Vitamin B 3 Supplementation for Optic Neuropathies: A Comprehensive Review

Abstract

Optic neuropathies, such as glaucoma, are some of the leading causes of irreversible blindness worldwide. There has been a lot of research for potential therapies that could attenuate and even reduce the impact of the pathological pathways that lead to the loss of retinal ganglion cells (RGCs). In recent years, vitamin B₃ (nicotinamide) has gained some interest as a viable option for these neurodegenerative diseases due to its fundamental role in enhancing the mitochondria metabolism of the RGCs. This review focuses on elucidating the impact of vitamin B₃ on retinal cells, especially when in a dysfunctional state like what happens in optic neuropathies, especially glaucoma. This review also summarizes the existing and future research on the clinical effects of vitamin B₃ in these optic neuropathies, and determines appropriate recommendations regarding its dosing, efficacy, and eventual side effects.

Introduction

The optic nerve is anatomically an extension of the central nervous system (CNS) and consists of the retinal ganglion cell (RGC) axons. Up to 90% of the RGCs extend to the lateral geniculate nucleus, which then transmits information to the visual cortex.¹

The RGCs are the main damaged neurons in glaucomatous optic neuropathy, which is the most common form of neurodegeneration involving the CNS and the leading cause of irreversible vision loss worldwide.² Major risk factors for glaucoma include increased intraocular pressure (IOP), age, and race (west African).³ However, in many individuals, glaucoma continues to progress despite significant IOP reduction.⁴

RGCs are responsible for transmitting visual information from the retina to the brain and, therefore, require a relatively large amount of energy.^5,6 Since lamina cribrosa is where the axons of the RGCs are unmyelinated, it is more susceptible to early microenvironmental changes such as mitochondrial dysfunction.^2,5

Studies in DBA/2J mice models (with chronic elevated IOP and glaucoma) and in an inducible model of ocular hypertension in rats have shown that these mitochondrial changes occur even before detectable neurodegeneration⁷ and, therefore, there is a need to investigate neuroprotective strategies targeting RGC health.

It has been shown that even after a prolonged period of functional loss due to acute and chronic elevated IOP insults, RGC function can be restored with nicotinamide supplementation.⁸ In contrast, there is also growing evidence supporting a link between vitamin intake and neuronal function, maintenance, and recovery.^9,10

Nicotinamide (or niacinamide or NAM), 1 of 3 vitamers of vitamin B₃ [along with niacin/nicotinic acid (NA) and nicotinamide riboside (NR)], is an essential nutrient that constitutes a precursor of the coenzyme nicotinamide adenine dinucleotide (NAD+), which is a critical molecule for mitochondrial health throughout the body, especially in neurons.^10,11 Note that intestinal flora can convert both NR and NAM to NA¹² and that NR has a slightly better bioavailability than NAM in humans.^13,14

NAD+ levels decrease with normal aging and are reduced in patients with primary open-angle glaucoma (POAG), which may correlate NAD+ levels with susceptibility to neurodegeneration.^2,5 Thus, the reduction of NAD may limit adenosine triphosphate (ATP) production, and result in an inability to provide sufficient energy to maintain cell health, ultimately leading to RGC degeneration.⁵

Currently, there is an unmet need for neuroprotective agents, and NAM/NAD+ supplementation would be a promising intervention in diseases such as glaucoma, alone or possibly in combination with other nutraceutical agents such as saffron, zeaxanthin, or pyruvate.^4,15

Discussion

Vitamin B₃ function in the retina

The retina is one of the most energy consuming tissues in the body and this energy is vital to maintain its homeostasis.

Optic neuropathies are diseases in which the function of retinal cells is compromised. Glaucoma is one of them and it is characterized by progressive dysfunction and loss of RGCs.¹⁶ Although elevated IOP is the main modifiable target, glaucomatous damage can arise in eyes with IOP in the normal range.¹⁷

Nicotinamide is a precursor of the coenzyme NAD+, which is an essential molecule for mitochondrial health throughout the body.¹¹ Mitochondrial dysfunction and decreased NAD+ levels are hallmarks of aging in many organs,^15,18 and their association with CNS impairment has been documented since the discovery of pellagra, in which the neurological dysfunction is one of the early manifestations of the disease caused by low levels of vitamin B₃.^13,15

