The Safety of Diclectin® in Breastfeeding

Cheuk Kiu Chow

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Pharmacology and Toxicology University of Toronto

Cheuk Kiu Chow

Master of Science

Department of Pharmacology and Toxicology University of Toronto

2015 Abstract

Background and Rationale: Diclectin® is a delayed-release medication indicated for nausea and vomiting of pregnancy. Some mothers who are concurrently pregnant and breastfeeding also take

Diclectin®. Hence, components of Diclectin® in breast milk may cause adverse events in the breastfed infants. This had not been studied before.

Objective: To determine the safety and clinical significance of doxylamine exposure through breast milk in infants breastfed by mothers taking Diclectin®.

Methods: 41 mothers concurrently taking Diclectin® and breastfeeding completed a telephone questionnaire regarding occurrence of adverse events in themselves and their breastfed infants.

Data were analyzed and compared with results from other studies.

Results: Maternal adverse events included sedation (29), dizziness (2), weakness (4), constipation (2), reduced lactation (2), abdominal pain (1), and increased appetite (1). Infant adverse events included sedation (5), constipation (1), slept less (1), and loose bowel (1).

Conclusion: Mothers taking Diclectin® and their breastfed infants may experience sedation. iii

Acknowledgments

Firstly, I would like to thank my family for their support and encouragement throughout my study.

Secondly, I would like to thank my mentor Ms. Caroline Maltepe. Her guidance and support throughout my study had proved essential and the lessons I learned from her will be indispensable for the rest of my life.

Thirdly, I would like to thank Dr. Shinya Ito for supervising my project during a time of hardship. Without his help, I would not be able to finish this study.

Fourthly, I would like to thank Dr. Bhushan Kapur and Dr. Irena Nulman, fellows at the Motherisk Program, and other graduate students at Motherisk for mentoring and allowing me to explore different facets of clinical pharmacology and toxicology.

Lastly and most importantly, I would like to sincerely thank Dr. Gideon Koren for taking me under his guidance and supervision at the Motherisk Program, which has provided me a multitude of experiences in clinical research.

I shall not forget the lessons from this experience.

Potential Conflict of Interest

This study was sponsored by Duchesnay Inc., the manufacturer of Diclectin®.

Dr. Gideon Koren is a paid consultant for Duchesnay Inc.

Table of Contents

Acknowledgments...... iii

Potential Conflict of Interest ...... iv

Table of Contents ...... v

List of Tables ...... viii

List of Figures ...... ix

List of Abbreviations ...... x

Chapter 1. Introduction ...... 1

1.1 Statement of the Problem ...... 1

1.2 Overall Objective ...... 2

1.3 Methods in Brief ...... 2

1.4 Research Question and Hypothesis ...... 2

Chapter 2. Literature Review of the Study Drug ...... 3

2.1 Diclectin® ...... 3

2.1.1 Teratogenicity of Doxylamine-Pyridoxine Combination Treatments for NVP ...... 3

2.1.2 Common Side Effects of Diclectin®/Bendectin®/Debendox® ...... 4

2.1.3 Bendectin®/Debendox® Overdose/Toxicity in Young Children ...... 5

2.1.4 Pharmacokinetic Studies of Diclectin® ...... 6

2.1.5 Doxylamine ...... 8

2.1.5.1 Physicochemical Properties of Doxylamine – How Likely Will It Enter Breast Milk? ...... 9

2.1.5.2 Pharmacokinetics of Doxylamine ...... 12

2.1.5.3 Metabolism of Doxylamine ...... 14

2.1.5.4 Common Side Effects of Doxylamine ...... 18

2.1.5.4.1 Overdose ...... 18

2.1.6 Vitamin B6/Pyridoxine ...... 20 vi

2.1.6.1 Pharmacokinetic and Physicochemical Properties of Vitamin B6/Pyridoxine ...... 22

2.1.6.2 Dietary Reference Intake ...... 26

2.1.6.3 Vitamin B6/Pyridoxine Toxicity ...... 28

2.1.6.4 Vitamin B6 deficiency in breastfed infants ...... 29

2.1.6.5 Vitamin B6 status of mothers and breast milk ...... 29

2.1.6.5.1 Using Vitamin B6 to Suppress Puerperal Lactation? ...... 31

Chapter 3. Other NVP Medications and Breastfeeding ...... 36

3.1 Diphenhydramine and Dimenhydrinate ...... 36

3.2 Metoclopramide ...... 37

3.3 Acid-Reducing Drugs ...... 38

Chapter 4. Methods of Assessing Infant Exposure to Drugs in Breast Milk...... 39

4.1 Milk/Plasma Ratio ...... 39

4.2 Relative Infant Dose ...... 41

4.3 Oral Bioavailability ...... 41

4.4 Infant Plasma Concentrations ...... 42

Chapter 5. Methods...... 43

5.1 Setting ...... 43

5.2 Study Design ...... 43

5.3 Study Group Subject Recruitment ...... 43

5.3.1 Prospective Recruitment ...... 43

5.3.2 Retrospective Recruitment ...... 44

5.3.3 Interview ...... 44

5.3.4 Inclusion and Exclusion Criteria ...... 44

5.3.5 Recruitment Outcome ...... 45

5.4 Data Collection and Target Endpoints ...... 45 vii

5.5 Analysis...... 47

5.5.1 Statistical Analysis ...... 47

5.5.2 Post-Hoc Comparisons...... 48

Chapter 6. Results ...... 50

6.1 Study Population ...... 50

6.2 Diclectin® and NVP-Related Data ...... 53

6.3 Maternal Other Exposures ...... 55

6.4 Dietary Patterns and Other Infant Characteristics ...... 56

6.5 Maternal Adverse Events ...... 58

6.6 Adverse Events amongst Breastfed Infants ...... 59

6.7 Correlations ...... 62

Chapter 7. Discussion ...... 64

7.1 Study Population ...... 64

7.2 Diclectin® and NVP-Related Data ...... 65

7.3 Maternal Other Exposures ...... 65

7.4 Maternal Adverse Events ...... 66

7.5 Adverse Events amongst Breastfed Infants ...... 67

7.6 Correlations ...... 69

7.7 Limitations of the Study...... 69

7.7.1 Limitations of Comparison with Previous Studies on Using Other Medications While Breastfeeding...... 71

7.7.2 Recall Bias ...... 74

Chapter 8. Conclusion and Future Directions ...... 76

References or Bibliography (if any) ...... 77

Appendix 1 – Questionnaire ...... 99

Appendix 2 – Approval from Research Ethics Board of the Hospital for Sick Children ...... 107 viii

List of Tables

Table 1 – Physicochemical properties of doxylamine ...... 9

Table 2 – Summary of vitamin B6 absorption, distribution, metabolism, and excretion60,68,74,75 23

Table 3 – Dietary Reference Intake Values (adopted from the RDA table by Health Canada77) . 27

Table 4 – Demographics Part 1 ...... 51

Table 5 – Demographics Part 2 ...... 52

Table 6 – Diclectin and NVP related data ...... 54

Table 7 – Other maternal exposures while breastfeeding and using Diclectin® ...... 55

Table 8 - Breastfeeding and infant-related data ...... 57

Table 9 – Maternal adverse events...... 58

Table 10 - Breastfed infant adverse events ...... 59

Table 11 – Demographics of sedated infants ...... 61

Table 12 – Demographics of infants who experienced non-sedation AEs ...... 62

Table 13 – Correlations between weight-adjusted maternal dose and maternal adverse events, infant adverse events, and total infant sleeping time per day ...... 63

Table 14 – Comparison of the rate of maternal sedation between this study and that of Atanakovic et al.19 with Fisher’s exact test ...... 66

Table 15 – Comparisons of the rate of infant sedation between Diclectin® and other medications with Fisher’s exact test...... 68

Table 16 – Comparison of cohort maternal age and parity between the current study and comparator studies ...... 73

List of Figures

Figure 1 – Predicted relative abundance of unprotonated, monoprotonated, and diprotonated doxylamine (from35)...... 11

Figure 2 – Doxylamine and its metabolites found in human urine ...... 16

Figure 3 – Structures of B6 vitamers and their interconversions ...... 21

Figure 4 – Flow Chart of Recruitment and Data Collection ...... 49

List of Abbreviations

AADC Aromatic-L-Amino-Acid Decarboxylase

AI Adequate Intake

AE Adverse Event

AUC Area Under (Concentration-Time) Curve

AUC0-t Area Under Curve from Time 0 to Last Measured Point

AUC0-∞ Area Under Curve from Time 0 to Infinite Time

B6 Vitamin B6

Cav Average Maternal Plasma Concentration

CI Confidence Interval

Cmax Maximal Concentration

Cmilk Drug Concentration in Breast Milk

CNS Central Nervous System

CV Coefficient of Variation

CYP Cytochrome P450

FDA Food and Drug Administration (United States)

Frel Relative Bioavailability

GC Gas Chromatography

HG Hyperemesis Gravidarum

HPLC High Performance Liquid Chromatography

kα Elimination Constant of the First Phase xi

kel Elimination Rate Constant

LC Liquid Chromatography

LOAEL Lowest-Observed Adverse-Effect-Level

M/P ratio Milk/Plasma Ratio

MS Mass Spectrometry

NAS National Academy of Science (United States)

ND Not Determined

NOAEL No-Observed Adverse-Effect-Level

NVP Nausea and Vomiting of Pregnancy

OR Odds Ratio

PA Pyridoxic Acid

PIF Prolactin Inhibiting Factor

PL Pyridoxal

PLP Pyridoxal 5’-Phosphate

PM Pyridoxamine

PMP Pyridoxamine 5’-Phosphate

PN Pyridoxine

PN-HCl Pyridoxine Hydrochloride

PNP Pyridoxine 5’-Phosphate

PNPO Pyridox(am)ine Phosphate Oxidase xii

PRL Prolactin

RID Relative Infant Dose

RR Relative Risk (also known as Risk Ratio)

