Abstract Parkinson disease (PD) is associated with cognitive impairment. We aimed to determine the effects of intranasal insulin (INI) on cognition and motor performance in PD. This was a proof of concept, randomized, double-blinded, placebo-controlled trial evaluating the effects of 40 international units (IU) of insulin or saline once daily for four weeks on cognitive and functional performance. Of 16 subjects enrolled, eight in the INI group and six in the placebo group completed verbal fluency (FAS), Unified Parkinson Disease Scale (UPDRS), and modified Hoehn and Yahr scale (HY, PD severity) at baseline and post-treatment and were included in the analyses. After treatment, the INI group had a better total FAS score (p = 0.02) (41 ± 8.2 vs. 30.8 ± 7.1, mean ±SD, p = 0.02) compared to the placebo group. The INI group also had improved HY (p = 0.04) and UPDRS-Motor (Part III) (p = 0.02) scores when compared to baseline. One INI treated patient with multiple system atrophy (MSA) remained stable and did not show disease progression. The placebo group had no change. INI administration was well tolerated and there were no hypoglycemic episodes or serious study related adverse events or medications interactions. INI is safe in PD and MSA patients and may provide clinically relevant functional improvement. Larger studies are warranted to determine the INI effect in treatment of cognitive and motor impairment in Parkinson disease. Trial Registration: ClinicalTrial.gov NCT02064166.

Citation: Novak P, Pimentel Maldonado DA, Novak V (2019) Safety and preliminary efficacy of intranasal insulin for cognitive impairment in Parkinson disease and multiple system atrophy: A double-blinded placebo-controlled pilot study. PLoS ONE 14(4): e0214364. https://doi.org/10.1371/journal.pone.0214364 Editor: J. Lucas McKay, Emory University, UNITED STATES Received: June 8, 2018; Accepted: March 12, 2019; Published: April 25, 2019 Copyright: © 2019 Novak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The data underlying the study are available at: http://alpha.physionet.org/content/inipdmsa/1/. Funding: This study was funded in part by The Langer Family Charitable Foundation, Chirag Foundation Investment Trust, Baker’s MSA fund, Mr. Yash R. Puri awarded to Dr. Peter Novak, and by Department of Neurology, University of Massachusetts. The analysis for the was in part supported by National Institute of Diabetes and Digestive and Kidney diseases R01DK103902 (https://www.niddk.nih.gov/) awarded to Dr. Vera Novak that was unrelated to this study. Competing interests: No competing interests.

Introduction Parkinson disease (PD) is associated with a decline in cognitive performance and about 26% of patients diagnosed with PD develop mild cognitive impairment (MCI), more commonly the non-amnestic type [1–3]. The MCI incidence increases with age, disease severity and duration. MCI increases the risk for dementia and disability in PD patients [1,2,4] as well as the care-giver’s burden [5]. Cognitive impairment in PD has been associated with various mechanisms [6–9] such as regional cerebral perfusion deficit [10] and microstructural [11] and anatomical abnormalities. The microstructural abnormalities manifest even in PD patients with normal cognitive performance and become widespread as cognitive function deteriorates [6,11]. PD patients have altered functional connectivity within the resting state default mode network (DMN) [12] which regulates memory and other complex cognitive behaviors. The most affected pathways are connections between the posterior cingulate cortex, prefrontal cortex and medial temporal lobe [7,8,12] and long-range connectivity to other regions [13,14] that also demonstrate gray and white matter atrophy [13,14]. Walking speed is an important indicator of overall functional health and is correlated with survival in older adults [15]. Slower walking may be an early indicator of deterioration of attention and executive function underlying progression of cognitive decline [16]. Insulin plays a key role in glucose metabolism in the brain where it exerts important neuromodulatory, neurotrophic, and neuroprotective effects [17]. Intranasal insulin (INI) administration acutely increased resting-state functional connectivity between hippocampal and DMN regions in patients with type 2 diabetes without affecting serum glucose [18]. INI improved verbal and visuospatial memory in older diabetic and healthy adults, likely via regional vasodilatation in the anterior cerebral circulation [19]. In patients with MCI and Alzheimer disease, INI treatment improved visuospatial working memory and verbal working memory [20,21]. The rationale is that central insulin resistance and consequently impaired insulin signaling in the brain may be the common pathways for cognitive decline with aging, diabetes and Alzheimer’s disease. In the brain, insulin has vasodilatatory and neurotrophic effects, and therefore INI potential benefits are mediated by other mechanisms than by improving peripheral glycemic control. The effects of INI administration on cognitive performance in patients with PD are yet to be elucidated. We evaluated the cognitive and functional effects of the daily administration of 40 international units (IU) of INI in adults with PD over a four week period as compared to placebo (sterile saline) administration. We hypothesized that INI would improve verbal cognition and motor disability in non-demented PD participants after the four weeks of treatment when compared to placebo.

