Short-Term Tolerance of Nasally-Administered NeuroEPO in Patients with Parkinson Disease

INTRODUCTION Parkinson disease (PD) is the second leading neurodegenerative disorder in the world after Alzheimer disease.[1] Disability caused by PD is a growing economic and social burden that parallels the aging of the world’s population.[2] PD is characterized by progressive loss of dopaminergic neurons. Dopamine precursors (such as L-3,4-dihydroxyphenylalanine, L-DOPA) are the fi rst line of therapy, but only provide a symptomatic remedy since they do not modify the course of the disease. For this reason, one of the main lines of investigation worldwide is the search for neuroprotective agents.[3,4]


INTRODUCTION
Parkinson disease (PD) is the second leading neurodegenerative disorder in the world after Alzheimer disease. [1] Disability caused by PD is a growing economic and social burden that parallels the aging of the world's population.
[2] PD is characterized by progressive loss of dopaminergic neurons. Dopamine precursors (such as L-3,4-dihydroxyphenylalanine, L-DOPA) are the fi rst line of therapy, but only provide a symptomatic remedy since they do not modify the course of the disease. For this reason, one of the main lines of investigation worldwide is the search for neuroprotective agents. [3,4] There is evidence that erythropoietin (EPO), an erythropoiesisstimulating agent important in tissue oxygenation, with roles originally indicated in anemia and kidney failure, has neuroprotective properties. [5,6] EPO has a demonstrated effect on mechanisms involved in cell death due to its antiapoptotic and antioxidant qualities, which prevent cell damage during the nitric oxide cascade. These qualities also lend themselves to EPO's neurotrophic effects and its effects on neurogenesis. It is believed that EPO protects neurons through a combination of these mechanisms. [7,8] EPO modulates neuroinfl ammation through neurotrophic actions on astrocytes, microglia and neurons. [9] It can pass through the blood-brain barrier and acts on the central nervous system through inhibition of apoptosis in microvascular endothelial cells and activation of astrocytes. [10,11] EPO has a high content of sialic acid, which protects it from hepatic degradation and enables its erythropoietic function, causing a rise in hemoglobin synthesis and blood pressure. [12] These effects are not present in NeuroEPO due to its low content of sialic acid. The molecule thus conserves its neuroprotective properties without its hematopoietic activity, which makes it a better candidate as a neuroprotective agent. [10,11] NeuroEPO is produced by the Molecular Immunology Center (CIM, CIMAB S.A., Havana, Cuba) and is under study at the Center for Drug Research and Development (CIDEM, Havana, Cuba.)

Original Research
Participants Participants were recruited from February 1, 2015 through July 31, 2015 at the outpatient services at the Movement Disorder Clinic, International Neurological Restoration Center (CIREN) in Havana, Cuba, 46 of whom were diagnosed with idiopathic Parkinson disease in accordance with the operational criteria of the Brain Bank of London (BBL). [15] Inclusion criteria A prior EPO-tolerance study by our group [16] using ior EPOCIM provided the framework for inclusion criteria: • at least one year since PD onset, so disease progression can confi rm diagnosis • no limit on number of years since disease onset of PD whenever patient's severity is between stages 1 and 2 on the Hoehn & Yahr (H&Y) Scale [14] • age 40-70 years • good response to dopaminergic stimulation with >30% change in score on the motor section of the Movement Disorders Society-Unifi ed Parkinson Disease Rating Scale (MDS-UPDRS) [17] • no cognitive decline (>26 points) as measured by the Mini-Mental State Examination (MMSE). [18] Exclusion criteria Because EPO stimulates erythrocyte formation and is a leading natural erythropoiesis-stimulating agent, the following individuals should be excluded from its use and were excluded from the study: pregnant and breastfeeding women; women of childbearing age who use contraceptives; patients with hematological disorders (sickle cell disease, myelodysplastic syndromes, active clotting and bleeding disorders), peripheral vascular diseases, renal or hepatic insuffi ciency, polycythemia, or hematocrit ≥50. Due to the intranasal administration, patients with allergic rhinitis or vasomotor rhinitis in its acute phase were excluded, as well as those with nasal cavity malformations. We also excluded patients with sepsis or active acute or chronic infl ammatory diseases; malignant tumors or cancer treatment; alcoholism or drug addiction in the two years prior to the study; high blood pressure or other decompensated chronic illnesses; or patients receiving immunosuppressant, androgen or anabolic steroid treatments in the month prior to recruitment.
The sample size was selected following the established international practice for tolerance clinical trials that emphasize the need to minimize the number of subjects exposed to an experimental product. [19,20] In order to assign patients to the groups, the Molecular Immunology Center (CIM, provider of the drug) automatically generated a single list of randomized numbers and labeled the vials containing the product and cases with the corresponding code. Although the initial study design called for only 20 patients, the decision was made to include more patients due to the satisfactory tolerance results obtained in a clinical trial on healthy subjects and subjects with ataxia. [13] The same coding method was applied until completion of the fi nal sample (n = 26), made up of 15 patients treated with NeuroEPO and 11 patients treated with placebo.
Criteria for removal from the study Accidents or another disease diagnosed during the study that would exclude a participant; patient's expressed interest in abandoning the study; treatment suspended for more than one dose, consecutively or in isolated instances; and death.