Vitamin B₃ role in optic neuropathies

Specifically, there is a strong association between NAD+ deficiency and some retinal neurodegenerative disorders. Leber hereditary optic neuropathy (LHON) is the most common disorder caused by mutations in mitochondria and it is caused by variants in genes that affect the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase system. Another disorder associated with nicotinamide deficiency is OPA1-related dominant optic atrophy. These 2 share an overutilization of nicotinamide to provide the redox cycle with enough electrons for NADH dehydrogenase, resulting in a deficiency of NAD+.¹⁹

A note on Leber's congenital amaurosis, an inherited disorder caused by mutations in NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1), which is a key NAD+ biosynthetic enzyme, underlining the importance of this molecule for retinal neurons.²⁰ In addition, in ischemic optic neuropathies (IONs), the physiopathology is not yet understood, but due to their ischemic mechanism, stress oxidation levels are known to be high.²¹ Therefore, this is another group of diseases in which NAD+ may potentially act (Fig. 1). Finally, glaucoma is also profoundly related to NAD+, mainly due to the high energy demand of RGCs (Fig. 2).^15,20

FIG. 1.

Overview of vitamin B₃'s potential role in ischemic optic neuropathy, resulting in anti-inflammatory, antioxidant, and antiapoptotic effects. IL1β, interleukin 1beta; Nf-κB, nuclear factor kappa B; Nrf2, nuclear factor erythroid-2-related factor 2; TNFα, tumor necrosis factor alpha.

FIG. 2.

Summary of the potential neuroprotective effects of nicotinamide supplementation in glaucoma. IOP, intraocular pressure; NAD+, nicotinamide adenine dinucleotide; RGCs, retinal ganglion cells.

Given the decrease of NAD+ with aging, retinal neurons become more vulnerable due to limited ATP production, resulting in mitochondrial dysfunction, and ultimately RGC degeneration.¹³

Nzoughet et al. demonstrated in a cohort of 34 patients with POAG a significantly lower plasma concentration of nicotinamide versus a control group.⁵ The combination of the decline in NAD+ levels with age¹¹ and the stress of elevated IOP causes mitochondria to become dysfunctional and predisposes to RGC loss.²⁰ Another study in mice corroborated that low NAD+ increases the sensitivity of RGCs to metabolic damage, particularly during periods of elevated IOP.¹¹

Because RGC death is preceded by several structural and functional changes that are not clinically defective, there may be a window of opportunity for neuroprotective interventions such as nicotinamide. Studies have focused on the potential protective effects of nicotinamide in glaucoma (NAMinG).

Vitamin B₃ supplementation in clinical studies

Animal studies

Williams et al. conducted a study on the correlation between nicotinamide and glaucoma using DBA/2J mice, a mouse model that simulated glaucoma. They identified mitochondrial abnormalities and decreased concentrations of NAD+ in these models compared with controls.¹² Subsequently, it was shown that the exogenous administration of NAD+ in the form of nicotinamide supplementation (at a dose equivalent to 2.5 g per day for a 60 kg person) was capable of restoring RGC function in aged and glaucoma-prone mice. This resulted in a 10-fold decrease in the likelihood of developing glaucoma.^7,12,15

Chou et al. and Zhang et al. also studied the effects of nicotinamide (equivalent dose of 2 g per day) on the RGC function in mice models using pattern electroretinography. They both observed an improvement in RGC density in the treated group compared with the control group.^22,23

Another study by Tribble et al. on mice with ocular hypertension revealed beneficial effects of nicotinamide treatment. They showed that at a cellular level, nicotinamide can improve mitochondrial function, and induce a neuroprotective effect on these impaired RGCs.²⁴

Chen et al. is the only study to focus on the effects of vitamin B₃ in anterior ION mice models. They found that vitamin B₃ treatment had a neuroprotective role, reducing inflammation and RGC death through the inhibition of oxidative stress and mitochondrial dysfunction.²¹

Human studies

In an observational study conducted in the United States of America with >5,700 patients, researchers identified a correlation between daily niacin consumption and glaucoma in individuals older than 40 years. The study also found that a greater intake of niacin (>21 mg per day, quartile 3–4) decreased the risk of glaucoma in comparison with those with a lower intake, regardless of IOP levels. They suggested that the protective effect of niacin is not through IOP reduction.¹³

Similarly, a large observational study conducted in Korea revealed that reduced niacin intake is linked to glaucoma, specifically normal tension glaucoma (NTG).²⁵ Although the pathophysiology of NTG remains incompletely elucidated, the mechanism of cellular damage bears certain similarities to those of other forms of glaucoma.¹⁷ Therefore, nicotinamide is believed to have potential benefits in this specific type of glaucoma.

