Rapid and Safe Correction of Vitamin B12 Deficiency using Novel Methylcobalamin Nasal Spray

Background & Aims: Methylcobalamin nasal spray (NasoB12® ) has been developed to overcome the drawbacks of currently available therapeutic options like the poorly absorbed oral route and the painful intramuscular route. The present study was designed to evaluate the safety and efficacy of NasoB12® in patients with Vitamin B12 deficiency, with serum vitamin B12 (svB12) <200 pg/mL (148 pmol/L).


Sci Forschen
The administration of B12 by the nasal route has a 70-year history, starting from nasal drops used for treating patients suffering from pernicious anemia [8] to cyanocobalamin nasal spray in the recent decades. Considering the potential advantages offered by a nasal spray over oral/injectable routes, and of methylcobalamin over other forms, a novel methylcobalamin nasal spray has been recently developed and received regulatory approval (Troikaa Pharmaceuticals Limited). In a first-in-human randomized, single-dose, two-way crossover comparative bioavailability study conducted at a CRO for global regulatory approvals, it was found that the relative bioavailability of NasoB12® was 20% that of intramuscular methylcobalamin (Clinical Trial Registry: CTRI/2015/04/005687). In this study we have evaluated the safety and efficacy of NasoB12® in patient with Vitamin B12 deficiency (svB12 <200 pg/mL (148 pmol/L)).

Material and Methods
This prospective, multicentric, open-label, clinical study was conducted in accordance with declaration of Helsinki (59 th WMA General Assembly, Seoul, October 2008); Schedule Y (amended version, 2005) of CDSCO, India; Ethical Guidelines for Biomedical Research on Human Participants, ICMR (2006); and ICH, E6-Guideline for GCP. Enrollment for this clinical trial began in late 2015, and ended in late 2018. The trial was conducted at 7 urban tertiary care centers. Electrochemiluminescence immunoassay (ECLIA) and Spectophotometry methods were used to determine serum vitamin B12 and hemoglobin respectively.
The trial protocol and informed consent form were reviewed, approved, monitored and overseen by a duly constituted Independent Ethics Committee (IEC) at each site. Informed consent was obtained from each patient. The contract research organization, QED clinical services private limited, was responsible for data management, statistical analysis, clinical study report writing. Full details of the trial design, conduct, oversight, and analyses can be found in the protocol and statistical analysis plan.

Treatment
NasoB12® (Methylcobalamin 250 mcg/0.05 mL/actuation) was administered as 1 actuation per nostril, i.e. 2 actuations/500 mcg dose. Patients were treated with 500 mcg intranasal methylcobalamin total 2 actuations at a time on alternate days for 2 weeks (7 doses) in treatment phase and with 500 mcg intranasal methylcobalamin, once in a week for 4 weeks (4 doses) in maintenance phase. Total 11 doses of the study drug were given to the patient during the study. Since all forms of Vitamin B12 are destroyed by light, precautions were taken to prevent photodegradation included the use of amber coloured USP type I glass vial as primary pack and specially coated outer-cartons as a secondary pack to protect the formulation from light.

Sample size
Assuming an overall success rate of 75% at treatment phase and 85% at maintenance phase compared to null hypothesis of 60% success rate, at least 32 efficacy evaluable subjects were required to complete the study. By assuming 40% drop out at treatment phase and 25% dropout at maintenance phase, 72 subjects were required to achieve 90% power with 5% level of significance. [Software: PASS (Power Analysis and Sample size) version 11]. However, as per local regulatory recommendations, 100 patients were to be enrolled in this study.

Efficacy analysis
For efficacy assessment, primary efficacy endpoint was evaluated as the proportion of patients achieving svB12 levels >200 pg/mL (148 pmol/L) after the 2 nd and 6 th week. The secondary endpoints included svB12 and hemoglobin levels after the 1 st , 2 nd , 4 th , 6 th weeks of treatment. Further the Physician's Global Assessment of Efficacy was assessed as secondary end point. Efficacy assessment was done on Per Protocol Population (PP) which consisted of the subjects who completed the study in compliance with the protocol and had no major protocol violations that impacted the efficacy assessments.