SD Standard Deviation

t1/2 Elimination Half-Life

t1/2,z Mean Terminal Exponential Half-Life

tmax Time to Reach Maximal Concentration

TLC Thin Layer Chromatography

UL Tolerable Upper Intake Level

US United States

UV Ultraviolet

Vmilk Average Volume of Milk Intake

Chapter 1. Introduction 1.1 Statement of the Problem

Nausea and vomiting of pregnancy (NVP) is the most common condition in pregnancy, affecting 50-85% of pregnancies, with symptoms ranging from mild to severe.1 Generally, women suffering NVP experiences nausea with/without vomiting, retching, gagging, dry heaving, odour aversion, and/or food aversion.1,2 Typically, NVP starts during 4-8 weeks gestational age, peaks at 7-9 weeks, and resolves by 16-22 weeks.1 The most severe form of NVP is known as hyperemesis gravidarum (HG). HG affects 0.3-3% of all pregnancies and may lead to maternal weight loss, electrolyte imbalance, dehydration, ketonuria, and vitamin or mineral deficiencies.2 Therefore, hospitalization is often necessary. NVP and HG have been associated with depression and anxiety, likely because they affect pregnant women’s life and health negatively.1,2

Doxylamine-pyridoxine combination is often used to treat NVP.3 In North America, this combination is produced by Duchesnay Inc., as a delayed-release formulation named Diclectin® in Canada and Diclegis® in the United States respectively.3 This formulation is the only medication approved for managing NVP by both Health Canada and the United States’ Food and Drug Administration (FDA). Doxylamine itself is also a widely available sedative antihistamine used either for allergies or to induce sleep.4 Vitamin B6 alone is sometimes used as a treatment for NVP also.5

The Motherisk Program is a teratogen information service—consisting of several helplines and a clinic—that provides evidence-based information to women, their partners, and their health care providers on the safety or risks associated with exposures, such as drugs, chemicals, radiations, and infectious diseases during pregnancy and lactation. During a call, Motherisk counsellors document clinical characteristics of the women/mothers, such as their medical and obstetrical history as well as details about medication/radiation/chemical exposure(s), and use of drugs of abuse on a standard Motherisk intake form. The Motherisk NVP Disease Management Helpline is one of the few service providers worldwide that counsel women about NVP management.

In our experience through the Motherisk Helplines, some pregnant women are willing to continue using Diclectin® for NVP, while nursing an infant. Although the safety of Diclectin® in pregnancy has been repeatedly shown, its safety during breastfeeding had not been investigated 2 prior to the current study. Indeed, the product monograph of Diclectin® states that the drug should not be used during breastfeeding.6 This puts a mother who is suffering NVP and breastfeeding in a dilemma – she has to discontinue either an effective antiemetic or breastfeeding.

Based on its physicochemical properties and data on similar ethanolamine antihistamines, doxylamine is expected to transfer from maternal blood into breast milk.7 In addition, studies have reported occurrence of adverse events in children and adults exposed to doxylamine/Diclectin®.4,6,8 Therefore, I believe the concern is valid.

1.2 Overall Objective

To determine the safety and clinical significance of doxylamine exposure through breast milk in infants breastfed by mothers taking Diclectin®.

1.3 Methods in Brief

Mothers who had used Diclectin® during breastfeeding were recruited and interviewed for this study. Information on the occurrence of adverse events and other factors in these mothers and their breastfed infants were collected. Results from this study were then compared with those of other studies using appropriate statistical tests.

1.4 Research Question and Hypothesis

To address the dilemma presented in Section 1.1, I posed the following research question: Would infants breastfed by mothers taking Diclectin® show higher risk of sedation (or other adverse events), when compared to infants breastfed by mothers taking other medications? I hypothesized that answer would be ‘Yes’. This was based on the high likelihood of doxylamine entering breast milk (see Section 2.1.6.1) and the fact that we know it can cause adverse events such as sedation in adults and children (see Sections 2.1.6.2 and 2.1.6.4).

Chapter 2. Literature Review of the Study Drug 2.1 Diclectin®

Diclectin® is a delayed-release medication indicated for managing symptoms of NVP. Each tablet of Diclectin® contains 10 mg doxylamine succinate and 10 mg pyridoxine hydrochloride6. In the United States (US), the same formulation was marketed as Bendectin® from mid-1950s to early-1980s, and contained dicyclomine hydrochloride from the beginning till 1978.9 During the 1970s, the National Academy of Sciences (NAS) conducted a peer review on Bendectin®, and recommended studies be performed on different combinations of the components.10 These unpublished studies reported that dicyclomine did not contribute to the efficacy of Bendectin®; consequently, dicyclomine was removed from the formulation.10,11

2.1.1 Teratogenicity of Doxylamine-Pyridoxine Combination Treatments for NVP

In the 1970s, Bendectin® was alleged to be teratogenic and lawsuits ensued.10 In early 1980s, the manufacturer of Bendectin®, Merrell Dow Pharmaceuticals, withdrew Bendectin® from the US market; as high insurance premiums due to litigation exceeded sales revenue from the medication.10 Shortly after Bendectin® was taken off the market, the rate of hospitalization due to NVP increased significantly in the US, while the rate of limb reduction deformities remained consistent. Whereas in Canada, after Bendectin® was withdrawn from the market, the rate of hospitalization due to excessive vomiting in pregnancy increased, but as the marketing of Diclectin® started and its prescription increased, the rate decreased.12

Subsequent to the withdrawal, multiple studies have shown that Bendectin® and Diclectin® are not associated with teratogenicities.13-16 In addition, Nulman et al.17 had found that exposure to Diclectin® in utero was not associated with negative neurodevelopmental outcomes in children aged 3-7.

In 2013, FDA approved the formulation to be re-marketed in the US for the treatment of NVP as Diclegis®, also manufactured by Duchesnay Inc.3

2.1.2 Common Side Effects of Diclectin®/Bendectin®/Debendox®

An unpublished, randomized, double-blind, multi-centre study in 2308 women with NVP compared placebo with different combinations of 10 mg doxylamine succinate, 10 mg dicyclomine hydrochloride, and/or 10 mg pyridoxine hydrochloride.6 Adverse events occurred in 8.7% of doxylamine-pyridoxine subjects, versus 11.2% in the placebo subjects. The most common adverse reactions were drowsiness (treatment: 15/265, placebo: 8/269), dizziness (treatment: 3/265, placebo: 2/269), fatigue or lethargy (treatment: 2/265, placebo: 3/269), gastric irritation or heartburn or indigestion (treatment: 2/265, placebo: 0/269), and headache (treatment: 2/265, placebo: 4/269).

In 1971, McGuinness et al. reported a double-blind study on the safety and efficacy of Debendox® (doxylamine succinate, pyridoxine hydrochloride, dicyclomine hydrochloride, 10 mg each) for treating NVP.18 Of the 81 NVP patients participated in this study, 41 were treated with Debendox® and 40 were treated with placebo. Patients were asked to take 2 tablets at bedtime every night for 14 consecutive nights. Debendox® patients reported 12 cases of side effects: feeling weak (2), tiredness (2), drowsiness (3), wind (1), furry sensation in mouth (1), lack of energy and funny feelings (1), constipation (1), and headache (1). Whereas placebo patients reported 6 cases of side effects: constipation, tiredness and giddiness, tiredness, sleepy, depression.

In 2001, Atanackovic et al. reported a study on the safety of using higher than standard dose (“supradose”) of Diclectin® to treat NVP.19 This study recruited 225 women, 123 of which used standard dose and 102 used supradose. Occurrences of sleepiness, tiredness, and/or drowsiness were not significantly different between the standard dose group (42/122) and the supradose group (31/97). There was no relationship between weight-adjusted dose and occurrence of adverse events. Note that adverse events were not recorded for all patients, hence the discrepancies in the denominators.

In 2015, Koren et al. reported a randomized, placebo-controlled trial on the safety of Diclegis® in women suffering NVP.20 Participants were randomized to take 2-4 tablets of Diclegis® (n = 131) or placebo (n = 125) daily for 14 days; and were monitored for adverse events. The most common adverse events possibly/probably/definitely related to Diclegis® treatment included: somnolence (14.5%), headache (6.1%), fatigue (4.6%), dizziness (4.6%), dry mouth (3.1%), and 5 diarrhea (1.5%). However, when compared to the placebo group, no significance was found between the incidences of adverse events. The authors quoted a Canadian study which reported that pregnant women in the first trimester––when NVP is at its peak and many women uses Diclectin® to alleviate it––did not have a higher rate of car crashes when compared to the general population.20,21

To summarize, the most common adverse events reported for Diclectin® were CNS-related, including somnolence/drowsiness, dizziness, fatigue, and headache. At standard doses (1-4 tablets), none of the adverse events reported constituted as serious or severe adverse events. Hence, one can conclude that standard use of Diclectin® is not expected to harm the patient. Even when used at 3 times the standard dose, the rates of adverse events were similar to those for the standard dose.19 However, the supradose study was limited by its small sample size. Future studies should investigate the safety of supradose further with a larger sample size. Linking these findings to the current study, if doxylamine in breast milk does reach clinically significant level, infants breastfed by mothers taking Diclectin®/doxylamine may experience the aforementioned adverse events, particularly CNS-related ones.

2.1.3 Bendectin®/Debendox® Overdose/Toxicity in Young Children

A search of the literature had not come up with Diclectin® or Bendectin® (doxylamine- pyridoxine) overdose/toxicity in children, hence reports regarding the doxylamine-pyridoxine- dicyclomine formulation are presented here.