In a clinical trial comprising 57 glaucoma patients, Hui et al. found that administering 3 g per day of NAM for a duration of 12 weeks led to a notable enhancement in inner retinal function, measured by electroretinography and standard automated perimetry.⁸

In a recent randomized clinical trial, De Moraes et al. treated 21 patients with POAG with a combination of nicotinamide and pyruvate and observed a statistically significant improvement in visual function based on the number of improving test locations on standard automated perimetry. In addition, they also noted a significant variation in the rate of change of visual fields (evaluating the pattern standard deviation), resulting in improved global perimetric sensitivity.⁴

All these studies suggest an association between nicotinamide and other vitamin B₃ precursors with neuroprotective effects in glaucoma. In contrast, a review highlighted that although there is some evidence suggesting that niacin may lower the risk of glaucoma, there is currently inadequate data to support vitamin supplementation.²⁶

Crucial details regarding the methods and results were extracted from selected articles and summarized in Table 1. To date, no previous systematic reviews with meta-analysis have been identified on this topic.

Table 1.

Summary of Selected Studies on Vitamin B₃ for Optic Neuropathies

Study	Design	Subjects (N)	Vitamin B₃ form	Human equivalent	Outcomes	Results
De Moraes et al.⁴	Randomized clinical trial	Early-to-moderate treated glaucoma (N = 42)	NAM 1,000 up to 3,000 mg/day + Pyruvate 1,500 up to 3,000 mg/day		Number of visual field test locations improvement and rate of change of visual field global indices	Improvement in visual field test locations and rate of change in pattern standard deviation
Hui et al.⁷	Randomized clinical trial	Early-to-moderate treated glaucoma (N = 57)	NAM 1,500 mg/day for 6 weeks followed by 3,000 mg/day for another 6 weeks		Inner retinal function measured by ERG photopic negative response	Better improvement in inner retinal function relative to control
Williams et al.¹¹	Animal study	DBA/2J mice—simulating glaucoma	NAM Oral 550 and 2,000 mg/kg per day	2,673 and 9,720 mg/day in 60 kg human	Optic nerve degeneration assessed by paraphenylenediamine staining	Dietary supplementation with NAM reduces vulnerability to glaucoma
Chen et al.¹⁸	Animal study	ION simulating mice model (N = 30)	Unspecified vitamin B₃ Oral 500 mg/kg per day	2,430 mg/day in 60 kg human	RGC function (and density) assessed by flash visual-evoked potential	Vitamin B₃ has neuroprotective properties by nerve regeneration and functional recovery of RGCs
					RGC density assessed by fluorogold retinal staining
					RGC survival assessed by TUNEL assay
Zhang et al.²⁰	Animal study	DBA/2J mice—simulating glaucoma (N = 60)	NR Injection of 1,000 mg/kg	N/A	RGC survival, assessed by counting cells in retinal flat mounts	Prophylactic systemic treatment with NR is protective in mouse models with RGC damage
					RGC function assessed by pattern ERG
					Suppressed retinal inflammation assessed by immunofluorescence
Tribble et al.²¹	Animal study	Glaucoma simulating mice model (N = 15)	NAM Oral 200, 400, and 800 mg/kg per day	900, 1,940, and 3,888 mg/day in 60 kg human	Loss of neuroretinal rim by OCT	NAM buffers against metabolic insufficiency and provides neuroprotection against glaucoma-related stresses
Tribble et al.²¹	Animal study	Glaucoma simulating mice model (N = 15)	NAM Oral 200, 400, and 800 mg/kg per day	900, 1,940, and 3,888 mg/day in 60 kg human	RGC loss and nuclear shrinkage assessed by counting cells in retinal flat mounts

ERG, electroretinogram; ION, ischemic optic neuropathy; NAM, nicotinamide; NR, nicotinamide riboside; OCT, optic coherence tomography; RGCs, retinal ganglion cells; TUNEL, terminal deoxynucleotidyl transferase dUTP nick and labeling.