Safety analysis
Incidence/severity of Treatment-Emergent Adverse Events (TEAEs), abnormality in vital signs/physical examination, and global tolerability were included in safety assessment. Safety analyses were performed on safety population where all subjects who received at least one dose of the study medication were included. Summary statistics were presented as numbers and percentages of patients experiencing treatment emergent adverse events.

Statistical analysis
In general, continuous data was presented by descriptive statistics i.e. n, mean, standard deviation, median, minimum and maximum values. Categorical data was presented as frequency (n) and percentage. Visit 1 (Day-7 to 0) was considered as the Baseline visit.
The statistical analysis was done in accordance with the ICH-E9 guidelines "Statistical Principles for Clinical Trials" and guidelines from CDSCO and USFDA guideline and applicable regulatory guidelines. All reports outputs were produced using SAS® version 9.4 in a secure and validated environment. (Statistical analysis plan, version 1.0:27NOV2017).

Patient enrollment
A total of 193 patients were screened, and 103 were enrolled ( Figure 1).
Eligibility was based on the Vitamin B12<200 pg/mL (148 pmol/L) & Folate levels > 3 ng/mL. 81 patients (78.6%) completed the study and were included in the efficacy analysis (Per Protocol Population). Total 103 patients were evaluated for safety analysis.

Demographics and other baseline characteristics and outcome
The mean age of the patient was 38.45 years with BMI of 23.83 kg/m 2 . There were 56.3% of male and 43.7 % were of female patients (Table 1A).  Figure 2).
A transient statistically significant change was observed in B12 levels after the 4 th week as compared to the 2 nd week, which was considered clinically acceptable considering the reduction in dosing frequency from alternate-day to once-weekly dosing. (Change at 4 th and 6 th weeks were: -90.93 pg/mL; (p= 0.027) and -41.99 pg/mL (p=0.683) respectively. Thus, by the end of the 6 th week, the decrease in B12 level was insignificant as compared to 2 nd week. The significant aspect of weekly therapy is that mean B12 levels were maintained well above 400 pg/mL.

02:
Due to suspected adverse event.

03:
Non-Compliance to drug treatment

12:
Patient request to be withdrawn.

04:
Any other

patients discontinued
The patient is instructed to self-administer drug at home on days 3 & 5.
The patient is instructed to self-administer drug at home on days 9, 11 and 13.
The patient is instructed to self-administer drug at home on day 22, based on the lab. report's ndings on day 18. The patient is instructed to self-administer drug at home on day 36.   A review of 'Prior and Concomitant Medication' confirmed that only 3.7% (3/81) patients received iron (Ferrous Sulfate) 200 mg/day (n=2/81) or 400 mg/day (n=1/81) for iron deficiency and all completed the study. An analysis of the 96.3% (78/81) subjects not receiving iron showed similar outcomes to the overall ("composite") population (Appendix: Supplementary Figure SF1).
Hemoglobin levels progressively increased from baseline hemoglobin of 11.92 to 12.39, 12.79, 13.28 and 13.63 g/dL after the 1 st , 2 nd , 4 th , and 6 th weeks respectively. In other words, the rise of hemoglobin was seen irrespective of iron supplementation. Reticulocyte count, demonstrated trend consistent with erythpoietic activity: a borderline higher value at 1 st and 2 nd weeks; and once hemoglobin approached 12.76 g/dL, the reticulocyte count started normalizing; but the hemoglobin kept rising till normal (Appendix: Supplementary Figure SF2).