In 1974, Meadow and Leeson reported two cases of Debendox® (doxylamine-pyridoxine- dicyclomine) poisoning in children.22 In the first case, an 18 months old boy consumed 23 Debendox® tablets. He was agitated, vomiting, and had myoclonic jerks and fits. Despite supportive treatment along with diazepam and paraldehyde, the child eventually died of cardiac arrest. The authors suggested that both dicyclomine and doxylamine might have contributed to the condition. It also appeared that the child was brought in too late as the parents had waited for symptoms to appear. In the second case, a 15 months old boy consumed up to 30 Debendox® tablets. In contrast to the first case, he was drowsy, confused, and had decreased level of consciousness. Aggressive purges and enemas, followed by peritoneal dialysis were utilized to remove as much of the medication from his system as possible. The child’s condition improved within the day and he appeared normal the next morning. 6

Also in 1974, Clarkson and Glenville reported a 3.5 years old boy who was thought to have ingested 57 tablets of Debendox® (doxylamine-pyridoxine-dicyclomine).23 No action was taken immediately after. Seven hours post-dose, he was agitated and restless. Upon arrival at the emergency, the boy was hallucinated, restless, had dilated pupils, dry mouth, hot dry skin, and tachycardia (anticholinergic symptoms). He was administered pilocarpine subcutaneously (cholinergic), chlorpromazine intramuscularly (antipsychotic), and magnesium hydroxide orally (purgative). After the boy had a generalized convulsion, he was sedated with diazepam intravenously. The boy had recovered fully by 24 hours post-admission.

In 1975, Bayley et al. reported a fatal case of Bendectin® (doxylamine-pyridoxine-dicyclomine) overdose in a 3 years old boy.24 The boy ingested about 100 tablets of Bendectin®. Initially, he showed signs of restlessness, disorientation, and ataxia, which then progressed into tonic-clonic seizures, followed by cardiorespiratory arrest, and eventually death. Gastric lavage was performed but was unable to recover any tablets. Postmortem blood, peritoneal fluid, and tissue homogenates were analyzed using gas chromatography-UV spectrometry. Doxylamine was detected in all specimens while dicyclomine and pyridoxine were only detected in peritoneal fluid and homogenates of liver, kidney, lung, and spleen.

To summarize, Bendectin®/Debendox® toxidrome in young children correlates with the anticholinergic toxidrome reported for antihistamine intoxication: gastrointestinal disturbance, dry mouth, hot dry skin, dilated pupils, tachycardia, hyper/hypotension, ataxia, nystagmus, drowsiness, agitation, convulsion, psychosis, prolonged coma, paralytic ileus, and urinary retention.25 However, we should note that dicyclomine is also an anticholinergic drug (specifically antimuscarinic).26 Therefore, anticholinergic symptoms observed in the cases above may be attributed to synergism of the anticholinergic effect of both doxylamine and dicyclomine. However, since the level of doxylamine in breast milk is not expected to reach toxic levels, we would not expect to see anticholinergic toxidrome in infants breastfed by mothers taking Diclectin®/doxylamine.

2.1.4 Pharmacokinetic Studies of Diclectin®

In 2009, Nulman and Koren reported a randomized, crossover, open-label study comparing the pharmacokinetics of Diclectin® to an oral solution of the same components.27 18 healthy, non- pregnant women of childbearing age, under fasting condition, were orally administered either 2 7 tablets of Diclectin® or a 20 mL solution containing 20 mg doxylamine succinate and 20 mg pyridoxine succinate. For each subject, the two treatments were separated by a washout period of at least 28 days. Blood samples were collected at multiple time points from 30 minutes pre-dose to 120 hours post-dose. Concentrations of doxylamine, pyridoxine, and pyridoxal were measured using LC-MS-MS, while that of pyridoxal 5’-phosphate was measured using LC-UV spectrometry. The following pharmacokinetic parameters were calculated for doxylamine, pyridoxine, pyridoxal, and pyridoxal 5’-phosphate: area under curve from time 0 to last measured point (AUC0-t), area under curve from time 0 to infinite time (AUC0-∞), maximal concentration (Cmax), time to reach maximal concentration (tmax), elimination rate constant (kel), ® elimination half-life (t1/2), and relative bioavailability of Diclectin -to-solution (Frel). As the Frel for doxylamine, pyridoxine, pyridoxal, and pyridoxal 5’-phosphate were within 100±10%, the authors concluded that the bioavailability of Diclectin® is similar to that of the solution. ® Meanwhile, the Cmax values tend to be lower and the tmax values tend to be higher for Diclectin , indicating a slower rate of absorption (as intended by the delayed-release formulation). The kel and t1/2 values were similar between the two formulations, suggesting similar elimination pharmacokinetics.

In 2013, Koren et al. reported a study on sex differences in pharmacokinetics and bioequivalence of Diclectin®.28 Twelve healthy males and twelve healthy non-pregnant females were administered 2 tablets of Diclecitn®. Blood samples were collected from 1-hour pre-dose to 72- hours post-dose. The study was repeated after 21 days. Doxylamine, pyridoxine, pyridoxal, and pyridoxal 5’-phosphate were measured using LC-MS-MS. AUC0-t and Cmax were calculated and compared between the two sexes. Results showed that females had significantly higher AUC0-t and Cmax for doxylamine and pyridoxal 5’-phosphate, and also significantly higher AUC0-t for pyridoxine. Bioequivalence was therefore not demonstrated between the sexes. The authors recommended that future pharmacokinetics studies on Diclectin® should account for sex differences. The authors also recommended that, since Diclectin® was prescribed for pregnant women only, future studies should be conducted in females.

In 2013, Matok et al. reported a study comparing the pharmacokinetics of Diclectin® in 18 non- pregnant women and 50 pregnant women in first trimester.29 Data for non-pregnant women were drawn from Nulman and Koren’s study27. For pregnant women, they had participated in a double-blind, randomized, multicentre, placebo-controlled study of Diclectin® for treating NVP 8 during 2008-2009. Women were directed to start with 2 tablets of Diclectin® per day and increase up to 4 tablets per day if deemed necessary. Diclectin® was administered for 14 days (days 1-14) and blood was collected on days 1, 4, 8, and 15. Apparent clearance of doxylamine and pyridoxal 5’-phosphate (active metabolite of pyridoxine) were calculated for these days. For both doxylamine and pyridoxal 5’-phosphate, there was no significant between-day difference in apparent clearance for pregnant women, and no significant difference in apparent clearance for pregnant versus non-pregnant women. Hence, the authors concluded that pregnancy and NVP did not affect the apparent clearance of doxylamine and pyridoxal 5’-phosphate during first trimester.

Pharmacokinetics studies of Diclectin® showed that it is well absorbed, as its components has 100 ± 10% relative bioavailability when compared to doxylamine-pyridoxine in solution.27 Studies have also suggested that future studies of Diclectin® in human should be performed in females, regardless of their pregnancy status. This is because the pharmacokinetics of Diclectin® differs between the sexes, but not between pregnant and non-pregnant females.28,29

2.1.5 Doxylamine

Doxylamine is a first generation H1 antagonist (i.e. antihistamine) that has anti-allergic, antiemetic, and sedative properties. It belongs to the ethanolamine class of H1 antihistamines, which also include other common over-the-counter drugs like diphenhydramine, clemastine, and dimenhydrinate.30 As a group, antihistamines are indicated for prophylactic and symptomatic treatment of allergies.31 Doxylamine alone is indicated as a sleeping aid.31 It is also a constituent of formations with antitussives and decongestants for temporary relief of cough and cold symptoms.32 For example (in Canada), Buckley’s Complete Nighttime Softgels, Nyquil Sinus Liquicaps, Tylenol Cold and Flu Nighttime.33

2.1.5.1 Physicochemical Properties of Doxylamine – How Likely Will It Enter Breast Milk?

Table 1 – Physicochemical properties of doxylamine

Property Values Reference

Average molecular mass 270.37 Da (doxylamine) 34

Water solubility 1000 g/L at 25°C 32 pKa (predicted) 3.23 (pyridyl N) and 8.87 (amino N) 35

LogD at pH 7.4 1.57 35 (predicted)

According to Rowe et al.36, medications excreted into breast milk have the following characteristics:

1. Molecular weight < 500 Da

2. Poorly bound to plasma proteins

3. pKa > 7.2 (since breast milk has a pH of 7-7.2, medications with pKa > 7.2 would become ionized and be retained in breast milk)

4. Selectively transported into breast milk, but few drugs are known to enter milk this way

In addition, Ito and Lee37 had suggested that the following characteristics would favour excretion of drugs into breast milk:

1. High lipophilicity

2. Cationic (studies had shown that some cationic drugs may be transported by organic cation transporters into breast milk)

Based on the physicochemical properties listed in Table 1, it is likely that doxylamine can cross into breast milk. Firstly, it has a moderate LogD at pH 7.4 (1.57), meaning it is sufficiently lipophilic to be retained in lipid fractions of breast milk. Doxylamine also has a relatively small 10 molecular mass of 270.37 Da, meeting the molecular weight requirement. Doxylamine’s highest pKa value is at approximately 9, meaning that in physiological pH (7.4), it will mostly be ionized and protonated (cationic) at the amino nitrogen (Figure 1). Based on this property, doxylamine has the potential to be transported by organic cation transporters into breast milk. However, being cationic may also limit doxylamine’s permeability across cell membranes by passive diffusion. Regardless, given that breast milk pH (7.0-7.2) is slightly lower than physiological pH (7.4), unprotonated doxylamine which entered breast milk may be further protonated and be retained in breast milk.

Figure 1 – Predicted relative abundance of unprotonated, monoprotonated, and diprotonated doxylamine (from35)

*x-axis = pH, y-axis = % abundance 12

2.1.5.2 Pharmacokinetics of Doxylamine

In 1989, Luna et al. reported a study comparing the pharmacokinetics of doxylamine (and diphenhydramine) in women taking low-dose estrogen oral contraceptive and age-matched drug- free control women.38 Fasting subjects received a single 25 mg doxylamine orally. No significant differences were found for Cmax, tmax (about 2 hours), t1/2 (about 10 hours), total area under curve (AUC), and total clearance (around 3-4 mL/min/kg). A small but insignificant difference was found for tmax (2.4±0.41 hours in controls and 1.9±0.25 in oral contraceptive users). The authors concluded that low dose estrogen contraceptives did not impair the clearance of doxylamine.

