Women's Health

The Journal of Clinical Pharmacology

Studying the Antiemetic Effect of Vitamin B6 54(12) 1429–1433
© 2014, The American College of
for Morning Sickness: Pyridoxine and Clinical Pharmacology
DOI: 10.1002/jcph.369
Pyridoxal Are Prodrugs

Ilan Matok, PhD1,2, Shannon Clark, MD3, Steve Caritis, MD4,

Menachem Miodovnik, MD5,6, Jason G. Umans, MD5,6, Gary Hankins, MD3,
Donald R. Mattison, MD7,8, and Gideon Koren, MD1

Abstract
Vitamin B6 has been known to possess antiemetic effects since 1942. This water soluble compound has several forms in the circulation including
pyridoxine, pyridoxal, and pyridoxal phosphate. The active antiemetic form of vitamin B6 is unknown. This was a pre-specified substudy of a randomized,
placebo-controlled trial comparing the antiemetic effect of the doxylamine–vitamin B6 combination (Diclectin1) (n ¼ 131) to placebo (n ¼ 126) in women
with nausea and vomiting of pregnancy. Serum concentrations of pyridoxine, pyridoxal, and pyridoxal 50 phosphate (PLP) and doxylamine were measured
on Days 4, 8, and 15. With Diclectin1 exhibiting a significant antiemetic effect in pregnancy, serum concentrations of pyridoxine were unmeasurable in
almost all patients and those of pyridoxal were undetectable in half of patients. In contrast, PLP was measurable at sustained, stable steady-state levels in all
patients. Our data suggest that there is a correlation between PLP levels and PUQE score of morning sickness symptoms when pyridoxine and pyridoxal
levels are undetectable, and hence they might be prodrugs of PLP, which may be the active antiemetic form of vitamin B6.