Vitamin B₃ safety profile

All supplementation products are well tolerated and safe when given within the therapeutic range. Mild side effects, such as flushing, chills, and gastrointestinal symptoms, may occur, but hepatotoxicity is a potential risk with toxic doses.¹⁴ Niacin is the precursor that is most likely to cause adverse effects, particularly at doses >50 mg per day. Daily doses of nicotinamide up to 3,000–3,500 mg are well tolerated, although chronic intake might predispose some gastric disturbances.^15,20,27 NMN (from 100 to 500 mg per day) and NR (up to 2,000 mg per day) have a similar safety profile, but their long-term effects in humans are yet to be determined.^15,20 Regarding ocular effects, there is only 1 case report of elevated IOP after taking 500 mg of oral niacin.²⁸

Upcoming clinical trials

Right now, 4 known ongoing clinical trials are worth mentioning.

Nicotinamide in glaucoma

This is a randomized, placebo-controlled, multicenter, phase 3 trial from the University College of London (in the United Kingdom, main investigator: Dr David Garthway-Heath). They estimate a total of 496 patients with open-angle glaucoma (OAG) who will receive 3,000 mg per day of nicotinamide (initial dose of 1,500 mg per day for 6 weeks) versus placebo over 27 months. The primary outcomes will focus on visual field progression and quality of life. The scheduled end date is November 2026.²⁹

The glaucoma nicotinamide trial

This is a prospective, randomized, placebo-controlled, double-masked trial from the Umea University (in Sweden) and the Centre for Eye Research Australia (in Australia). They estimate a total of 660 patients with OAG who will receive 3,000 mg per day of nicotinamide (initial dose of 1,500 mg per day for 6 weeks) versus placebo >2 years. The outcome will focus on visual field progression. The scheduled end date is December 2026.³⁰

NR as a neuroprotective therapy for glaucoma

This is a prospective, randomized, placebo-controlled trial from the Chinese University of Hong Kong (in China). A total of 125 patients with OAG will receive 300 mg per day of NR versus placebo over 2 years. The main outcome will be the progression of the retinal nerve fiber layer in the optic coherence tomography (OCT) and visual field progression. The scheduled end date is March 2023.¹⁴

Nicotinamide and pyruvate for open angle glaucoma: a randomized clinical study

This is an interventional, randomized, clinical trial from Columbia University (in the United States of America). They estimate a total of 188 patients with OAG who will receive nicotinamide and pyruvate (doses not specified) versus placebo over 20 months. The primary outcomes are changes in the central visual field and in the retinal nerve fiber layer and ganglion cell layer by OCT. The scheduled end date is January 2028.³¹ All these trials will recruit patients with early-to-moderate OAG, including those with POAG, normal-tension glaucoma, and pseudoexfoliation glaucoma, who have not undergone prior glaucoma surgery. Note that the final 3 studies will exclude patients with IOP >25–30 mmHg, and the third study will exclude patients who have experienced visual field progression. In addition, the last study will exclude patients with any previous incisional glaucoma surgery. Throughout all trials, the patients will continue their antihypertensive drops.

Summary

To conclude, it is crucial to elucidate the connection between the homeostasis of mitochondrial metabolism and nicotinamide in retinal cells, therefore, linking their dysfunction with optic neuropathies such as LHON or glaucoma. Ongoing trials will help to better determine the role of nicotinamide in healthy, untreated, and treated patients with optic neuropathies. Further research is needed to establish the most effective doses of vitamin B₃ supplementation and the optic neuropathies that may benefit the most from such treatment.

Footnotes

Acknowledgments

This study was developed in the context of the project Smart Drug Delivery Device for Glaucoma Treatment: SmartGlauco (PTDC/CTM-REF/2679/2020). I am thankful for the opportunity to be part of such an interesting and stimulating project.

