically relevant single dose of 90 mg, pridopidine acts as a selective S1R ligand showing near to complete S1R occupancy with negligible occupancy of the D2/D3R. The dose S1R occupancy relationship suggests cooperative binding of pridopidine to the S1R. Our findings provide significant clarification about pridopidine’s mechanism of action and support further use of the 45-mg twice-daily dose to achieve full and selective targeting of the S1R in future clinical trials of neurodegenerative disorders. Clinical Trials.gov Identifier: NCT03019289 January 12, 2017; EUDRA-CT-Nr. 2016-001757-41. Electronic supplementary material The online version of this article (10.1007/s00259-020-05030-3) contains supplementary material, which is available to authorized users.


Subjects
Twenty male healthy volunteers (HVs) between 25 and 55 years of age, without a clinically significant neuropsychiatric disorder (Diagnostic and Statistical Manual of Mental Disorders, DSM-5), without a history of alcohol, or any other substance dependence in the past 2 years, in good physical and mental health as determined by medical and psychiatric history, suicidality assessment, physical examination, 12-lead ECG, vital signs, clinical laboratory tests, and a MRI scan and without a CYP2D6 poor metabolizer genotype were enrolled in the study.
Three male patients with Huntington disease (HD) were enrolled who were diagnosed with HD based on clinical features and the presence of ≥36 cytosine-adenine-guanine (CAG) repeats in the huntingtin gene. Patients were at least 25 years of age (symptom onset >18 years), had a body weight ≥50 kg, and a sum of ≥25 points on the Unified Huntington Disease Rating Scale-Total Motor Score (UHDRS-TMS) at the screening visit. Patients were without a clinically significant psychiatric disease, such as major depressive disorder or anxiety according to the DSM-5, without suicidal ideation or attempt at any time in the past or as measured by suicide ideation score of ≥3 on the Columbia-Suicide Severity Rating Scale (C-SSRS), without a history of alcohol, or any other substance dependence in the past 2 years, without a CYP2D6 poor metabolizer genotype, without a prolonged QTcF interval in the ECG (QTcF interval >450 ms), without a clinically significant heart disease or other severe medical illness. This was assessed by medical and psychiatric history, suicidality assessment, physical examination, 12-lead ECG, vital signs, clinical laboratory tests, and an MRI scan. For the patients with HD taking allowed antipsychotic, antidepressant, or other psychotropic medication, the dosing of medication must have been kept constant for at least 6 weeks before the baseline PET and had to be kept constant during the study.