Keywords
pyridoxine, morning sickness, nausea and vomiting of pregnancy, prodrug

Vitamin B6 has a wide range of physiological roles, Patients and Methods

including a variety of enzymatic processes associated
mostly with amino acid metabolism.1 Systemically, the
pyridoxine molecule undergoes a complex range of
metabolic changes to form pyridoxal and pyridoxal 50
phosphate (PLP)1 (Figure 1).
The antiemetic effect of vitamin B6 was first
documented by Willis et al in 1942.2 Later, this effect
was confirmed through randomized, double-blind place-
bo-controlled studies.3–5 The original eight-way random-
ized trial has shown that B6 has an independent
antinauseant effect to that of doxylamine.6 A recent
systematic review reported that the evidence of pyridox-
ine effectiveness in reducing nausea and vomiting of
pregnancy (NVP) is not strong, however, it describes a
study showing that pyridoxine reduced nausea, and
another study documenting that high dose pyridoxine
reduced symptoms of NVP more than a low dose.7
To date, no study has addressed the question of
whether pyridoxine itself and/or one or more of its
metabolites possess the antiemetic effects.
We used pharmacokinetic and pharmacodynamic data
generated during a randomized controlled trial of women
with NVP to address this question. At the present time the
mechanisms leading to the antiemetic effects of vitamin
B6 have not been elucidated. We aimed to study whether
pyridoxine and pyridoxal are prodrugs of PLP, as the
active antiemetic molecule.
This was a double-blind, randomized, multicenter,
placebo-controlled study of the delayed-release combina-
tion of doxylamine succinate (10 mg) and pyridoxine
hydrochloride (10 mg) (Diclectin1) in the treatment of
NVP. Women were recruited at three university centers:
The University of Texas Medical Branch in Galveston,
1
Motherisk Program, Hospital for Sick Children, University of
Toronto, Toronto, Ontario, Canada
2
Division of Clinical Pharmacy, Institute of Drug Research, School of
Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem,
Jerusalem, Israel
3
Department of Obstetrics and Gynecology, Medical Branch, Univer-
sity of Texas, Galveston, TX, USA
4
Department of Obstetrics and Gynecology, Medical Center, Univer-
sity of Pittsburgh, Pittsburgh, PA, USA
5
Medstar Health Research Institute, Hyatsville, MD, USA
6
Georgetown-Howards University Center for Clinical and Transla-
tional Science, Washington, DC, USA
7
University of Ottawa, Ottawa, Ontario, Canada
8
Risk Sciences International, Ottawa, Ontario, Canada
Submitted for publication 12 June 2014; accepted 21 July 2014.
Corresponding Author:
Gideon Koren, MD, Division of Clinical Pharmacology/Toxicology,
Hospital for Sick Children, 555 University Avenue, Toronto, ON,
Canada M5G 1X8
Email: gkoren@sickkids.ca
1430
Figure 1. Pyridoxine biotransformation. PM, pyridoxamine; PN,
pyridoxine; PLP-5, pyridoxal phosphate; PL, pyridoxal; 4-PA, 4-
pyridoxal.
Texas, University of Pittsburgh, Pittsburgh, Pennsylva-
nia, and Georgetown University, Washington, DC. The
women were pregnant, at least 18 years of age, in the
gestational age range of 7–14 weeks, suffering from NVP,
had a Pregnancy-Unique Quantification of Emesis
(PUQE) score 6 (ie, moderate NVP; 7–9), and had
not responded to conservative management consisting of
dietary/lifestyle advice according to the 2004 American
College of Obstetrics and Gynecology (ACOG) Practice
Bulletin.10 Women treated by other antiemetics, suffering
from chronic medical conditions, or those who could not
communicate in either English or Spanish were excluded.
The current study is a secondary analysis of de-identified
data from a published study (clinical trial registration no.
NCT00614445) where IRB approval and patient consent
were obtained from all participating sites.11 The study
protocol was approved by the Ethics Review Committee.
All participants signed an informed consent prior to
initiation of study procedures.
After normal physical examination and laboratory
testing (hemoglobin and blood count, liver function tests,
electrolytes, amylase), and after confirming in utero
singleton pregnancies by ultrasound, women were
randomized through a computerized program to receive
Diclectin1 (doxylamine succinate 10 mg and pyridoxine
hydrochloride 10 mg) or placebo of a similar look. Two
tablets of study drug (Diclectin1 or placebo) were
administered at bedtime on Day 1. If symptoms of nausea
and vomiting persisted into the afternoon hours of Day 2
(ie, PUQE Score above 3), the subject was directed to take
her usual dose of 2 tablets at bedtime and an additional
tablet the next morning on Day 3. Based upon assessment
in the clinic on Day 4 (1 day), the subject could have
been directed to take an additional 4th tablet in mid-
The Journal of Clinical Pharmacology / Vol 54 No 12 (2014)
afternoon to control evening symptoms. The minimum
assigned study medication was 2 tablets daily at bedtime,
increasing when indicated to the maximal dosage of 4
tablets per day according to the timing, duration, severity,
and frequency of the symptoms experienced by the
participant. The study had a 15-day period consisting of
14 dosing days. Telephone contact was made on Days 2,
6, 12, and 14 in order to assess subject diary information,
adverse events (AEs), concomitant medications, and
compliance with the study medication. Patients returned
to the clinic in the morning prior to their morning dose on
Day 4 (1 day), Day 8 (1 day), and on Day 15 (1 day;
end of study visit) to collect diary report and complete all
study related data.
Subjects completed the PUQE score and the study
diary (once daily every morning prior to study dose at
approximately the same time each day). The primary
effectiveness criterion for evaluation consisted of change
from baseline in the PUQE score. The PUQE score
incorporates the number of daily vomiting episodes,
number of daily retching, and length of daily nausea in
hours, for an overall score of symptoms rated from 3 (no
symptoms) to 15 (most severe).7–9
Blood samples were drawn (5 mL) before the start of
therapy, and then on Days 4, 8, and 14 before the morning
dose of the drug, to measure plasma concentrations of
doxylamine, pyridoxine, pyridoxal, and PLP.
Sample Analysis
Plasma pyridoxine, pyridoxal, and PLP were assayed at
Anapharm, Inc. (Quebec City, QC, Canada) using a high
performance liquid chromatographic method-tandem
mass spectrometric (HPLC–MS–MS), described below.
Briefly, pyridoxine, pyridoxal, and PLP were extracted
from a 0.05-mL aliquot of human NaF/K2C2O4 plasma
using protein precipitation procedure, then injected into a
liquid chromatograph equipped with a C1 8.3 mm column.
The mobile phase A was a mixture of Milli-Q type water,
acetonitrile, ammonium formate 5 mM, and trifluoro-
acetic acid 0.05% (v/v) and the mobile phase B was a
mixture of Milli-Q type water, acetonitrile, ammonium
formate 5 mM, and trifluoroacetic acid 0.05% (v/v) and
the mobile phase B was a mixture of Milli-Q type water,
acetonitrile, ammonium formate 5 mM, and trifluoro-
acetic acid 0.05% (v/v). The detection method used was
tandem mass spectrometry. Quantitation is by peak area
ratio method. A weighted (1/C2) linear regression is
performed to determine the concentration of the analyte.
For pyridoxine this method yielded a linear standard
curve (r2 > 0.99) from 1.00 to 200 ng/mL, with, between
run precision of 5.7%, within run precision of 4.27–14.8%
and 92% recovery.
PLP standard curve was linear (r2 > 0.99) over the
range of 2.03–101.52 ng/mL. Between-run precision (QC
coefficients of variation was 3.69–7.08%.
Matok et al 1431