Authors' Contributions

J.A. contributed to conceptualization (equal), writing—original draft (lead), review, and editing (equal); T.C., M.V., and C.M. were involved in writing—original draft (equal); F.T.V. and L.A.P. carried out writing—review and editing (equal) and supervision (equal); Q.F. and I.P. were involved in writing—review and editing (equal) and validation (equal); and J.T.F. was in charge of conceptualization (lead), writing—original draft (equal), review and editing (lead), and supervision (lead).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

References

Yücel

. Central nervous system changes in glaucoma. J Glaucoma, 2013; 22(5 SUPPL.1):55–56; doi: 10.1097/IJG.0b013e3182934a55

Liebmann

, Cioffi

. Nicking glaucoma with nicotinamide?. N Engl J Med, 2017; 376(21):2079–2081.

Williams

, Harder

, John

SWM

, et al. Glaucoma as a metabolic optic neuropathy: Making the case for nicotinamide treatment in glaucoma. J Glaucoma, 2018;(12):1161–1168; doi: 10.1097/IJG.0000000000000767.Glaucoma

De Moraes

, John

SWM

, Williams

, et al. Nicotinamide and pyruvate for neuroenhancement in open-angle glaucoma: A phase 2 randomized clinical trial. JAMA Ophthalmol, 2022; 140(1):11–18; doi: 10.1001/jamaophthalmol.2021.4576

Nzoughet

, de la Barca

JMC

, Guehlouz

, et al. Nicotinamide deficiency in primary open-angle glaucoma. Invest Ophthalmol Vis Sci, 2019; 60(7):2509–2514; doi: 10.1167/iovs.19-27099

Calkins

. Critical pathogenic events underlying progression of neurodegeneration in glaucoma. Prog Retin Eye Res, 2012; 31(6):702–719; doi: 10.1016/J.PRETEYERES.2012.07.001

Williams

, Harder

, Foxworth

, et al. Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice. Science (1979), 2017; 756:1–18; doi: 10.1126/science.aal0092.Vitamin

Hui

, Tang

, Williams

, et al. Improvement in inner retinal function in glaucoma with nicotinamide (vitamin B3) supplementation: A crossover randomized clinical trial. Clin Exp Ophthalmol, 2020; 48(7):903–914; doi: 10.1111/ceo.13818

Ramdas

, Wolfs

RCW

, Kiefte-De Jong

, et al. Nutrient intake and risk of open-Angle glaucoma: The Rotterdam Study. Eur J Epidemiol, 2012; 27(5):385–393; doi: 10.1007/s10654-012-9672-z

10.

Fricker

, Green

, Jenkins

, et al. The influence of nicotinamide on health and disease in the central nervous system. Int J Tryptophan Res, 2018; 11:1178646918776658; doi: 10.1177/1178646918776658

11.

James

. The role of NAD+ and |(Vitamin B3) in glaucoma: A literature review. J Nutri Sci Vitaminol, 2022; 68(3):151–154.

12.

Williams

, Harder

, Cardozo

, et al. Nicotinamide treatment robustly protects from inherited mouse glaucoma. Commun Integr Biol, 2018; 11(1):e1356956; doi: 10.1080/19420889.2017.1356956

13.

Taechameekietichai

, Chansangpetch

, Peerawaranun

, et al. Association between daily niacin intake and glaucoma: National health and nutrition examination survey. Nutrients, 2021; 13(12):1–13; doi: 10.3390/nu13124263

14.

Leung

CKS

, Ren

, Chan

PPM

, et al. Nicotinamide riboside as a neuroprotective therapy for glaucoma: Study protocol for a randomized, double-blind, placebo-control trial. Trials, 2022; 23(1):45; doi: 10.1186/s13063-021-05968-1

15.

Cimaglia

, Votruba

, Morgan

, et al. Potential therapeutic benefit of nad+ supplementation for glaucoma and age-related macular degeneration. Nutrients, 2020; 12(9):1–21; doi: 10.3390/nu12092871

16.

Levin

. Neuroprotection in optic neuropathy. Asia Pac J Ophthalmol (Phila), 2018; 7(4):246–250; doi: 10.22608/APO.2018299

17.

Killer

, Pircher

. Normal tension glaucoma: Review of current understanding and mechanisms of the pathogenesis. Eye, 2018; 32(5):924; doi: 10.1038/S41433-018-0042-2

18.

Verdin

. NAD+ in aging, metabolism, and neurodegeneration. Science (1979), 2015; 350(6265):1208–1213; doi: 10.1126/SCIENCE.AAC4854

19.