Product and administration
NeuroEPO is presented in a singledose 1-mL vial, containing 1 mg/mL of low sialic acid, recombinant human erythropoietin (rHu-EPO) produced in cultures of the ovarian cells of Chinese hamsters (CHO cells). Each vial also contains polysorbate 80, sodium edetic acid, sodium chloride, medium molecular weight hydroxypropyl methylcellulose thickener and water for injection. The placebo formula is identical except for absence of the active ingredient. The drug was administered intranasally once a week for fi ve consecutive weeks with the patient lying in a supine position. During the fi rst application, the patient's head was inclined at a 45-degree angle below the horizontal plane of the body, to ensure that the product arrives to its site of action. A dose of 0.5 mL was administered slowly in each nostril as two doses of 0.25 mL 15 minutes apart. This ensured arrival at the superior turbinate while keeping in mind the maximum capacity for intranasal medications. [21,22] The initial maximum dose was based on preclinical and clinical studies, as well as safety guidelines. [23][24][25][26][27][28] Hematological and biochemical tests Blood tests were conducted prior to start of treatment and one week after completion. These included: hemoglobin, hematocrit, platelet count and erythrocyte-sedimentation rate, creatinine, uric acid, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, gamma-glutamyl transpeptidase, cholesterol and triglycerides. For statistical analysis, calculations were made of the relative change in all hematological and biochemical parameters, defi ned as the difference between the two measurements divided by the initial value.
Clinical evaluation Vital signs were measured before treatment and one hour after drug application. Tolerance was evaluated during the entire study through investigation of adverse events via patient surveys after each treatment session. In the case of an adverse event, the medical researcher acted in accordance with its nature and severity.
Defi nition and classifi cation of adverse events An adverse event was considered as any unfavorable medical event presenting in patients receiving the product without necessarily having a causal relation with treatment. Adverse events were classifi ed as local (site of application) or systemic, [29] in accordance with guidelines provided by the Center for State Control of Medicines and Medical Devices (CECMED). An event was considered mild if it was well tolerated, caused minimum bother, and did not interfere with daily activities; moderate if it was a bother but did not interfere with daily activities; and severe if it did interfere with daily activities. [30] The causal relation was classifi ed as: "Extremely likely/sure," "Likely," "Possible," "Unlikely," "Not related," or "Not able to be evaluated/unclassifi able," according to WHO causality criteria. [31] The participants' code was opened after study completion, which enabled identifi cation of patients belonging to each group.

Ethical considerations
The study adhered to the ethical considerations of the Declaration of Helsinki. [32] Written informed consent was obtained from all participants; and confi dentiality was ensured.
Statistical procedures Information was summarized in means and standard deviations for quantitative data and in percentages for qualitative data. To compare distribution of frequencies of the

Original Research
two groups' adverse events, the Chi square (X 2 ) test was used. For comparisons of the groups with respect to the continuous variables, two analytically equivalent procedures were used: (a) an analysis of variance for repeated observations in the readings of systolic and diastolic blood pressure (taking the pretreatment value as a covariant); and (b) for all other variables, comparison of means for independent groups based on relative change.