For pyridoxal this method was linear (r2 > 0.99) over Table 2. Proportion of Patients With Undetected versus Detected
Levels of Vit. B6 Species Over Time
the range of 10.13–405.12 ng/mL. Between-run precision
had coefficients of variation between 4.28% and 13.40%. Concentration
Within-run precision had coefficients of variation
between 2.46% and 7.64%. Analysate Day ¼0 >0 Total

Pridoxine Day 4 (1 day) 121 10 131

Statistical Analysis Day 8 (1 day) 125 6 131
To compare between women whom their pyridoxal levels Day 15 (1 day) 123 8 131
were higher than zero and those with zero and the change Pyridoxal 50 -phosphate Day 4 (1 day) 10 121 131
in PUQE we used two way repeated ANOVA (Days 1, 4, Day 8 (1 day) 18 113 131
Day 15 (1 day) 11 120 131
8, and 15 were looked into). To compare between women
Pyridoxal Day 4 (1 day) 64 67 131
whom their PLP levels were higher than zero to those with Day 8 (1 day) 53 78 131
zero (Days 1 and 15 were looked into). Day 15 (1 day) 63 68 131

Results
Two hundred and eighty-three pregnant women
experiencing NVP were enrolled and randomized. After
written informed consent, 9 subjects randomized to
Diclectin1 (6.4%) and 18 randomized to placebo (12.9%)
withdrew consent. Therefore, 131 women on Diclectin1
and 125 on placebo were included in the Intent to Treat
Effectiveness analysis.
This PK/PD analysis included only 131 women who
received vitamin B6 (combined with doxylamine) in the
the Diclectin1 arm. Measured concentrations of vitamin
B6 and its metabolites revealed that median pyridoxine
steady levels were zero (ie, below the limit of detection)
on Days 4, 8 and 15, pyridoxal was undetectable in half of
the patients, whereas pyridoxal 5-phosphate (PLP) levels
were measurable at all time points and stable from Days 4
to 15 (Tables 1 and 2). During the 15 day trial there was a
steady and significant improvement in the PUQE score in
general. In the presence of unmeasurable levels of
pyridoxine and an apparent steady state concentrations
of PLP (Figure 2). In women whose PLP concentrations
were greater than zero, the mean PUQE difference was
higher than those who had zero concentration (n ¼ 121,
mean PUQE difference ¼ 5.3 (3.4 to () 11.1);
n ¼ 10, mean PUQE difference ¼ 3.5 (2.0 to ()
5.6) respectively, P < 0.001). There was no statistical
difference between those who had pyridoxal concentra-
tion higher than zero to those who had concentration of
zero (n ¼ 67, mean PUQE difference ¼ 5.2 (2.4 to ()
11.1); n ¼ 64, mean PUQE difference ¼ 5.4 (3.2 to
() 10.1), P ¼ 0.333).
Discussion
Vitamin B6, a water-soluble compound, was discovered
in the 1930s during nutrition studies on rats. Several forms
of vitamin B6 (pyridoxine, pyridoxamine and pyridoxal,
PLP, and other) have been identified in the blood. PLP is a
cofactor of a large number of essential enzymes in the
human body.1 Enzymes dependent on PLP mainly
involve amino acids metabolism, including transfer of
the amino group, decarboxylation, racemization, and
beta- or gamma-elimination or replacement. PLP cova-
lently binds to substrates and then acts as an electrophilic
catalyst, thereby stabilizing different types of carbanionic
reaction intermediates.1 Overall, more than 140 PLP-
dependent enzymatic activities have been identified.12
The potential antiemetic role of vitamin B6 has been
first reported in 1942 in the context of nausea and
vomiting in pregnancy.2 A number of randomized,
placebo-controlled trials have corroborated these original
observations,3–5 although the mechanism of action has not
been elucidated. Vitamin B6 has been used in many