Inclusion and exclusion criteria
Inclusion criteria: healthy subjects a. Male subjects between 25 and 55 years (inclusive) of age, with a body mass index (BMI) ≥18.0 to ≤30 kg/m 2 and a body weight of at least 50 kg (inclusive).
b. Good physical and mental health as determined by medical history and psychiatric history, suicidality assessment, physical examination, 12-lead ECG, vital signs, and clinical laboratory tests. A subject with a clinical abnormality in the laboratory profile or BP can be included only if the investigator or his designee considers that the abnormality does not introduce an additional risk factor for the subject's health, or interfere with the study objectives.
c. Ability to understand the requirements of the study; are willing to comply with the requirements of the study (eg, imaging procedures, all dietary, exercise, and alcohol restrictions) and provided their written informed consent to participate in the study. d. Willingness to provide a blood sample for genetic analyses (including CYP2D6 status, S1R polymorphs, genetic long QT syndrome in patients who had QT prolongation following study drug administration or any other genetic analyses related to pridopidine response) at the screening visit. i. Ability and willingness to provide written informed consent prior to any study related procedure being performed at the screening visit. Patients with a legal guardian should be consented according to local requirements.
j. Willingness to provide a blood sample for genetic analyses (including CAG analysis, CYP2D6 status, S1R polymorphs, genetic long QT syndrome in patients who had QT prolongation following study drug administration or any other genetic analyses related to pridopidine response or Huntington disease) at the screening visit.
k. Willingness and ability to take oral medication and able to comply with the study specific procedures.
l. Ability to travel to the study center for the duration of the study. m. Availability and willingness of a caregiver, informant or family member to accompany the patient to the clinic at study visits. The suitability of the caregiver should be judged by the investigator.
n. For patients taking allowed antipsychotic, antidepressant or other psychotropic medication, the dosing of medication must have been kept constant for at least 6 weeks before baseline (visit 2, day -1) and must be kept constant during the study.
o. Ability to understand the requirements of the study; willingness to comply with the requirements of the study (eg, imaging procedures, all restrictions) and provided their written informed consent to participate in the study. x. Current or history of heart condition or increased pro-arrhythmic risk, including: -History of cardiovascular disease (eg, coronary artery disease, stroke, arrhythmias, congestive heart failure, uncontrolled hypertension, deep vein thrombosis, pulmonary embolism, family history of thrombophilia) -History of Long QT Syndrome or a first degree relative with this condition -Family history of sudden death/Brugada syndrome -Prolonged Fridericia-corrected QT (QTcF) interval (defined as a QTcF interval of >450 msec) at the screening or admission (baseline) visit(s). If there is evidence of a prolonged QTcF interval from the initial (single) measurement, then the ECG can be repeated twice, and the mean of the 3 screening measurements will be used to determine whether or not the subject is suitable for inclusion in the study.
-Repolarization deficits -Untreated hypokalemia and/or untreated hypomagnesaemia -Unclear syncopes -Other clinically significant abnormal ECG as judged by the investigator.
y. Creatinine clearance <90 mL/min at screening, calculated using the Cockcroft-Gault equation. It is permitted to repeat the test once, if clinically appropriate.
z. Hemoglobin value below the lower limit of the reference range and evaluated by the investigator to be clinically significant.
bb. Presense or history of clinically significant diseases of the renal, hepatic, gastrointestinal, cardiovascular, musculoskeletal, immunological, endocrine (including diabetes even if controlled by diet), metabolic diseases, or other condition known to interfere with the absorption, distribution, metabolism or excretion of drugs, as judged by the investigator. dd. History of clinically significant psychiatric diseases, such as major depressive disorder or anxiety, as judged by the investigator. ee. Adverse events of suicidal ideation or attempt at any time in the past or as measured by suicide ideation score of ≥3 on the C-SSRS (screening version), or subjects who answer "Yes" on any of the 5 C-SSRS Suicidal Behavior Items (actual attempt, interrupted attempt, aborted attempt, preparatory acts, or behavior) if the attempt or acts were performed at any time in the past, or subjects who, in the opinion of the investigator, present a risk of suicide. ff. One of the following conditions: -major trauma or surgery in the 2 months before screening or at any time between screening and admission -acute/chronic infection within 2 weeks before screening or at any time between screening and admission -malignancy within the last 5 years -epilepsy, seizure, convulsions or syncope (including febrile seizures) -history of tuberculosis -BP outside the range of 90 to 139 mmHg (systolic) or 55 to 89 mmHg (diastolic); or pulse rate outside the range of 45 to 99 bpm; all measured after 5 min rest in seated or supine position. Vital signs may be retested twice at intervals of 5 min. gg. Missing willingness or ability to refrain from intensive physical exercise during the study.
hh. Current or history of clinically significant allergy or known hypersensitivity to any ingredients of the study medication (pridopidine, silicified microcrystalline cellulose, magnesium stearate).
ii. Planned medical treatments (excluding dental care) during the study period, which may interfere with the study.
jj. Use of one of the following prohibited drugs, substances, or foods as follows: -an investigational drug (new chemical entity) within 6 weeks prior to the first day of study drug administration or within 5 half-lives (whichever is longer) -any monoamine oxidase inhibitors within 14 d before the first day of study drug administration -any other medications (including over-the-counter [OTC] medications, vitamins, or herbal or nutritional supplements) within 7 d before the first day of study drug administration (except paracetamol/acetaminophen or ibuprofen used occasionally, up to 24 h before the first day of study drug administration) -drugs known to significantly inhibit CYP2D6 enzyme drug metabolism within 21 d prior to the first day of study drug administration or within 5 half-lives (whichever is longer) before the first day of study drug administration, or drugs known to significantly induce CYP enzyme drug metabolism within 28 d before the first day of study drug administration -drugs known to cause significant QT-prolongation such as anti-arrhythmic drugs or antidepressants within 28 d before the first day of study drug administration -daily consumption of more than 6 units of caffeine and/or xanthine-containing products, within 2 weeks before the first dose of study drug, or not able to refrain from consumption of more than 2 units of caffeine-containing foods or drinks from 48 h prior to admission visit and discharge. One caffeine unit is contained in the following items: 1 cup of coffee, 2 cans of cola, 1 cup of tea, ½ cup of energy drink (eg, Red Bull) or 3 chocolate bars. Subject should be able to abstain from caffeine intake for 20 h during any day.