Materials and methods This was a pilot single-center, double-blinded, placebo-controlled study with parallel design thatevaluated the safety of INI on cognitive function in patients with PD. The study was conducted at the Autonomic Laboratory at the University of Massachusetts Medical School in Worcester, Massachusetts, United States. The study was registered at www.clinicaltrials.gov, number NCT02064166. Participants Subjects were recruited from the Movement Disorders Clinic at the University of Massachusetts Memorial Medical Center. The Institutional Review Board at the University of Massachusetts approved the study. Participants were screened over the phone and eligible participants signed the informed consent form, as approved by the Institutional Review Board at the University of Massachusetts. Inclusion criteria were: men and women older than 17 years with a clinical diagnosis of PD or MSA. Exclusion criteria were: pregnant or lactating women, patients with significant systemic illness that may interfere with the trial, a history of dementia, participants unable to walk for more than one minute, a history of allergic reaction to insulin, and nasal cavity inflammation that may prevent the absorption of insulin. Intervention and randomization Participants were treated with 40 IU of human insulin (Novolin R Novo Nordisk, Bagsvaerd, Denmark) or placebo (sterile saline) delivered using the Via Nase device (Kurve technologies, Seattle, WA) once daily before breakfast for four weeks. The Via Nase device is an electronic atomizer that delivers the drug into the upper nasal cavity olfactory region, thus enhancing penetration into the brain. The principal investigator (P.N.) who enrolled the participants, the study staff (D.A.P) who conducted the outcome assessment, and participants and their healthcare providers were blinded to the randomization assignment. V.N. was not involved in data collection. Subjects were randomized into the INI or placebo groups by the research pharmacy using the randomly permuted blocks method with two subjects per block (http://randomization.com). Insulin and placebo were distributed using the identical vials by the research pharmacy, thus blinding the participants and investigators. Protocol Participants completed a screening visit, a baseline assessment, two follow-up visits, and an end-of treatment assessment over a four week treatment period. All participants completed detailed medical histories, neurologic physical exams and laboratory investigations (basic metabolic panel and pregnancy test in women of childbearing age). Functional assessments included neuropsychological testing, disease severity scales (to evaluate motor function and disease progression) and a walking test. Functional assessments at baseline and post-treatment were conducted while participants took their usual medications. The last INI/placebo dose administration was on the day of post treatment assessment. Neuropsychological, clinical and motor assessments The Montreal Cognitive Assessment (MoCA) test was used to assess symptoms of cognitive impairment [22]. The verbal fluency FAS test was used to assess phonemic fluency and verbal memory [23]. For FAS, participants were asked to name words starting with letters F, A and S over a one-minute interval. The Beck Depression Inventory (BDI) is a 21-item scale that was used to evaluate depressive symptoms [24]. The clinical and motor assessments included the modified Hoehn and Yahr Scale (HY) to evaluate the severity of PD and treatment response [25] and the Unified Parkinson Disease Rating Scale (UPDRS, version modified by the Movement Disorders Society) to clinically assess PD effects on motor, cognitive, and other functions [26]. UPDRS, a widely-used outcome measure in clinical trials, is a sensitive indicator of motor progression and has satisfactory interrater reliability [27,28]. Motor score was calculated as proposed by Van Rooden et al. [29]. Bradykinesia score was calculated from the UPDRS item 23 + 24 + 25 +26 + 31 bilaterally. Motor asymmetry was estimated using lateralized UPDRS scores (item 20–26) (UPDRS I-III) as suggested by Jankovic et al. [26]. The UPDRS sub-scores summarize: UPDRS-I intellectual, mood and motivation impairment; UPDRS-II eating, activities of daily living, walking and balance; UPDRS-III speech, tremor; bradykinesia finger tapping, postural stability, and body bradykinesia and dyskinesia. Neuropsychological tests (MoCA, FAS, BDI) were administered by the research fellow trained in the study procedures (D.A.P.) and clinical tests (HY, UPDRS) were administered by the study clinician (P.N.). Gait test All participants completed a standard four meter walking test [30] at their preferred walking speed to assess normal