In 2012, Videla et al. reported a study on how food affects the pharmacokinetics of doxylamine administered as oral tablets.39 This was a cross-over study where the pharmacokinetics of doxylamine were measured at both fed and fasting states. 24 subjects were included (12 male, 12 female). For the fed state study, patients fasted for 10 hours prior to a high-fat, high-calorie breakfast. 30 minutes after the breakfast, patients were administered a 25 mg doxylamine tablet orally. For the fasting state study, patients fasted for 10 hours prior to drug administration. Blood samples were collected before and after drug administration for measuring the levels of doxylamine. One subject withdrew before second treatment and data was collected for fed conditions only. Somnolence was reported in 70.8% of subjects under fed conditions and 56.6% of subjects under fasting conditions. (My calculation using Fisher’s exact test showed that the difference was not significant.) Mean Cmax were similar between fed and fasting conditions

(120.99 vs 118.21 ng/mL). Median tmax was similar between the two states, though slightly longer in the fed state (2.50 h versus 2.00 h). Doxylamine use in both states were shown to be bioequivalent based on the confidence intervals for Cmax and AUC. Mean t1/2 and hence mean kel were almost the same between fed and fasting states (t1/2: 13.49 vs 13.11; kel: 0.0544 versus 0.0553). No significant differences were found between the sexes. Note that in female, fed state tmax was insignificantly higher than fasting state tmax.

In 2013, Videla et al. reported a randomized, laboratory-blinded, crossover study comparing the pharmacokinetic profiles of one oral dose of 12.5 mg or 25 mg doxylamine in 12 healthy volunteers (3 male, 9 female).40 Subjects were randomized to one of the doses on days 1 and 8 under fasting condition. Mean Cmax and AUC0-t were proportionally higher for 25 mg than 12.5 mg. Mean tmax was the same for both groups (1.67 h). Mean t1/2 was also similar for both groups 13

(12.5 mg: 12.23 h; 25 mg: 12.45 h). 2 subjects reported somnolence after taking 12.5 mg doxylamine, while 6 subjects reported somnolence after taking 25 mg doxylamine. The authors concluded that the two doses exhibited linear pharmacokinetics with low intra-subject variability.

In 2013, Balan et al. reported a study on the pharmacokinetics of doxylamine in children of 2-17 years.8 Subjects were given a single dose of doxylamine succinate solution, for which the dose increased with the subjects’ age. 40 subjects completed the study. Blood samples were collected pre-dosing, and at multiple time points (up to 76 hours) post-dosing. Subjects were also monitored for adverse events. In the analysis, subjects were categorized into 3 age groups: 2-5,

6-11, 12-17. Small, insignificant increases in Cmax and AUC were noted with age, especially when comparing the 2-5 age group to the 12-17 age group. Median tmax were also earlier in younger children when compared to older children (1 hour in 2-5 group versus 2 hours post-dose in 6-11 and 12-17 groups). The mean terminal exponential half-lives (t1/2,z) were not significantly different across the age groups (14.8 hours (CV 55.7%) for 2-5 group, 17.5 hours (CV 30.5%) for 6-11 group, 15.5 hours (CV 34.3%) for 12-17 group). Oral clearance, both adjusted and unadjusted for body weight, significantly increased with age. There were 19 cases of mild/moderate adverse events (AEs) experienced by 16 subjects. 16 of the 19 AEs were sedation or somnolence (in 15 subjects). The remaining three cases were headache, otitis media, and dizziness. Incidences of somnolence did not differ between different age groups.

Based on these studies, one may conclude that the pharmacokinetics of doxylamine has a linear relationship with the dose; and is not significantly affected by the use of estrogen oral contraceptives, fed or fasting state. In adults, the tmax of doxylamine was approximately 2 hours, and the t1/2 ranged from 10 to 13 hours. Note that these values agreed with those in Nulman and Koren’s study27, in which subjects were orally administered a solution of doxylamine and pyridoxine. In children, the tmax of doxylamine was 1 hour in young children and 2 hours in older children, and the overall t1/2 ranged from 14-17 hours. Concluding from limited evidence, tmax of doxylamine is earlier in younger children than in older children and adults, and t1/2 of doxylamine is longer in children than in adults.

® In Diclectin , doxylamine tmax and t1/2 in adult women averaged 6.1 hours and 11.76 hours respectively.27 Compared to the values of non-slow-release formulations in the studies above, the ® tmax was greatly lengthened and the t1/2 was similar for doxylamine in Diclectin . 14

2.1.5.3 Metabolism of Doxylamine

There are only a few studies on the metabolism of doxylamine, suggesting a lack of study in this area.

In 1987, Ganes and Midha reported a study identifying desmethyldoxylamine (nordoxylamine), N,N-didesmethyldoxylamine (dinordoxylamine) and their N-acetyl conjugates in human urine samples using gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC).41 A healthy male volunteer was administered 50 mg doxylamine succinate orally at bedtime. Urine samples were obtained up to 12 hours post-dose. Results showed that a large portion of doxylamine consumed were excreted unchanged. In addition, the authors also proposed that doxylamine would undergo either one or two N-demethylation, the product of which would then be N-acetylated.

In 1991, Luo et al. reported a study investigating N+-glucuronide metabolites in the urine of volunteers who took different antihistamines.42 Two healthy volunteers were administered 75 to 100 mg doxylamine succinate orally. Urine was collected for 36 hours from initial dosing. HPLC was used to analyze the samples. Results showed that 0.8-1.0% of the dose was excreted as N+- glucuronide. Therefore, N+-glucuronidation is not a major metabolic pathway for doxylamine.

In 1993, Siek and Dunn reported the levels of doxylamine, nordoxylamine, and dinordoxylamine in blood and urine samples of a person died of doxylamine overdose.43 HPLC, high performance- thin layer chromatography (HP-TLC) and GC/MS were used to measure the levels. Unchanged- doxylamine was the major species in both blood and urine, this was followed by nordoxylamine (in both blood and urine) and dinordoxylamine (found in urine only). Future studies should investigate whether the second demethylation occurs at kidney or if there could be other explanations for this finding.

In 2014, Remane et al. reported a study in which a 24 years old female was orally administered 30 mg doxylamine, 43 urine samples were collected for 11 days post-dose.44 Based on the AUC for doxylamine and nordoxylamine, the concentration of doxylamine in urine was about 2 times that of nordoxyamine. This suggests that a large portion of doxylamine was excreted unchanged. Both doxylamine and nordoxylamine were detectable for at least 10 days post-dose. 15

To summarize, doxylamine has been suggested to undergo one to two N-demethylation, forming N-desmethyldoxylamine (nordoxylamine) and N,N-didesmethyldoxylamine (dinordoxylamine). These metabolites may then undergo N-acetylation, forming N-acetyl conjugates. Additionally, in very small amounts, doxylamine may undergo N+-glucuronidation at the nitrogen atom of the dimethylaminoalkyl tail. Nevertheless, majority of the doxylamine would be excreted unchanged in urine, under normal dosing as well as overdosed situations. Figure 2 summarizes doxylamine metabolism.

Figure 2 – Doxylamine and its metabolites found in human urine

*Modified from Figure 1 in Ganes and Midha41 17

In spite of the aforementioned studies on doxylamine metabolites in humans, no study on enzymes mediating doxylamine metabolism could be found. The following paragraphs summarize a few studies possibly related to enzyme(s) metabolizing doxylamine.

In 1996, Bookstaff et al. reported that doxylamine had induced enzymes involved in thyroxine metabolism in mice.45 Mice of 45-52 days old were treated with diets containing 0, 40, 375, 750, or 1500 ppm doxylamine, or 375 ppm phenobarbital. 50% of the animals in each group were treated for 7 days; with the remaining 50% treated for 15 days. Liver microsomes were prepared and used to test for cytochrome P450 (CYP) activities with various substrates. All doxylamine and phenobarbital treatments were associated with significant increases in CYP concentrations, with the exception of doxylamine 40 ppm for 7 days. Doxylamine treatments induced CYP2B markedly, CYP3A and 2A modestly, and thyroxine-glucuronosyltransferase by 2.1-2.8 folds, but not CYP1A, 2E, or 4A. It is not uncommon that an enzyme induced by a particular drug also metabolizes the drug (known as auto-induction). Some examples include: phenytoin and CYP2C9 and 2C19,46 alcohol and CYP2E1,47 and carbamazepine and CYP3A448,49. Therefore, CYP2B, 3A, and 2A may potentially metabolize doxylamine. We should also be aware that as this study was performed in mice, the results may not necessarily apply to humans.

Also in 1996, Thompson et al. reported a randomized open-label, positive and placebo- controlled, parallel-designed study on the influence of doxylamine and phenobarbital on mixed function oxidase activity, as measured by antipyrine pharmacokinetics and 6β-hydroxycortisol urinary excretion in 48 healthy male human subjects.50 Subjects were randomly assigned one of three treatments every 6 hours for 17 days (16 subjects per treatment): 12.5 mg doxylamine succinate, 30 mg phenobarbital, or placebo. Blood and urine samples were collected. While phenobarbital had induced mixed function oxidase activity; doxylamine and placebo did not show any evidence of enzyme induction. The authors interpreted that the lack of change in antipyrine measures after doxylamine treatment as a lack of change in CYP1A2, CYP2C9/18, and CYP3A4 isozymes.