Table 1. Plasma Level of Vitamin B6 Species and of Doxylamine

Visit Statistics Pyridoxine (ng/mL) Pyridoxal 50 -Phosphate (ng/mL) Pyridoxal (ng/mL) Doxylamine (ng/mL)

Day 4 N 131 131 131 131

Median 0.000 42.290 10.260 64.980
Min, Max 0.00, 32.78 0.00, 127.42 0.00, 255.55 0.00, 152.43
Day 8 N 131 131 131 131
Median 0.000 45.100 13.110 64.240
Min, Max 0.00, 179.96 0.00, 107.83 0.00, 186.83 0.00, 196.53
Day 15 N 131 131 131 131
Median 0.000 39.110 10.380 52.550
Min, Max 0.00, 38.45 0.00, 126.28 0.00, 214.71 0.00, 200.38
1432
Figure 2. Median PUQE score versus median serum concentrations of pyridoxine, pirydoxal, and PLP.
countries singly, or in combination with doxylamine
(Bendectin1, Diclectin1, Diclegis1) for nausea and
vomiting of pregnancy. While it has been used for this
indication for 70 years now, no attempts have been made
to identify its active pharmacological species. To the best
of our knowledge, the present study is the first attempt to
synthesize pharmacokinetic and pharmacodynamic data
in defining the active antiemetic form of this vitamin.
Our data, based on 131 women with mild and moderate
morning sickness, reveal that the antiemetic effect is
achieved in the absence of measurable concentrations of
pyridoxine. This is not surprising, as the elimination half-
life of pyridoxine has been previously shown by us to be
short (1–2 hours) even in the delayed release formulation
used in Diclectin1.13 Moreover, pyridoxal concentrations
were also unmeasurable during steady state in about half
of our patients (Table 2). In contrast, PLP plasma
concentrations plateaued and were measurable in most
patients on Days 4, 8, and 15, in association with the
increase in the antiemetic effect (Figure 2).
It could be argued that the observed antiemetic effect
was due to solely doxylamine, the second agent in the
Diclectin1 tablet. However, previous studies have shown
the additive effect of doxylamine and vitamin B6, and
other studies have shown the antiemetic effects of vitamin
B6 over placebo in a variety of conditions 3–5. Put
together, our results indicate that pyridoxine, and possibly
pyridoxal, are likely prodrugs, and PLP is likely the main
active antiemetic molecule.
Our study suggests that, similar to many known
enzymatic activities, PLP, and not pyridoxine or
pyridoxal is the active antiemetic form of vitamin B6.
The Journal of Clinical Pharmacology / Vol 54 No 12 (2014)
This observation may have major implications for
pharmacokinetic and bioequivalence studies of com-
pounds containing vitamin B6 for nausea and vomiting of
pregnancy, as well as studies investigating the mecha-
nisms of its antiemetic effects.
Funding
The study was supported by Duchesnay, Inc., Blainville,
Quebec, Canada and executed by Premier Research Group,
Philadelphia, PA. GK is holder of the Research Leadership for
Better Pharmacotherapy during Pregnancy and Lactation.
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