Safety analysis
NasoB12® was well-tolerated. The most common adverse events were gastrointestinal, metabolism and nutrition disorders (3.9% [n=4] patients each). No deaths/ serious/ severe TEAEs were reported. Overall 14% patients reported 26 events of which 11 were considered treatment-related. One patient experienced a mild non-serious TEAE (nasal congestion) which resolved without any treatment being required (Table 2 and 3).
The physicians' global assessment of tolerability: Except for 1 patient, the investigators rated the global tolerability as "excellent" (no adverse event reported) or "good" (mild adverse event; resolved spontaneously; no stoppage of treatment) for all patients in the study (86.4% [n=76] and 12.5% [n=11] patients, respectively).
The SCCD concept was not well established when this study was designed and hence the protocol used the cut-off of svB12<200 pg/ mL (148 pmol/L) which was prevailing at that point of time (in 2015). Current scientific consensus (e.g. AAFP) targets the svB12 >296 pmol/l (400 pg/ml and above) as normal levels; [10] hence we would like to utilize this perspective while discussing our results.
Our study did not target symptomatic adults, but included all types of individuals with low B12 levels, because researchers today believe in the existence of a wide spectrum of patients with no symptoms to mild to severe manifestations which could be neuropsychiatric (e.g. neuropathic pain; cognitive impairment), hematologic (megaloblastic anemia; pancytopenia), obstetric (Neural Tube Defects; growthretardation) and pediatric (cognitive; anemia) [2,[11][12][13][14].
A positive outcome was achieved during the study, as 100% and 95% of patients achieved the primary efficacy endpoint of B12 >200 pg/mL (148 pmol/L) after the 2 nd and 6 th week respectively.
From the very first week of NasoB12® therapy, the svB12 levels crossed well above 400 pg/mL, into the "normal range" [2]. Subsequently normal svB12 levels were maintained at all time-points, including the 500 mcg once-weekly maintenance therapy. This makes NasoB12® an attractive alternative for both: the treatment as well as the maintenance therapy of B12 deficient patients.
The mean Hemoglobin of B12 deficient patients consistently increased each week from baseline hemoglobin of 11.94 g/dL to 13.58 g/dL at the end of the study (Figure 2), and where only 3.7% patients received a concomitant iron supplementation. Our study corroborates the concept that a rapid rise in B12 levels is associated with a rise in hemoglobin. Andrès E, et al. [13] previously demonstrated this trend in hematological outcomes in 10 patients with B12 malabsorption and cobalamin deficiency treated over 3 months. In contrast, administration of NasoB12® in the current work gave the rise in Hemoglobin (0.45 g/dL, p=0.0002) as early as the 1 st week and this trend was sustained till the 6 th week, when normal hemoglobin levels were attained (corresponding to a rise of 1.64 g/dL, p <0.0001).  Irrespective of the baseline iron stores, the baseline hemoglobin values were similar and patients experienced a consistent weekly rise in Hemoglobin, both in the treatment phase (alternate-day NasoB12) as well as in the maintenance phase (once-weekly NasoB12).
In our trial 4.9% (n=4) patients did not experience a rise in B12 levels. This appears modest compared to 33% (n=17/51) non-responders reported, after patients received hydroxocobalamin nasal drops for 3 months [14]. The authors suggested that the non-responders had either low B12 protein saturation or a very rapid urinary excretion. In this study the authors used 5,000 mcg one nasal drop in each nostril, twice per week, [14] while the current work employs the scheme with administration of a smaller dose i.e. 500 mcg every alternate day for seven doses, followed by weekly dosing. Small but frequent doses of B12 are known to be more efficient for upload of B12 into the tissues, compared to large but rare doses because low doses do not overload the specific transport system of B12 (in intestine and blood). Excessive vitamin B12 in the circulation, e.g., such as after injections, usually exceeds the binding capacity of specific protein, (Transcobalamin) and is excreted in the urine [1]. NasoB12® was found to be safe, well tolerated and no serious adverse events were reported..

The limitations of other approaches
It is now known that there are at least four forms of vitamin B12 (cobalamin), i.e. cyanocobalamin, hydroxocobalamin and two coenzymes forms which are biochemically active, namely methylcobalamin and adenosylo-cobalamin [1,[15][16][17].
Nasal formulations using Cyanocobalamin/Hydroxocobalamin: Cyanocobalamin nasal spray, (Nascobal, USA) is approved only for weekly maintenance therapy, a month after receiving 100 mcg intramuscular B12. The current usage pattern of Cyanocobalamin is that all hematologic parameters should be normal when beginning treatment with Cyanocobalamin nasal spray [18].
Scientific data suggest that the relative bioavailability of the nasal formulations of cyanocobalamin and hydroxocobalamin (versus intramuscular) is 2% [19] to 6.1% [18] and 2% to 5% [20] respectively. Interestingly, authors have concluded that it was time to drop cyanocobalamin [30].
Oral Formulations: Oral vitamin B12 supplementation is considered to be less reliable. For example, a pharmacokinetic study, conducted by Castelli et al, on a "conventional" commercial oral formulation demonstrated an absolute bioavailability of only 2.16% [21].