In 2006, Akutsu et al. reported an in vitro study on human liver microsomal CYP and diphenhydramine N-demethylation.51 In this study, human liver microsomes and recombinant insect cells expressing CYPs were treated with diphenhydramine. Liquid chromatography-mass spectroscopy (LC-MS) was then to identify diphenhydramine, N-demethyl diphenhydramine, 18 and orphenadrine (internal standard) in the solutions extracted from the microsomes or cells. These procedures had also been repeated with individual CYPs chemically inhibited. Results identified CYP2D6 as a major contributor, and CYP1A2, 2C9, and 2C19 as minor contributors to N-methylation of diphenhydramine. As diphenhydramine and doxylamine are structurally similar, and both undergo N-demethylation, the aforementioned enzymes may also be involved in the N-demethylation of doxylamine. However, this proposition should be treated with care, as in vitro findings may not represent the in vivo mechanism. CYP2D6 has also been reported to metabolize some other antihistamines such as promethazine52 and chlorpheniramine53.

Based on the above studies, CYP1A2, 2A, 2B, 2C9, 2C19, 2D6, and 3A are possible candidates of doxylamine metabolizing enzyme. Further studies in human liver and kidney is needed to identify the enzymes responsible for doxylamine metabolism.

2.1.5.4 Common Side Effects of Doxylamine

As reported by some studies in section 2.1.6.2 (Pharmacokinetics of Doxylamine), the most common side effect of doxylamine was sedation/somnolence. Other side effects may include headache, weakness, fatigue, and dizziness.

Antihistamines as a class has been associated with multiple side effects. After usual dose, the following side effects may occur: (CNS effects) drowsiness, fatigue, somnolence, dizziness, impairment of cognitive function/memory/psychomotor performance, headache, dystonia, dyskinesia, agitation, confusion, and hallucination; (other effects) mydriasis, dry eyes/mouth, urinary retention/hesitancy, decreased gastrointestinal motility, constipation, peripheral vasodilation, postural hypotention, and appetite stimulation and weight gain.30 For a full list of antihistamine-related adverse events, please refer to Simons and Simons’ review.30

2.1.5.4.1 Overdose

In 1987, Köppel et al. reported an evaluation of 109 cases of doxylamine overdose.54 In about 60% of the cases, 10-40 times the therapeutic dose (25 mg) was ingested. In about 50% of the cases, plasma doxylamine exceeded maximum therapeutic level by 8-50 times. Symptoms of doxylamine toxicity included impaired consciousness (most common), seizures, anticholinergic symptoms like tachycardia and mydriasis, and psychotic behaviour similar to catatonic stupor. No dose-response correlation was observed for the symptoms, suggesting differential 19 susceptibility to doxylamine toxicity amongst individuals. Based on their clinical communications, the authors suggested that rhabdomyolysis was an uncommon, but not rare, serious complication of doxylamine toxicity. If severe enough, rhabdomyolysis may impair renal function to the point of acute renal failure.

Also in 1987, Mendoza et al. reported a case of rhabdomyolysis associated with doxylamine overdose in a 16 years old male.55 At home, the patient was disoriented, staggering, agitated, and vomiting. In the emergency room, the patient was combative, febrile, had petechiae on face and upper chest (capillary bleeding, rash-like appearance), hallucinating, demonstrated nystagmus (involuntary eye movement), trembling, and mydriasis. Other symptoms included hypertension, sinus tachycardia, and urinary retention. Urine screening showed that he had rhabdomyolyis. Together with toxic screening, urine screening also showed that he had doxylamine toxicity. The authors had investigated the cause of rhabdomyolysis, for which they had ruled out other possibilities (infection, metabolic and/or hereditary, myopathy, idiopathic, other rhabdomyolysis- causing medications) and concluded that doxylamine overdose likely caused the non-traumatic rhabdomyolysis observed. In support of this conclusion, the peak serum doxylamine was reported to be 7.5 µg/mL (75 times the therapeutic level) and the half-life was prolonged to 22 hours.

In 2007, Jo et al. reported a study on the risk factors for rhabdomyolysis associated with doxylamine overdose.56 27 patients with doxylamine monotoxicity (no other overdose/toxicity) admitted to a teaching hospital during July 2000-September 2005 were recruited. Incidence of doxylamine toxidrome symptoms were as follows: somnolence (12/27), tachycardia (11/27), hypertension (7/27), dizziness (7/27), nausea and vomiting (7/27), seizures (5/27), hyperthermia (3/27), and irritability (1/27). 16/27 patients developed rhabdomyolysis, 3 of which also developed acute renal failure. When the dose ingested exceeds 20 mg/kg, development of rhabdomyolysis could be predicted with high sensitivity and specificity. However, the study did not detect a correlation between ingested amount and peak serum creatinine phosphokinase level, an indicator of muscle injury. Although doxylamine-induced seizure had been proposed to be the cause of rhabdomyolysis, the authors suggested that reported serum creatinine phosphokinase levels were too high to be attributed to seizure alone. The authors concluded that high dose of doxylamine was directly toxic to striated muscle, though again there were varied susceptibility amongst individuals. 20

In 2015, Cantrell et al. reported a retrospective study of unintentional ingestion of doxylamine in children of 6 months-5 years old.57 Using a poison system database, 140 cases from 1997-2012 were identified. Cases with non-doxylamine ingestions were excluded. Based on the record, 22 patients experienced self-limiting symptoms including lethargy (17/22), tachycardia (12/22), and agitation (4/22). Again, study results showed that individuals differ in susceptibility to doxylamine toxicity. The lowest weight-based dose which caused drowsiness was 1.5 mg/kg, yet a child who took 4.2 mg/kg showed no symptoms. No life-threatening symptoms such as seizures or arrhythmias were reported. Only one case of hospitalization was reported, in which a 2 years old boy ingested up to 150 mg (13.2 mg/kg). He was presented to the emergency department with agitation and mydriasis. Activated charcoal was administered and the boy was admitted to the intensive care unit. All symptoms had resolved by the 8th hour post-ingestion.

In summary, symptoms of doxylamine toxidrome include CNS symptoms (e.g. impaired consciousness, seizures, psychotic behaviour, disorientation, staggering or trembling, agitation, nystagmus, somnolence, lethargy, dizziness, nausea and vomiting, and irritability), anticholinergic/antimuscarinic symptoms (e.g. mydriasis, febrile, tachycardia, hypertension, hyperthermia, petechiae, urinary retention), and systemic complications (e.g. rhabdomyolysis, and renal impairment/failure secondary to rhabdomyolysis). It has been proposed that antihistamines may injure sarcolemma, cause leakage of intracellular contents and subsequently activate proteolytic enzymes, leading to progressive injury of the muscle cell, which causes rhabdomyolysis.58,59

2.1.6 Vitamin B6/Pyridoxine

Vitamin B6 (B6) is a group of interconvertible compounds with 7 known forms: pyridoxine (PN), pyridoxine 5’-phosphate (PNP), pyridoxal (PL), pyridoxal 5’-phosphate (PLP), pyridoxamine (PM), pyridoxamine 5’-phosphate (PMP), and 4-pyridoxic acid (PA). Figure 3 denotes their structures and interconversions.

Figure 3 – Structures of B6 vitamers and their interconversions

PN and PLP are commonly found in over the counter supplements, by themselves or in combination with other vitamins or minerals. PN is also used by itself or combined with doxylamine for nausea and vomiting of pregnancy. PLP is the main active form of B6 in our body and is a cofactor/coenzyme in many reactions in humans.60 For instance, metabolism of many brain neurotransmitters and amino acids are PLP-dependent.60 22

2.1.6.1 Pharmacokinetic and Physicochemical Properties of Vitamin B6/Pyridoxine

While multiple studies61-64 have suggested that B6 vitamers are absorbed through passive diffusion in intestines, some studies64,65 have suggested that carrier-mediated intestinal absorption is possible. The bioavailability of B6 is about 75-85% for general diet.63,66,67 Absorbed B6 vitamers are mainly metabolized in liver, where they are converted into PLP, the active form that participates in bodily reactions.68 PLP is then distributed to tissues and organs through blood, in which it is bound to albumin and hemoglobin.68 On cell membranes, membrane phosphatase converts PLP to PL, which is then absorbed (and converted) for use.68,69 Skeletal muscle stores 75-80% of the body’s B6, mainly as PLP.68,70,71 B6 vitamers are mainly excreted in urine, with PA being the major excretory product.63,72,73 However, other pathways of excretion may exist73. Table 3 is a summary of B6 absorption, distribution, metabolism, and excretion.

Table 2 – Summary of vitamin B6 absorption, distribution, metabolism, and excretion 60,68,74,75

Organ Main Reactions/Processes

Stomach Free or phosphorylated forms of B6

Intestine Phosphorylated forms (PLP, PMP, PNP) dephosphorylated by intestinal phosphatases before absorption  PL, PM, PN are the main absorption forms

Liver PL, PM, PN re-phosphorylated by pyridoxal kinase  PLP, PMP, PNP

PMP and PNP further converted into PLP by pyridox(am)ine phosphate oxidase (PNPO)  PLP enters blood

PL also converted into PA for excretion  PA enters blood

Blood PLP bound to albumin, distributed throughout body

Cell membranes or PLP dephosphorylated by membrane-associated phosphatases  PL choroid plexus

Inside cells or brain cells PL re-phosphorylated by pyridoxal kinase  PLP

PL also converted into PA for excretion  PA enters blood

Urinary system PL, PN, and PA excreted in urine

In 1990, Speitling et al. reported a study of the pharmacokinetic properties of B6 vitamers after single (600 mg) or chronic high doses (300 mg/day for 14 days) of oral PN-HCl (Hexobion® Forte).72 The study was performed in 10 healthy male volunteers aged 20-30 years old. The dosing schedule was as follows: Day 1 – 600 mg, Days 2-13 – 300 mg, Day 14 – 600 mg. Blood and urine samples were collected to measure the concentrations of B6 vitamers. Cmax of PN was reached at about 1.3 hours after the first dose, and it was totally eliminated from plasma at about 9 hours (range: 6-13 hours). Estimating from values provided on the first 600 mg dose –1 concentration-time graph, the kel of PN was approximately 1.06 h and the t1/2 was approximately 0.65 hours. Similar blood time-courses were determined for PL and PA. Although PLP had lower maximal concentration than other B6 vitamers in plasma after an acute dose (Day 1), it was the highest at steady-state (during chronic dosing), suggesting there was conversion from other B6 vitamers. The concentration-time graph for chronic use also showed that while there was maintenance of plasma PLP, PL, and PA between doses, PN quickly disappeared before dose administrations, suggesting rapid conversion to other forms and/or rapid elimination. After chronic intake, the dominant B6 vitamer in plasma was PLP. About 57% of the administered PN was excreted in urine, 28% of which was unmetabolized, 64% was PA, 4% was PLP, and 4% was PL. Steady state was reached for PLP, PL, and PA within 2 days of treatment.