walking speed, number of steps and average stride length. The time used to complete a four meter walk was recorded. Average stride length was calculated by dividing the walked distance by the recorded number of steps. Statistical analysis The analyses were performed using JMP 13 (SAS, Cary NC). Baseline demographic characteristics were compared between the groups using the Kolmogorov-Smirnov and Chi-squared tests. Primary endpoints were cognitive function measures (FAS) and secondary endpoints were MoCA, HY, UPDRS and BDI. The study outcomes (cognitive function measures FAS, MoCA), depression score (BDI), PD severity (HY scale and UPDRS-Motor), vital signs, and laboratory measures were compared between the placebo and insulin groups at baseline and at the end of treatment using repeated measures MANOVA adjusted for age. We have determined that data came from a normal distribution using the Shapiro-Wilk test, p>0.05. The treatment effect (baseline vs. post-treatment) was also compared within the INI and within the placebo group using a paired t-test. The effect size of the total FAS score was measured using Cohen’s test for post-treatment data. A level of p <0.05 was considered significant. The FAS was used as the primary cognitive outcome measure. Based on our current study, with 14 subjects total, we would have 79% power to detect a post-treatment difference of 10.2 points in the FAS total score between the insulin and placebo groups, standard deviation = 7.1, alpha = 0.05. Analyses were conducted by the original assigned group.

Discussion This prospective, placebo-controlled, double-blinded study compared the effects of the daily administration of 40 IU of Novolin R to sterile saline intranasally over a four-week period in PD patients on cognitive and motor performance. The administration of intranasal insulin was safe, as it has also been shown in the prior studies [18,19,21,32]. There were no significant changes in serum glucose, no hypoglycemic episodes and no serious study-related adverse events. Participants were able to adhere to the protocol and administer study medications. At baseline, the INI and placebo groups had similar degrees of cognitive and motor performance that were within normal range for participants in the sixth/seventh decades [31]. Insulin administration over a four-week period improved the total FAS score, which is a measure of verbal fluency, when compared to the baseline and placebo group in the repeated measure design. The FAS score increased in the INI group by 5.6% but decreased in the placebo group by 6.4%. However, paired comparisons between baseline and post-treatment assessments were not significant in the insulin and in the placebo group due to a small sample size. Verbal fluency involves successful retrieval of information from memory. It requires attention and concentration, as well as the accomplishment of other executive cognitive tasks, which are the cognitive domains affected by PD. Further studies are needed to determine whether INI may provide benefits to PD patients even before they develop clinically significant cognitive impairment. The treatment effects on motor performance and functionality were evidenced by the insulin group participants’ lower disability score on the HY scale as compared to the placebo group, which is a clinically significant finding. There was also an improvement in the UPDRS-Motor score (part III) in the insulin group when compared to baseline, but not in the placebo group. The placebo group showed a tendency toward decreased verbal fluency, HY score, and UPDRs score over a one month period. One patient in the treatment group was also diagnosed with probable MSA, which is a rapidly progressing degenerative disease that has much shorter survival than PD [33]. The patient remained stable during the treatment period and did not show a progression of symptoms, but perhaps displayed a trend toward improvement on UPDRS scores. Although this is a single case of INI treatment in MSA, it warrants further investigation as there are no therapies available to modify disease progression. Patients with PD are known to have a worse cognitive performance when compared to the general population. Mild cognitive impairment (MCI) is common in PD patients without dementia, affecting about 25% of patients. The cognitive domains affected are memory, visuospatial, and attention/executive function impairment, with memory most commonly affected (13% of patients) [4,13]. Non-amnestic MCI observed in patients with PD affects different cognitive domains, such as verbal memory, concentration and visuospatial memory [1]. Although our cohort was not diagnosed with PD-related MCI, they still demonstrated an improvement in verbal memory after the