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Safety, tolerability, and pharmacokinetics of allopregnanolone as a regenerative therapeutic for Alzheimer's disease: A single and multiple ascending dose phase 1b/2a clinical trial

Abstract Introduction Allopregnanolone is an endogenous neurosteroid with the potential to be a novel regenerative therapeutic for Alzheimer's disease (AD). Foundations of mechanistic understanding and well‐established preclinical safety efficacy make it a viable candidate. Methods A randomized, double‐blinded, placebo‐controlled, single and multiple ascending dose trial was conducted. Intravenous allopregnanolone or placebo was administered once‐per‐week for 12 weeks with a 1‐month follow‐up. Participants with early AD (mild cognitive impairment due to AD or mild AD), a Mini‐Mental State Examination score of 20–26 inclusive, and age ≥55 years were randomized (6:2 to three allopregnanolone dosing cohorts or one placebo cohort). Primary endpoint was safety and tolerability. Secondary endpoints included pharmacokinetic (PK) parameters and maximally tolerated dose (MTD). Exploratory endpoints included cognitive and imaging biomarkers. Results A total of 24 participants completed the trial. Allopregnanolone was safe and well tolerated in all study participants. No differences were observed between treatment arms in the occurrence and severity of adverse events (AE). Most common AE were mild to moderate in severity and included rash (n = 4 [22%]) and fatigue (n = 3 [17%]). A single non‐serious AE, dizziness, was attributable to treatment. There was one serious AE not related to treatment. Pharmacokinetics indicated a predictable linear dose‐response in plasma concentration of allopregnanolone after intravenous administration over 30 minutes. The maximum plasma concentrations for the 2 mg, 4 mg, 6 mg, and 10 mg dosages were 14.53 ng/mL (+/−7.31), 42.05 ng/mL (+/−14.55), 60.07 ng/mL (+/−12.8), and 137.48 ng/mL (+/−38.69), respectively. The MTD was established based on evidence of allopregnanolone‐induced mild sedation at the highest doses; a sex difference in the threshold for sedation was observed (males 10 mg; females 14 mg). No adverse outcomes on cognition or magnetic resonance imaging–based imaging outcomes were evident. Conclusions Allopregnanolone was well tolerated and safe across all doses in persons with early AD. Safety, MTD, and PK profiles support advancement of allopregnanolone as a regenerative therapeutic for AD to a phase 2 efficacy trial. Trial registration ClinicalTrials.gov‐NCT02221622

[17%]). A single non-serious AE, dizziness, was attributable to treatment. There was one serious AE not related to treatment. Pharmacokinetics indicated a predictable linear dose-response in plasma concentration of allopregnanolone after intravenous administration over 30 minutes. The maximum plasma concentrations for the 2 mg, 4 mg, 6 mg, and 10 mg dosages were 14.53 ng/mL (+/−7.31), 42.05 ng/mL (+/−14.55), 60.07 ng/mL (+/−12.8), and 137.48 ng/mL (+/−38.69), respectively. The MTD was established based on evidence of allopregnanolone-induced mild sedation at the highest doses; a sex difference in the threshold for sedation was observed (males 10 mg; females 14 mg). No adverse outcomes on cognition or magnetic resonance imagingbased imaging outcomes were evident.
Conclusions: Allopregnanolone was well tolerated and safe across all doses in persons with early AD. Safety, MTD, and PK profiles support advancement of allopregnanolone as a regenerative therapeutic for AD to a phase 2 efficacy trial.

K E Y W O R D S
allopregnanolone, Alzheimer's disease, maximally tolerated dose, neurogenesis, pharmacokinetics, phase 1 clinical trial, regenerative therapeutic, translational research