Unpredictable and unreliable absorption pattern
3. Not reliable for the treatment of pernicious anemia 4. Not suitable for patients with nausea, vomiting or who are unable to tolerate oral medication    Regulatory Activities (MedDRA) dictionary (version: 20.0) Patients having multiple events within same system organ class were counted only once in respective system organ class under any event. Patients having multiple events with same preferred term were counted only once within the respective PT. System organ class has been sorted alphabetically and preferred terms have been sorted within each system organ class alphabetically. Sublingual Formulations: While, oral therapy may not be appropriate for those with swallowing difficulties, an alternate route of therapy for vitamin B12 deficiency is by the sublingual mucosa [24]. There is less clinical data on sublingual formulations of Vitamin B12. However, since vitamin B12 is a large molecule, which is hydrophilic, the absorption needs to be confirmed. Absorption of vitamin B12 from sublingual formulation may actually take place from the GI tract, after saliva has cleared the dose from the oral cavity. Thus oral or sublingual supplementation requires a higher dose of vitamin B12 to be administered and does not provide a predictable increase of vitamin B12 in the body.

Parenteral administration; intramuscular (IM) or intravenous (IV):
IM/IV is often preferred to reliably treat vitamin B12 deficiency by clinicians. Patients with previous stomach and terminal ileum resections are often treated with IM or IV vitamin B12 injections. Usually, the patient is put on a lifetime regimen of monthly maintenance injections. From a clinical perspective, vitamin B12 injection is painful and difficult to administer to disabled or elderly patients. Besides, the patient needs to visit a health professional repeatedly, leading to inconvenience and hence, compromised compliance.

Limitations of parenteral therapy:[25-27]
1. Not ideal route for vitamin B12 supplementation in elderly due to loss of muscle mass (Sarcopenia) and they are often on anticoagulants 2. Frequent need of health professional's assistance, makes patient dependent. 3. Very inconvenient due to frequent visits to the clinics, hence promotes non-compliance. 4. Painful and long term injection lead to inconvenience and noncompliance 5. Injection site related adverse reactions. 6. Allergic reactions and severe anaphylactic reactions are reported. 7. Cost of overall treatment is high.
In contrast to oral Vitamin B12 therapy, Naso B12® is associated with rapid, consistent, predictable and reliable absorption. Naso B12® can be self-administered with high compliance, even by patients with swallowing problems, nausea or vomiting, or with absorption issues, including pernicious anemia, post-bariatric surgery and malabsorption.
In contrast to injectable vitamin B12 therapy, Naso B12® has lower cost, lower manpower burden, does not require a patient of an accompanying person to visit a healthcare facility, is self-administered and painless, has few adverse events, and has high compliance.
NasoB12® is the world's first Methylcobalamin nasal spray, designed and delivered keeping light-sensitivity in mind.
NasoB12® was well tolerated and associated with a rapid rise of hemoglobin.
Our data opens the door for more research on how the formulation delivery system and packaging, which protect vitamin B12 from light, would lead to clinically meaningful outcomes in the real world.

Study limitations and directions for future research
Our intention was not to study the underlying cause for low Vitamin B12 levels, but rather, to correct them, irrespective of the underlying causes. Future researchers may well study the effect on various dietary patterns and etiologic subtypes. This study excluded patients with 5. Concerns about swallowing difficulties 6. Non compliance due to long term daily dosing 7. Due to poor absorption, cost to benefit ratio does not favor oral therapy in future research these parameters will be included. Given the rise of Hemoglobin with NasoB12®, future studies could be designed for pernicious anemia, and also focus on the children, adolescents, women, especially pregnant women and the elderly [29,30,35].

Conclusion
In the present clinical study, at the end of treatment phase, a statistically significant rapid rise in mean serum vitamin B12 levels was achieved and maintained till the end of the maintenance phase. NasoB12® was associated with a favorable hematopoietic response. Therefore, NasoB12® can be beneficial for the treatment and maintenance of serum vitamin B12 levels in patients with low serum Vitamin B12 levels. Thus, NasoB12® offers a fast and consistent alternative to oral or sublingual B12 therapy; and a simpler and convenient alternative to B12 injections, without safety concerns.