Bocca

, Le Paih

, Chao De La Barca

, et al. A plasma metabolomic signature of Leber hereditary optic neuropathy showing taurine and nicotinamide deficiencies. Hum Mol Genet, 2021; 30(1):21–29; doi: 10.1093/hmg/ddab013

20.

Jadeja

, Thounaojam

, Bartoli

, et al. Implications of NAD+ metabolism in the aging retina and retinal degeneration. Oxid Med Cell Longev, 2020; 2020:12; doi: 10.1155/2020/2692794

21.

Chen

T-W

, Wu

P-Y

, Wen

Y-T

, et al. Vitamin B3 provides neuroprotection via antioxidative stress in a rat model of anterior ischemic optic neuropathy. Antioxidants, 2022; 11(12):2422; doi: 10.3390/ANTIOX11122422

22.

Chou

, Romano

, Amato

, et al. Nicotinamide-rich diet in dba/2j mice preserves retinal ganglion cell metabolic function as assessed by perg adaptation to flicker. Nutrients, 2020; 12(7):1910; doi: 10.3390/nu12071910

23.

Zhang

, Zhang

, Chrenek

, et al. Systemic treatment with nicotinamide riboside is protective in two mouse models of retinal ganglion cell damage. Pharmaceutics, 2021; 13(6):1–16; doi: 10.3390/pharmaceutics13060893

24.

Tribble

, Otmani

, Sun

, et al. Nicotinamide provides neuroprotection in glaucoma by protecting against mitochondrial and metabolic dysfunction. Redox Biol, 2021; 43:101988 ; doi: 10.1016/j.redox.2021.101988

25.

Jung

, Kim

, Park

. Dietary niacin and open-angle glaucoma: The Korean National Health and Nutrition Examination Survey. Nutrients, 2018; 10(4):387; doi: 10.3390/NU10040387

26.

Fahmideh

, Marchesi

, Barbieri

, et al. Non-drug interventions in glaucoma: Putative roles for lifestyle, diet and nutritional supplements. Surv Ophthalmol, 2022; 67(3):675–696; doi: 10.1016/J.SURVOPHTHAL.2021.09.002

27.

Hwang

, Song

. Possible adverse effects of high-dose nicotinamide: Mechanisms and safety assessment. Biomolecules, 2020; 10(5):1–21; doi: 10.3390/biom10050687

28.

Tittler

, de Barros

DSM

, Navarro

JBVK

, et al. Oral niacin can increase intraocular pressure. Ophthal Surg Lasers Imaging, 2008; 39(4):341–342; doi: 10.3928/15428877-20080701-17

29.

Anonymous. Nicotinamide in Glaucoma—Full Text. View—ClinicalTrials.Gov. n.d. Available from: https://clinicaltrials.gov/ct2/show/NCT05405868 [Last accessed: March 26, 2023].

30.

Anonymous. The Glaucoma Nicotinamide Trial—Full Text. View—ClinicalTrials.Gov. n.d. Available from: https://clinicaltrials.gov/ct2/show/NCT05275738 [Last accessed: March 26, 2023].

31.

Anonymous. Nicotinamide and Pyruvate for Open Angle Glaucoma: A Randomized Clinical Study—Full Text. View—ClinicalTrials.Gov. n.d. Available from: https://clinicaltrials.gov/ct2/show/study/NCT05695027 [Last accessed: June 12, 2023].

Vitamin B 3 Supplementation for Optic Neuropathies: A Comprehensive Review

Abstract

Introduction

Discussion

Vitamin B3 function in the retina

Vitamin B3 role in optic neuropathies

Vitamin B3 supplementation in clinical studies

Animal studies

Human studies

Vitamin B3 safety profile

Upcoming clinical trials

Nicotinamide in glaucoma

The glaucoma nicotinamide trial

NR as a neuroprotective therapy for glaucoma

Nicotinamide and pyruvate for open angle glaucoma: a randomized clinical study

Summary

Footnotes

Acknowledgments

Authors' Contributions

Author Disclosure Statement

Funding Information

References

Vitamin B₃ function in the retina

Vitamin B₃ role in optic neuropathies

Vitamin B₃ supplementation in clinical studies

Vitamin B₃ safety profile