INTRODUCTION
Thus far no interventions have demonstrated meaningful therapeutic efficacy to treat Alzheimer's disease (AD) resulting in a 99.6% clinical trial failure rate with several accelerating disease progression. [1][2][3][4] Current thinking in the field supports the complexity of AD pathophysiology, which has enabled a more diverse therapeutic pipeline. 1,5 An innovative approach is to target the regenerative system of the brain while simultaneously activating systems that reduce burden of AD pathology. [6][7][8] Allopregnanolone (Allo), 3α-hydroxy-5α-pregnan-20-one, is an endogenous pregnane neurosteroid and a reduced metabolite of progesterone. 9,10 Unlike its precursor, Allo is inactive at nuclear progesterone receptors and instead promotes neurogenesis through activation of GABA A receptor complex on neural stem cells. 6,[9][10][11][12][13][14][15] Allo is a first-in-class regenerative therapeutic aimed at treating AD, with a strong foundation of human safety exposure. 11,[16][17][18][19][20][21][22] Intravenous infusion of Allo has been proven safe and tolerable in healthy adults, 16 both adults and children with super-refractory status-epilepticus, 19,23 men with fragile-X associated tremor/ataxia syndrome (FXTAS), 18,24 and women with postpartum depression. [25][26][27] The most common side effect induced by high doses of Allo is sedation/somnolence, which has been previously reported [16][17][18][25][26][27] and is an expected effect due to its known positive allosteric modulation of GABA A receptors. FXTAS patients demonstrated improvement in executive functioning, episodic memory, and learning after treatment with Allo. 18 In patients with postpartum depression, Allo (brexanolone) was superior to placebo in improvement of depressive symptoms. [25][26][27] Both females and males are exposed to Allo during fetal development and throughout their lifetime. During their reproductive years, women are chronically exposed to Allo. During pregnancy, blood concentration of Allo is highest during the third trimester reaching, on average, 157 nmol/l (50 ng/mL), which is not associated with adverse effects and is safe for mother and fetus. 9,[28][29][30] In the aged and degenerated brain, Allo content is diminished, and both the pool of neural stem cells and proliferative capacity are markedly reduced. 9,31,32 The primary objective of the phase 1b/2a trial was to assess the safety and tolerability of Allo in persons with early AD after a singledose administration and after chronic exposure over a 12-week period of once-per-week dosing. Pharmacokinetic (PK) profile and maximally tolerated dose (MTD) were established, and cognitive, imaging, and blood-based biomarkers explored. This is the first report describing the weekly administration of Allo in this patient population.

HIGHLIGHTS
• Targeting the regenerative system of the brain while simultaneously activating systems that reduce burden of pathology is an innovative therapeutic approach in treating Alzheimer's disease (AD). • Allopregnanolone is a first-in-class regenerative therapeutic with a strong foundation of human safety.
• Allopregnanolone was safe and well tolerated when administered intravenously in an early AD cohort.
• Pharmacokinetics and maximally tolerated dose were established for a regenerative dosing regimen in early AD.
• Outcomes support advancement of allopregnanolone to later stages of clinical development.
The study was approved by the US Food and Drug Administration and the University of Southern California (USC) Institutional Review Board and was registered with clinicaltrials.gov (NCT02221622). All participants provided written informed consent prior to participation.

Participants
Eligible participants were at least 55 years of age, met criteria for MCI due to AD or probable AD, 33 Participants who met eligibility requirements were randomly assigned to Allo or placebo in a 6:2 allocation ratio. Computergenerated random allocation was programmed by the study statistician. Randomization was stratified on sex to ensure balance across cohorts.
The sample size of six participants per active dose cohort and six total placebo participants was selected to identify major safety and tolerability signals related to dose. In terms of estimation of PK and other continuously measured parameters, selected sample sizes allowed estimation with a 95% confidence interval with limits of ±0.8 standard deviation (SD). Confidence intervals on mean group differences allowed interval limits of ±1 SD.

Interventions and study drug
Allopregnanolone or placebo were administered via an intravenous (IV) infusion once weekly for 12 consecutive weeks. The infusion

Multiple ascending dose adjustment
The first and second cohort received 2 mg and 4 mg of IV Allo, respectively, for 12 weeks. Symptoms of sedation were monitored during the infusions using a combination of a validated self-administered visual analogue scale, the Mood Rating Scale (MRS), 13 and a clinician administered questionnaire, the Stanford Sleepiness Scale (SSS). 37 These assessments showed no symptoms of sedation in either the 2 mg or 4 mg cohorts. Consequently, a dose escalation regimen was implemented for the participants in the third cohort to ensure that a sedative dose would be identified. Dose escalation for the last cohort occurred in the following manner: 6 mg-10 mg-14 mg-18 mg. If any participant met the criteria for qualified sedation at any dose, higher doses were not tested for that participant and the lower dose was evaluated during the next infusion. Participants continued at a sub-sedative maintenance dose for the remainder of the study. Additionally, if any of the pre-established critical laboratory criteria were met within a dose cohort, dose escalation was temporarily suspended until safety data across all participants and dose cohorts was thoroughly evaluated.

Safety assessments
The primary objective of the trial was to evaluate the safety and toler- Safety data from the intent-to-treat study population was analyzed by the DSMB during and at the end of the study. Safety data from perprotocol study population is reported and analyzed here. The incidence and severity of TEAEs was tabulated for participants randomized to each dose and compared to placebo using Fisher's exact tests. Summary and descriptive statistics were used to further detail the safety assessment data that were measured on a continuous scale. Mean values on these continuous safety measures were compared by randomized group using mixed effects models, with randomized group and visit (follow-up) as fixed effects and patient as a random effect. For these safety analyses, the primary comparisons of interest were differences in each dose group relative to placebo.