In 1994, Zempleni and Kübler reported a study on the pharmacokinetics of intravenously administered pyridoxine in 10 healthy male volunteers.76 Volunteers were supplemented with multivitamin (including a total of 32 mg PN-HCl) for 6 days to prevent superimposed distribution phenomena during PN-HCl infusion. This was followed by a 3 days washout to remove excess B6. Volunteers were then infused with 100 mg PN-HCl over 6 hours. Blood and urine samples were collected for 10 days and B6 vitamer concentrations were measured using HPLC. In samples collected within a few hours after administration started, concentration of PN was consistently the lowest amongst all B6 vitamers measured. After infusion stopped, PN level dropped abruptly to <50 nmol/L within 1-2 hours. The volume of distribution for PN was 45.8 ± 19.9 L, similar to the subjects’ body water volume (45.6 ± 3.7 L). The authors suggested that as PN was not bound to plasma proteins, it was free and could easily penetrate cell membrane. Hence, steady state concentration was quickly achieved after initiating infusion. Since the concentration of PN was consistently low shortly after infusion stopped, only the elimination –1 constant of the first phase (kα) was calculated, with kα = 6.049 ± 2.062 h . Using kα, the authors 25

derived that t1/2 = 0.12 hour. About 73.6% of the administered PN was excreted in urine, approximately 9.1% of which was unmetabolized, 86.5% was PA, and 4.5% was PL. There was no report of PLP in urine. The study also found that PLP and PL concentrations in erythrocytes were 1.8- and 2.6-folds higher than in plasma respectively. The authors suggested that erythrocytes may act as a buffer for excess PL, and that erythrocytes have the ability to convert other B6 vitamers to PLP.

Regarding the two studies above, since their routes and duration of administration were different,

Cmax cannot be compared. It is also interesting to note the difference in t1/2 between the two studies: 0.65 hours in Speitling et al.’s study and 0.12 hours in Zempleni and Kübler’s study. Since Speitling et al. used a very high dose of PN-HCl, the elimination/conversion mechanisms 27 might have been saturated, leading to the longer t1/2. In Nulman and Koren’s study, where subjects were administered a solution of doxylamine and pyridoxine, the average tmax and t1/2 were 0.618 hours and 0.26 hours respectively. While the t1/2 was close to that in Zempleni and

Kübler’s study (0.12 hours), the tmax was different from that in Speitling et al.’s study (1.3 hours). We suspect that the tablet in Speitling et al.’s study took some time to dissolve and be absorbed while the PN in solution in Nulman and Koren’s study could be directly absorbed by the intestines.

® In Diclectin , PN’s tmax and t1/2 in adult women averaged 3.81 hours and 0.34 hours 27 respectively. Compared to the tmax of the normal-release PN tablet in Speitling et al.’s study ® ® (1.3 hours), the tmax of PN in Diclectin was greatly lengthened. This is expected as Diclectin is ® a slow-release formulation. The t1/2 of PN in Diclectin (0.34 ± 0.15 hours) as reported by Nulman and Koren27 was also similar to that reported by Zempleni and Kübler76 (0.12 hours). This is again expected, as the rate of elimination should not be affected by the formulation (as long as it is sufficiently lower than the rate of absorption).

Comparing the urinary excretion profile between Speitling et al.72 and Zempleni and Kübler’s76 studies, one could first see that a higher portion of the dose was excreted in urine in Zempleni and Kübler’s study. Given the high dose used in Speitling et al.’s study, it was possible that some of the excess dose was not absorbed, or shunted to other pathways of elimination such as biliary or fecal excretion. We also saw a higher portion of PN and lower portion of PA in Speitling et al.’s study, when compared to Zempleni and Kübler’s study. This may suggest that the high level 26 of PN had saturated the conversion to the other forms, so that more of it was excreted unchanged.

2.1.6.2 Dietary Reference Intake

In 1998, the Institute of Medicine of the United States compiled a report on dietary reference intakes of some vitamins.63 Please refer to Table 3 for a list of Recommended Dietary Allowance (RDA) and Tolerable Upper Intake (UL) values of vitamin B6. The report also suggested that, while a few studies with significant methodological/reporting flaws have reported toxicity in the 100-200 mg range, other studies did not associate this dose range with adverse events. Additionally, majority of adverse events were reported to occur at chronic use of 500 mg or more. Based on these evidences, the committee recommended 200 mg vitamin B6 per day to be the no-observed adverse-effect-level (NOAEL), and 500 mg to be the lowest-observed-adverse- effect level (LOAEL). NOAEL was further divided by an uncertainty factor of 2, based on the quality of evidence, to obtain a tolerable upper intake (UL) of 100 mg. Based on the UL, women taking standard doses of Diclectin® (1-4 tablets) chronically are not expected to experience side effects from the PN component.

Table 3 – Dietary Reference Intake Values (adopted from the RDA table by Health Canada77)

Age RDA UL

0-6 months 0.1* ND

7-12 months 0.3* ND

1-3 years 0.5 30

4-8 years 0.6 40

9-13 years 1.0 60

14-18 years male 1.3 80

14-18 years female 1.2 80

≤ 18 years pregnant female 1.9 80

≤ 18 years lactating female 2.0 80

19-50 years 1.3 100

19-50 years pregnant female 1.9 100

19-50 years lactating female 2.0 100

≥ 51 years male 1.7 100

≥ 51 years female 1.5 100

ND – Not Determined; RDA – Recommended Dietary Allowance; UL – Tolerable Upper Intake

* Values for 0-12 months are Adequate Intake (AI) 28

2.1.6.3 Vitamin B6/Pyridoxine Toxicity

In general, consumption of ≤ 100 mg vitamin B6 in the form of pyridoxine hydrochloride (PN- HCl) by adults is unlikely to cause toxicity. B6 toxicities due to extreme chronic overdose have been reported in adults since 1980s. Some recent cases are presented below. For earlier cases, please refer to the Dietary Reference Intake report.63

In 2006, Silva and Cruz reported a case of peripheral sensory neuropathy in a patient who had been taking a vitamin B supplement for 10 years.78 The neuropathy was first attributed to her underlying lupus condition, which seemed to be in remission. Nutrition facts on the supplement showed that each capsule contains megadoses of multiple vitamins, including 100 mg of PN- HCl. After she stopped taking the supplement, her neuropathy slowly improved within months.

In 2007, Thompson et al. reported an 80-year-old female who had worsening disequilibrium (loss of balance) over 18 months, attributed to dorsal column sensory neuropathy.79 She reported to have used 200 mg B6 per week. Investigation did not come up with other cause for the dorsal column sensory loss. Serum B6 was measured to be 171 nmol/L, higher than the highest level described by a previous overdose report (120 nmol/L). Symptoms reversed completely within weeks after stopping B6 supplementation.

In 2007, Rankin et al. reported a familial case series of 3 children with pyridoxine-dependent seizures (due to homozygous mutations in the antiquitin gene).80 These children had been administered high dose PN since 2 months postpartum (case 1) or since antenatal period (cases 2 and 3, administered to mother during pregnancy, continued PN therapy shortly or immediately after birth). All three cases suffered mild axonal neuropathy, which may be related to high PN supplementation, ranging from 11 to 30 mg/kg/day.

In 2008, Gdynia et al. reported a 75-year-old male who had ingested 9.6 grams of PN per day for 3 years prior to hospitalization.81 The patient also overdosed himself with multiple other vitamins and minerals. He had had a slowly progressive gait disturbance for 1 year and paresthesias in lower extremities for 2 years, and was wheelchair bound. The patient also showed muscle weakness and loss of senses to “touch, temperature, pinprick, vibration, and joint-position at all distal limbs.” The skin colour was yellowish-brown. The patient was also diagnosed with a sensorimotor mixed axonal-demylelinating polyneuropathy. Blood PN was measured to be 1850 29

μg/L (normal range 40-120 μg/L). 1 year after the patient was instructed to stop taking B6, the patient did not need the wheelchair anymore, and could walk without a cane; his skin colour also restored. However, the patient was still showing some ataxic and motor signs, with electrophysiological examinations still showing some abnormalities. Although the patient also overdosed himself with other supplements, since the symptoms improved after he stopped B6, the authors concluded that the symptoms were B6-related.

In general, chronic B6 overdose would lead to sensory neuropathy, which may lead to loss of balance, gait disturbance, paresthesia, loss of senses, and muscle weakness. In one severe case, the patient was wheelchair bound. In most cases, symptoms resolve within months after cessation of B6 overdose. However, in the last case, i.e. the most severe case, the patient had not fully recovered even after he stopped taking B6 for a year. Most, if not all, of the cases on B6 toxicity were related to chronic overdose of at least a year, as no case report was found for acute overdose.

Given that the highest dose reported for Diclectin® was 12 tablets, i.e. 120 mg PN-HCl, and that pregnant women would use it for at most throughout their pregnancy (about 40 weeks), using Diclectin® for NVP is not expected to cause B6 toxicity in its users.