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-cigarette smoking (defined as >5 cigarettes per week) or use of other nicotine-containing products (eg, snuff, nicotine patch, nicotine chewing gum, mock cigarettes, or inhalers). Ex-smokers should have ceased smoking at least 6 months before screening.
-any food or drink/beverage containing alcohol, grapefruit or grapefruit juice, apple or orange juice, vegetables from the mustard green family (eg, kale, broccoli, watercress, collard greens, kohlrabi, brussel sprouts, mustard), and charbroiled meats within 7 d before the first day of study drug administration until after the last day of pharmacokinetic sampling.
-a positive urine drug test or a positive alcohol breath analyzer test at admission visits, or not willing or able to refrain from illicit drugs during the study.
kk. Donation or reception of any blood products (eg, plasma, platelets, etc) in the 1 month before the first day of study drug administration, or has made more than 2 donations within the 12 months preceding the first day of study drug administration, or plans to donate during the study or during the 1 months after the last study visit.
ll. Any of the following reasons: -The subject is mentally or legally incapacitated, or unable to give consent for any reason.
-The subject is in custody due to an administrative or a legal decision, or under tutelage, or being admitted to a sanitarium or social institution.
-The subject is unable to be contacted in case of emergency.
-The subject is an employee of the site or a relative of an employee at the site.
-Any other reason, at the discretion of the investigator. If there is evidence of a prolonged QTcF interval at screening from the initial (single) measurement, then the ECG will be repeated twice, and the mean of the 3 screening measurements will be used to determine whether or not the patient is suitable for inclusion in the study.
k. Clinically significant heart disease at the screening visit, defined as follows: (i)  u. Adverse events of suicidal ideation or attempt at any time in the past or as measured by suicide ideation score of ≥3 on the C-SSRS (screening version), or patients who answer "Yes" on any of the 5 C-SSRS Suicidal Behavior Items (actual attempt, interrupted attempt, aborted attempt, preparatory acts, or behavior) if the attempt or acts were performed at any time in the past, or patients who, in the opinion of the investigator, present a risk of suicide.
v. Known intracranial neoplasms, vascular malformations, history of cerebrovascular accident, or intracranial hemorrhage.
w. Current or history of clinically significant allergy or known hypersensitivity to any ingredients of the study medication (pridopidine, silicified microcrystalline cellulose, magnesium stearate).
y. Treatment with any investigational product within 6 weeks of screening prior to the first day of study drug administration or within 5 half-lives (whichever is longer) or patients planning to participate in another clinical study assessing any investigational product during the study.
z. Use of one of the following prohibited substances, or foods as follows: any food or drink/beverage containing alcohol, grapefruit or grapefruit juice, apple or orange juice, vegetables from the mustard green family (eg, kale, broccoli, watercress, collard greens, kohlrabi, brussel sprouts, mustard), and charbroiled meats within 7 d before the first day of study drug administration until after the last day of pharmacokinetic sampling.
a positive urine drug test or positive alcohol breath analyzer test at admission visits, or not willing or able to refrain from illicit drugs during the study.

Supplementary Materials and Methods (continued) [ 18 F]Fluspidine or [ 18 F]Fallypride PET/MR image acquisition and processing
During the dynamic PET acquisition, arterial radioactivity concentration of [ 18 F]Fluspidine in plasma was measured in up to 38 samples taken from the radial artery. Eleven to 15 blood samples (2 ml) were acquired in the first 3 min after tracer injection followed by 23 samples between 3 and 210 min. One ml plasma was obtained by centrifugation and its activity concentration was measured using a Wizard gamma counter. All activity measurements were decay-corrected to the start time of the PET/MR acquisition. Tracer binding to plasma proteins was determined by ultracentrifugation as described previously. The amount of non-metabolized [ 18 F]Fluspidine was determined in 7 additional arterial blood samples (10 ml) taken at 3, 10, 20, 50, 90, 150, and 210 min post injection by high performance liquid chromatography separation of the parent compound fraction (fPC) followed by gamma counting. Proteinfree plasma was obtained from plasma (centrifugation at 4000 U, 5 min) by centrifugation (10 000 U, 10 min) after addition of acetonitrile (2/3; V/V). The fraction of non-metabolized [ 18 F]Fluspidine fPC was fitted by a sum of two exponential functions and used to compute the arterial input function for kinetic modeling [S3].

MRI morphometric analysis
MRI scans (T1-MPRAGE), acquired at baseline PET, were evaluated morphologically by a specialist in radiology and semiquantitatively analyzed for brain atrophy of the caudate head in HVs and patients with HD. Non-HD-pattern or other relevant parenchymal defects were excluded. A progressive atrophy of the caudate head in the patient with HD is the most striking radiological feature and will result in a decrease of the frontal horn width (FH) to intercaudate distance (CC) ratio (abnormal FH/CC ratio < 2.2) or an increase of the intercaudate distance to inner width ratio (threshold for pathologic CC/IT ratio > 0.12) [S4].

Test-retest PET study
The

Pharmacokinetic parameters
Cavg2-4h, Cmax and AUC0-24h increased with increasing dose of pridopidine in the HVs (Supplementary       and RO, median in the case of tmax, and COV in % in the case of Cmax, AUC0-24h and t1/2. Geometric mean is given for Cmax, AUC0-24h and t1/2.