Pharmacokinetic assessment
A 24-hour PK profile was established after a single administration of

Imaging biomarkers
MRI scanning of the brain without IV contrast was performed at baseline and end-of-study using a 3T whole-body scanner (General Electric Signa HDxt). The imaging protocol used was a modification of the Alzheimer's Disease Neuroimaging Initiative 3T MRI protocol. T1 weighed images were acquired and volumetric analysis was done using Freesurfer 6.0 longitudinal version. Automated segmentation was used to calculate hippocampal volumes. Scans were also evaluated using Corlnsights MRI to assess overall baseline characteristics.

Study population
Overall, 47 participants were screened and of these, 26 participants were randomized ( Figure A.1

Safety and tolerability
Overall, Allo IV administration was safe and well tolerated by all participants. AEs were reported by 83% of participants in the placebo arm and 61% of participants in the Allo arm (

Pharmacokinetics
Allo PK parameters are summarized as mean values and standard deviations (Table 3) In dosing cohort three, all participants received 6 mg infusions at study week 1. All participants were individually dosed to their MTD, which was found to be 6 mg for the three male participants and 10 mg for the three female participants. Differences in exposure at study week 1 across three doses (2, 4, and 6 mg) show a less than dose-proportional concentration response at the lower dose. Differences in exposure at study week 12 across four doses (2 mg, 4 mg, 6 mg, and 10 mg), demonstrates that C max was consistent with a linear dose-concentration relationship. The AUC 0-last, data support a linear dose-exposure relationship after repeated dosing over 12 weeks (Table 3).  (Table 3).  Table 4. MRS scores are 1 to 100, with lower scores indicating increased sedation. There was a statistically significant difference in post-infusion MRS scores among dosing cohorts in alertness, and mental and physical sedation that was dose dependent (P = 0.006, 0.002 and 0.01, respectively). Overall, incidence of SSS scores consistent with sedation (≥7) was directly proportional to Allo dose. The highest rated SSS score for participants in cohort one (2 mg) was 2; the majority had a score of 1, which denotes a person who is "feeling active, vital, alert, or wide awake." The highest rated SSS score in cohort two (4 mg) was 4, which indicates that a person is "somewhat foggy or let down." For cohort three (6-18 mg) the highest rated SSS score was ≥7, defining a person with "sleep onset soon" or who is asleep.

Pharmacodynamics and target engagement: sedation
In all female participants, no sedation was observed per the SSS and MRS at either the 6 mg or 10 mg but was observed at 14 mg and 18 mg.
In contrast, male participants demonstrated sedation at doses ≥ 6 mg.

Exploratory endpoints
All 24 participants completed pre-and post-treatment cognitive assessments. After 12 weeks of treatment, no statistically signifi-

DISCUSSION
Safety and tolerability were the primary outcome measures in this first-in-human study of Allo in persons with early AD. As anticipated, both the single dose IV administration and the weekly dosing over the course of 12 weeks were safe and well tolerated in this study population. Safety outcomes are consistent with previously reported safety data in both animals and humans, 11,[16][17][18][19]23,24,39 and from an openlabel study 25 and two randomized-controlled trials 26,27 of Allo in the treatment of severe postpartum depression.   ). b MRS range is 0-100 with lower scores indicating increased sedation. c Linear mixed model with random subject effect was used to compare mean MRS scores among treatment cohorts. P-value represents overall difference across all treatment cohorts. Dunnett's method was used for multiple comparisons between each Allo cohort with placebo. The mean difference in mental sedation between Allo 6-18 mg and placebo is significant (P = 0.007). The difference in alertness between Allo 6-18 mg placebo is marginally significant (P = 0.07). All other P-values >0.17.  beta (Aβ) plaques from the brain, which are associated with ARIA, 40 the mechanisms by which Allo reduces Aβ load in the brain are related to its reduced generation. 6 Sedation was dose dependent (Table 4)  The dosing regimen is a critical factor for the neuro-regenerative effect of Allo, which occurs at nanomolar concentrations and is suppressed at higher doses. 6,9,12 Allo exhibits an inverted U-shaped dose-response which at low concentrations results in promotion of neural stem cell regeneration and at high concentrations suppresses proliferation, which protects against unchecked cell division. 6,9,12 Previous preclinical data indicated that Allo administered once per week was maximally efficacious for increasing neurogenesis and markers of white matter generation while simultaneously reducing multiple indicators of AD pathology. 11

CONCLUSIONS
Allopregnanolone is a first in class regenerative therapeutic for early AD that targets endogenous neural stem cells and disease-modifying mechanisms. The results of this phase 1b/2a clinical trial demonstrated that Allo was well tolerated and safe in an AD study population. Pharmacokinetic profile and MTD obtained will guide dose selection for a randomized-controlled phase 2 trial to investigate the long-term safety and efficacy of this novel therapeutic for AD.