2.1.6.4 Vitamin B6 deficiency in breastfed infants

Severe B6 deficiency in infants has been associated with tonic-clonic seizures and microlytic hypochromic anaemia.82 B6 deficiency has also been associated with reversible growth retardation,83 lesser performances on behavioural tests,84,85 anaemia,86 high protein level in spinal fluid,86 hyperactivity,87 and electroencephalogram abnormalities.86 It has been suggested that B6 consumption of less than 0.1 mg/day (0.591 µmol/day) would lead to seizure.86 According to Yagi et al.88, the level of B6 in breast milk usable by the breastfed infant was 1.01 ± 0.32 μmol/L. Assume a 6 months old infant drinks 1 L of mature breast milk per day with no other sources of B6, he/she would consume about 1 μmol of B6 daily, meeting both the AI for 0-6 months and the requirement to prevent seizure.

2.1.6.5 Vitamin B6 status of mothers and breast milk

Several studies have shown that maternal B6 consumption is related to the level of B6 in breast milk. In one study where mothers were provided vitamin supplements, the level of vitamins 30

(including B6) in breast milk were higher in supplemented mothers than non-supplemented (control) mothers, with concentrations correlated to consumptions.89 In another study, where 19 subjects who had been breastfeeding for 3 weeks-30 months were recruited.90 Study results again showed that there was a positive correlation between B6 intake and B6 in breast milk. Also, intra-individual variations in B6 levels (daily and weekly) were small.

In 1985, Styslinger and Kirksey reported a study where 24 lactating mothers received either 0, 2.5, 10, or 20 mg PN-HCl per day for 3 days (6 mothers per treatment).91 Dietary B6 intake were also estimated. Concentrations of B6 in breast milk increased significantly with each elevation in PN-HCl dose. The positive correlation between total maternal B6 intake and breast milk B6 concentration was shown to be highly significant (r = 0.79, p < 0.0001; linear equation: y = 89 + 15x, where y = milk B6 (µg/L) and x = maternal B6 intake (mg/day)). Therefore, if we apply the equation for a mother who takes 4 tablets of Diclectin® per day (maximum standard dose, 40 mg of PN-HCl) and the baby drinks 1 L of breast milk per day (a high estimate), then the baby will consume about 0.7 mg of B6 per day, above the AI but not concerning, as much higher doses had been used to treat infants with B6 deficiency safely. Hence, for mothers who take standard doses of Diclectin® and breastfeed, the amounts of B6 in their breast milk are not expected to cause adverse events in their infants.

In 2002, Chang and Kirksey reported a study on the relationships between maternal B6 supplementation, B6 status of breastfed infants, and their growth during 0-6 months.92 47 healthy term infants and their mothers participated in the study. Mothers were assigned to take different doses of PN-HCl supplementation: 2.5 mg/day (11 mothers), 4.0 mg/day (13 mothers), 7.5 mg/day (12 mothers), or 10.0 mg/day (11 mothers). Assessments and breast milk collection were performed once per month. B6 concentrations in breast milk generally correlated with the dose of PN-HCl supplement. Volume of milk intake and numbers of feeding in 24-hour periods did not differ significantly between the groups. The highest B6 intake calculated was slightly below 0.5 mg – for the 10 mg group at 6 months (see Figure 2 in the publication). Regardless of maternal B6 intake, breast milk B6 increased gradually from 0 to 6 months. While only infants from the 10.0 mg group met or exceeded the 1989 RDA for 4-6 months olds (0.3 mg B6/day), all infants met the 1998 AI for 0-6 months and most infants grew normally. The authors concluded that maternal supplement of 2.5 mg daily would be sufficient to provide breastfed infants of 0-6 months old adequate B6 through breast milk. 31

In 1983, Ford et al. reported a study where milk samples were obtained from 35 mothers with term deliveries and 26 mothers with preterm deliveries during postpartum days 1-244.93 Concentrations of various vitamins, including vitamin B6, were measured. This study found that B6 levels in breast milk had increased as breast milk matured over time, concurring with multiple previous studies (see Figure in the publication). In addition, at the same number of days postpartum, breast milk of mothers with pre-term babies contained less B6 than breast milk of mothers with term babies.

To conclude, studies have shown that B6 concentration in breast milk is correlated with maternal B6 intake. In addition, infants breastfed by mothers taking standard doses of Diclectin® are not expected to experience adverse effects due to the PN-HCl component. Studies had also observed a positive correlation between breast milk B6 and postpartum age.

2.1.6.5.1 Using Vitamin B6 to Suppress Puerperal Lactation?

Currently, there are conflicting evidences as to whether Vitamin B6 suppresses lactation.

In the 1970s, B6 had been proposed to suppress lactation through the dopaminergic system. However, the exact mechanism was unclear.94 It was not until early 1980s was dopamine recognised as the prolactin inhibitor factor (PIF), which allowed us to understand mechanistically how B6, prolactin (PRL), dopamine/PIF, and lactation may be related.95-97 In dopaminergic neurons, PLP is known to be a coenzyme for aromatic-L-amino-acid decarboxylase (AADC), which converts L-Dopa into dopamine. Consequent to the increase in PLP, the rate of conversion would be expected to increase. Dopamine/PIF acts on the anterior pituitary to suppress PRL secretion. During breastfeeding, PRL stimulates mammary glands to produce breast milk. As the amount of dopamine/PIF increases, PRL secretion decreases and stimulation on mammary glands reduces, in turn reducing lactation. L-Dopa has been reported to suppress serum PRL and in some cases, galactorrhea.98-100

It had been hypothesized that by promoting conversion of L-Dopa into dopamine with pyridoxine, lactation can be suppressed. An early study94 assessed this hypothesis in 254 puerperal mothers, where mothers were orally administered either placebo (86 patients), 5 mg stilbestrol (68 patients), or 200 mg pyridoxine 3 times per day for six days (75 patients blinded, 25 patients unblinded), beginning on postpartum days 2-3. State of the breasts and amounts of 32 milk secretion were observed and reported by nurses for six days. Nurses were allowed to administer analgesics as needed. Within 1 week postpartum, lactation ceased in 17% of the placebo group, 83% of the stilbestrol group, 93% of the blinded pyridoxine group, and 100% of the unblinded pyridoxine group. Symptom relieve was achieved within 10-12 hours for the pyridoxine group, over 24 hours for the stilbestrol group, and several days for the placebo group. However, it was unclear what analgesics were administered and if they had affected lactation in any ways.

Another study compared the effect of PN (300 mg twice daily), quinestrol (4 mg once), methylergometrine (unknown dose 3 times daily), oxytocin (buccal 200 IU once), and placebo on plasma PRL and milk secretion in puerperium.101,102 50 mothers were divided into five groups. Participants received treatments and were monitored for 8 days. Plasma PRL was measured with radioimmunoassay, and milk volume was measured by measuring weight difference before and after breastfeeding. None of the treatments suppressed plasma PRL and milk secretion. In addition, 5 of 10 mothers in the B6 group had pain in breast, and 4 mothers had to be treated with bromocriptine.

In a double-blind study, Neurobion® (100 mg vitamin B1 + 200 mg vitamin B6 + 200 μg vitamin B12) and placebo were administered to 52 and 43 mothers respectively for 7 days at 1 tablet 3 times daily from postpartum day 1.103 All mothers were asked to bind their breasts, but 2 in the placebo group and 3 in the Neurobion® group did not, and 1 in the placebo group did not record any information. Lactation was not established in these 6 mothers. 2 of 52 Neurobion® mothers showed lactation, compared to 10 of 42 placebo mothers (χ2 test with Yates correction: p < 0.02). The authors concluded that Neurobion® successfully inhibited lactation, and suggested that the high incidence of lactation inhibition in the placebo group was likely due to avoidance of nipple stimulation. No side effects were observed in the Neurobion® group while 1 patient in the control group developed urticarial rash. Note that co-administration of B1 and B12 precludes us from directly associating lactation suppression with B6.

In 1976, Canales et al. reported a study comparing the effect of bromocriptine and B6 on serum prolactin and lactation. All women treated in this study had breastfeeding contraindicated for medical reasons. For their first 7 days postpartum, 14 women were administered 150 mg PN 3 times daily while 20 women were administered 7.5 mg bromocriptine daily. Note that the PN 33 daily dose is lower than previous studies (450 versus 600 mg/day). Blood samples were collected daily for PRL measurement. The study also included 23 untreated breastfeeding mothers (controls) who provided blood samples for 7 days. All PN women suffered painful breast engorgement while all bromocriptine women had lactation suppression. PN had no significant effect on serum PRL (similar to untreated group) while bromocriptine suppressed serum PRL sharply on day 2 postpartum. The authors concluded that PN did not suppress lactation.

In 1976, Macdonald et al. reported a double-blind controlled trial on the efficacy of pyridoxine in suppressing puerperal lactation.104 82 women were assigned to the placebo group and 93 were assigned to the pyridoxine group. Women were instructed to take tablets of placebo or 200 mg pyridoxine 3 times daily. The treatment group was not significantly different from the placebo group with regards to breast engorgement, breast discomfort, analgesics required, and persistence of lactation. The authors concluded that PN did not suppress lactation.

In another double-blind trial, puerperal patients were administered either 200 mg PN, 5 mg stilbestrol, or sucrose every 8 hours for 5 days (total 15 times).105 For the 5 days of treatment, patients were assessed daily for breast tenderness, engorgement, and lactation. Lactation was inhibited in 45/155 PN patients, 151/165 stilbestrol patients, and 33/162 sucrose/placebo patients. The authors concluded that PN 200 mg every 8 hours for 5 days did not inhibit puerperal lactation.

In 1977, Masala et al. reported a study where 10 healthy women who did not want to breastfeed their newborns were treated with 200 mg PN 4 times per day (800 mg/day) for 4 days.106 Prior to PN administration, volunteers mechanically emptied their breast. Blood was drawn at 30 and 15 minutes before and 0, 10, 20, 30, and 60 minutes after emptying. After PN treatment, volunteers emptied their breast and had blood collected in the same manner. Serum PRL was measured. Mechanical breast emptying induced a rise in PRL compared to basal levels before emptying. Although PRL was consistently lower in the pyridoxine group, the difference was not significant and no lactation inhibition was observed in the participants. However, it cannot be ascertained whether paired or unpaired t-test was used.