RESULTS
Participants' demographic and clinical characteristics were collected during enrollment ( Table 1). All participants remained in the study through its completion.
Adverse events occurred in both groups: four adverse events in three patients in the NeuroEPO group (20.0%) and two adverse events in one patient in the placebo group (9.1%), with no signifi cant differences between groups (p = 0.22) ( Table 2). All events were mild and likely related to the patient's positioning during product administration. One patient in the placebo group presented polyuria and nasal irritation in the second application, apparently unrelated to the product, as this effect was not reported for recombinant EPO or in the NeuroEPO tolerance study involving healthy subjects. [13] Three patients in the NeuroEPO group presented systemic events: immediate nausea on fi rst administration and in one case, vomiting. Event duration was three to ten hours, with a steady decline in symptom intensity over time. All events required only observation, and patients experiencing the adverse event spontaneously recovered without sequelae or need for medication.
Blood pressure readings were within normal range for participants in both groups, both before application of the product and in the fi ve post-application measurements ( Table 3). The hematological and biochemical parameters remained steady and with-

Original Research
in normal limits (Tables 4 and 5), and for this reason statistical tests (intra-and inter-group) were not performed. The means of each parameter for each group were similar before treatment and one week after treatment completion (Tables 3-5).

DISCUSSION
This clinical trial confi rms PD patients' short-term tolerance to intranasally administered NeuroEPO at the doses employed.
There is an imbalance in sex and severity in both groups. In our opinion, this disparity did not infl uence the results of the trial.

Sex
The prevalence of PD is lower in females than in males, in a proportion 1:3. [32,33] In our study, the placebo group had fewer women (n = 3) than the NeuroEPO group (n = 8). This is an effect of general randomization; it does not have the same implication as in a drug-effi cacy study. Due to its angiogenic effect, EPO could act in females as a hormone, playing a role in reproductive organ function; [34,45] this effect is not present in NeuroEPO, a form of EPO without the hematopoietic effect.
In addition, women of childbearing age/pregnant women were excluded in the recruitment process.
Severity PD severity was evaluated using the H&Y scale. The placebo group had only one patient in H&Y stage 1, while the NeuroEPO group had four. This imbalance was present in recruited participants (only 10 of the 46 patients who met the BBL criteria were in H&Y stage 1 while 36 were in H&Y stage 2). In our experience the symptoms of stage-1 patients are mild and patients rarely seek treatment, while stage-2 patients present bilateral motor compromise so they often volunteer to participate in clinical trials.
Tolerance We did not fi nd signifi cant changes in the hematological parameters. Four adverse events occurred in three patients treated with NeuroEPO (20%); all were mild and happened after the fi rst intranasal administration. The frequency of adverse events reported here is lower than that found in the NeuroEPO clinical trial conducted in healthy individuals, [13] where 80% of the subjects who received NeuroEPO reported at least 1 mild adverse event. This can be related to the lower doses and frequency employed in our study (1 mg, once per week for fi ve weeks), where for the fi rst time, patients with Parkinson were exposed to this molecule, in comparison with 1 mg every eight hours for four days. [13] Evidence showed good local tolerance. The mild adverse events at site of administration are similar to those observed in preclinical studies with both NeuroEPO and controls in the nasal irritation test and could be considered to be common when using this route of administration. [23] The nausea reported by three patients in the NeuroEPO group was not reported in the study with healthy volunteers. This adverse event could be explained by the positioning of the patient's head for half an hour during the administration of the drug (lowered at an angle to the body). These events occurred in the fi rst administration. In subsequent applications of the drug, head position was modifi ed, adopting a horizontal position, and this effect disappeared.
The data indicate an appropriate dose level at which NeuroE-PO can be safely nasally administered to PD patients. A phase 2-3 clinical trial of NeuroEPO is now underway to evaluate its possible benefi cial effects.

CONCLUSIONS
Nasal administration of NeuroEPO in patients with Parkinson disease (stages 1 and 2 on the Hoehn & Yahr Scale) is well tolerated. These results endorse further clinical study of this product for PD treatment.

ACKNOWLEDGMENTS
We thank the patients and their providers who volunteered to participate in this clinical trial, as well as all those who cooperated with the study: nurses, physicians, laboratory technicians and employees of CIREN, and the CIM specialists who provided the product and methodology used in the study.