administration of INI over four weeks of treatment. The cognitive decline observed in PD population has been attributed to impairment within the connectivity DMN as well as to impairment in regional perfusion [10]. PD patients with MCI show selective decline in interconnectivity in the non-motor networks, and specifically between the bilateral lentiform nuclei and superior parietal lobules and precuneus, primarily affecting the dorsal attention network [14] as well as the orbitofrontal cortex and other regions [7]. PD mainly affects long-range connectivity, as well as topological features of the brain networks related to memory, executive function, and visuospatial orientation [14]. Prior studies have demonstrated that INI administration acutely increased resting-state functional connectivity between hippocampal and DMN regions and improved verbal and visuospatial memory in type 2 diabetes and healthy adults [18]. This cognitive enhancement has also been attributed to vasodilatation in the middle cerebral artery [19], which perfuses cortical regions involved in language and attention performance. This pilot study evaluated safety and feasibility of INI treatment in PD patients. The INI administration was safe; patients were able to follow the protocol and complete daily administration of INI without difficulties. However, the sample size of our study was small and therefore a larger study is needed to validate the results. In addition, our PD population was relatively well-functioning with only mild cognitive deficits. The study sample did not allow enrolling participants with more rapidly progressing MCI or dementia. Our battery of tests were selected to overlap with assessment of cognitive domains diagnostics for MCI (general cognition, verbal fluency and visuospatial orientation) [3] but also with tests that have successfully shown the effects of intranasal insulin therapy in other studies [19]. Therefore, in future studies we plan to include a larger patient population with more widespread motor and cognitive deficits. A longer duration of treatment may be needed to achieve long-term effects. Limitations This was a pilot study and thus has several limitations, such as a small number of participants, relatively short duration of the study, and the administration of only one dose (40 IU) per day. There were also no participants with significant cognitive impairments, who may benefit the most from the insulin treatment. PD medications e.g. pramipexole, amantadine, and ropinirole may have adverse effects on mood and cognition, however they cannot be stopped for ethical reasons, and their doses were stable during treatment period. The sample size of this study was small and therefore, we could not conduct extensive statistical adjustments for medications, disease duration, glucose levels or other potential confounders. We can only presume that observed changes were the treatment effect rather than test–retest effect, because the placebo group has shown a trend toward lower cognitive and motor scores. Several studies have shown that INI is safe, is not associated with severe side-effects and does not affect glycemic control or causes hypoglycemic episodes [18,19,21,32]. Therefore, INI can be safely used in nondiabetic population, such as PD, without the risk of hypoglycemic episodes. Our PD patients had normal fasting serum glucose and did not have diagnosis of diabetes or prediabetes, and therefore hemoglobin A1c was not measured in this study. Insulin in the brain has vasodilatory and neurotrophic effects, and therefore potential benefits of INI action are mediated by other mechanisms than by improving peripheral glycemic control.

Conclusions This pilot longitudinal study has shown that INI administration may improve functional motor skills in PD and may potentially preserve cognitive performance as compared to baseline and the placebo group. This proof-of-concept approach has shown that the use of INI in treatment of cognitive and motor impairment related to PD is safe and feasible without the risk of hypoglycemia. The lack of disease progression in the MSA case warrants further investigation. Our study provided preliminary data that suggested an improvement of functional skills after four weeks of daily INI treatment that paves the way toward a larger cohort study to evaluate long-term safety and potential efficacy of intranasal insulin administration for potential treatment and prevention of functional decline in patients with Parkinson disease.

Acknowledgments P.N. designed the study and contributed to all aspects of the manuscript preparation; D.A.P. contributed to study conduct, analyses and manuscript preparation. V.N. contributed to the study design and manuscript preparation.