In another study, 9 women were administered 200 mg PN immediately postpartum, followed by 200 mg 3 times daily for 7 days.107 Blood was sampled on days 1 and 5 and lactation status was assessed on day 5 (as controls were discharged on day 5). Blood samples from another 9 34 breastfeeding women served as controls. There was no significant difference in serum PRL between the two groups. For the PN group, difference between day 1 and day 5 serum PRL was also not significant. None of the PN patients had suppressed lactation.

In 1980, Boes reported a double-blind trial which compared the efficacy of bromocriptine and PN in inhibiting puerperal lactation.108 There were 49 patients in the bromocriptine group and 48 patients in the PN group. All patients received a packet of 42 tablets and were instructed to take 1 tablet 3 times daily for 14 days. The actual regimen for bromocriptine was one 2.5 mg tablet twice per day for 14 days (one tablet per day was inactive); while the actual regimen for PN was one 200 mg tablet 3 times per day for 6 days (tablets for remaining days were inactive). Patients were instructed to take the tablets in the correct order. It is unclear when exactly were the treatments initiated, although the author mentioned that the treatments were intended to inhibit puerperal lactation. Efficacy of the treatments were assessed by the ward sister and doctor in charge. No major side effect was recorded. Bromocriptine effectively suppressed lactation in all 49 patients while PN effectively suppressed lactation in 29 patients. However, 14 PN patients and 1 bromocriptine patient required other medication to suppress lactation, which was usually stilbestrol.

A large study in Italy studied the effect of injecting 600 mg/day PN-HCl (Benadon®) intramuscularly for 5-7 days on lactogenesis.109 Treatment started on the first day after delivery. 1592 patients were recruited and treated. Mammary engorgement, breast pain, and milk secretion were assessed each day. Patients returned for assessments after 20 and 70 days. 90% of the patients came back on day 20 and 70, with normal menstrual cycle restarting at 41-63 day postpartum. Overall, 89.3% of the patients responded and none of them reported adverse effects. Amongst the non-responders, 81 suffered mammary engorgement, 60 suffered breast pain, and 18 had galactorrhea. For the non-responders, PN treatment continued for 6 days with 25 mg furosemide or compressive bandage. At the end, symptoms persisted only in 11 non-responders. The authors also did a review of past studies on PN for lactation suppression, for which they found that when PN was administered intramuscularly, the success rate was high, ranging from 77 to 98%.

In 1985, Andon et al. reported that at low doses (0.5 mg and 4.0 mg), B6 supplementation does not decrease plasma PRL or suppress lactation.110 In this double-blind randomized study, 20 35 lactating mothers were randomly assigned to take a supplement containing the same composition of vitamins and minerals, with either 0.5 or 4.0 mg PN. Blood and milk samples were collected at 1-2 weeks postpartum and 1, 3, 6, and 9 months. Plasma PLP and breast milk B6 concentrations were significantly higher in the 4.0 mg than the 0.5 mg group.

In 1992, Kang-Yoon et al. reported a study where: 6 mothers received 27 mg PN-HCl/day with their infants unsupplemented; 14 mothers received 2 mg PN-HCl/day, 7 of their infants were administered 0.4 mg PN-HCl/day while the remaining 7 were unsupplemented. Treatments lasted from days 1 to 28 postpartum.111 Daily milk volume intake was measured on day 7, 14, and 28. When adjusted for infant weight, daily milk volume intake did not differ between the groups.

To conclude, majority of the studies had shown that high doses of oral PN failed to suppress puerperal lactation. Meanwhile, as reported by Scaglione and Vecchione’s study and review109, intramuscular administration of high dose PN appeared to be effective in suppressing puerperal lactation. Based on these findings, the PN component of Diclectin®––when Diclectin® is used at normal dosages (1-4 tablets daily, i.e. 10-40 mg PN-HCl daily)––is not expected to suppress maternal lactation.

Chapter 3. Other NVP Medications and Breastfeeding

Other than doxylamine-pyridoxine combinations, other medications have also been used to treat NVP. Some common ones (based on Motherisk NVP helpline’s experience) include: diphenhydramine, dimenhydrinate, metoclopramide, and acid-reducing drugs such as omeprazole and ranitidine.

3.1 Diphenhydramine and Dimenhydrinate

Diphenhydramine is also a first generation ethanolamine antihistamine, very similar in structure to doxylamine; whereas dimenhydrinate is the salt of diphenhydramine and 8-chlorotheophylline (2006 Scharman).112 Diphenhydramine is sometimes used with other NVP drugs such as droperidol or metoclopramide to manage hyperemesis gravidarum.113,114

No published literature reported measuring the concentration of diphenhydramine in breast milk using modern assay methods.115 A study reported in 1951 injected 100 mg of diphenhydramine intramuscularly to 4 women, followed by measuring diphenhydramine concentrations in breast milk.115,116 At 1-hour post-dose, diphenhydramine was detected in the milk of 2 women (42 and 100 μg/L). At 5-hours post-dose, diphenhydramine was again detected in the milk of only 2 women (20 and 100 μg/L).

In 1993, Ito et al. reported a follow-up study on the occurrence of side effects in infants exposed to medications through breastfeeding.117 There were 7 infants exposed to dimenhydrinate and 1 reported irritability. Occurrence of side effects for the antihistamine class overall was 10% for irritability/colic and 1.6% for drowsiness.

Overall, there is a lack of information on the safety of diphenhydramine and dimenhydrinate in children. Although their concentration in breast milk had been measured, these measurements were not necessarily accurate. Future studies should use modern assays (e.g. LC-MS-MS) to confirm these findings. Given that diphenhydramine and dimenhydrinate could be found in breast milk, there exists a risk that infants who drink such breast milk may experience side effects such as irritability or drowsiness, as reported by Ito et al.’s study.117 Future follow-up studies should attempt to recruit larger numbers (preferably > 50) of mother-infant pairs exposed to a single drug through breastfeeding to improve study power. 37

3.2 Metoclopramide

Metoclopramide is recommended by the American College of Obstetricians and Gynecologists as an alternative for treating NVP.118 In addition, metoclopramide has been used as a galactagogue in mothers with poor lactation, albeit studies have reported conflicting results on its efficacy.119-124

In studies on the efficacy of metoclopramide in improving lactation, most of the breastfed infants did not experience any adverse events.119,121,122,125,126 However, one infant had experienced gastrointestinal discomfort.125 In addition, one study also reported that some neonates exposed to metoclopramide through breastfeeding had higher plasma PRL than age-matched control samples.127

Two studies have assessed the intake of metoclopramide through breast milk by infants.

In 1980, Lewis et al. reported the levels of metoclopramide in breast milk from mothers given a single dose of 10 mg metoclopramide.120 Blood and breast milk were sampled at 2-hours post- dose, and metoclopramide concentrations were measured in these samples using gas-liquid chromatography. Mean metoclopramide concentration in breast milk was 125.7 ± 41.7 ng/mL. Therefore, an infant who drinks 1 L of milk per day would consume no more than 130 µg metoclopramide per day (or 45 µg/kg/day) on average, a dose the authors considered subtherapeutic.

In 1983, Kauppila et al. reported a study measuring the level of metoclopramide in breast milk and infant plasma, as well as the level of infant plasma PRL.127 5 mothers with 3-9 days old neonates and 18 mothers with 8-12 weeks old infants had participated. Mothers were required to take 10 mg metoclopramide 3 times per day for 2 weeks. While metoclopramide was found in all breast milk samples, it was found only in the plasma of 1 neonate. Metoclopramide intake were 6-24 μg/kg/day for the neonates and 1.3-13.2 μg/kg/day for the infants. The authors suggested that these doses were much less than the therapeutic dose (500 μg/kg/day) for children.

To summarize, studies have suggested that metoclopramide intake through breast milk is low and hence unlikely to cuase adverse effects. Regardless, future studies should investigate whether neonate exposure to metoclopramide through breast milk can cause elevated PRL (as reported by Kauppila et al.127), and if this would have any long-term consequence. 38

3.3 Acid-Reducing Drugs

Acid symptoms such as heartburn and acid reflux have been associated with severe NVP. A study has shown that acid-reducing drugs, aside from relieving acid symptoms, also reduce NVP.128 Commonly used acid-reducing drugs in NVP include omeprazole (proton pump inhibitor) and ranitidine (H2 receptor antagonist).129

In 1998, Marshall et al. reported a mother who had taken 20 mg omeprazole for gastroesophageal reflux starting from 29th week of gestation and continued into lactation.130 Omeprazole successfully treated her symptoms. At 3 weeks postpartum, blood and breast milk were sampled to measure omeprazole concentrations using high performance liquid chromatography (HPLC). Peak omeprazole concentration in breast milk (58 nM) was measured to be less than 7% that of maternal serum (950 nM). The authors suggested that the low concentration in breast milk may be due to the fact that omeprazole is highly protein-bound in plasma. The authors further proposed that, since uncoated omeprazole would be inactivated by breastfed infants’ stomach acid, it will unlikely affect their stomach acid secretion.

In 1985, Kearns et al. reported the level of ranitidine in breast milk and serum samples from a mother.131 The mother had taken 150 mg ranitidine hydrochloride every 12 hours for 60 hours (5 doses). Breast milk and blood samples were collected at 1.5, 5.5, and 12 hours after the 5th dose. The highest level of ranitidine in breast milk was 2610 μg/L, collected at 5.5 hours post-dose. Given that the breastfed infant was 3.4 kg, we can assume that he drinks about 0.51 L of breast milk per day, leading to a maximum daily oral dose of 1.33 mg or 0.39 mg/kg. This is relatively low when compared to doses previously reported for critically ill preterm and term neonates (0.5-1.5 mg/kg).132

To summarize, omeprazole intake through breast milk is not expected to cause adverse events in the breastfed infant as uncoated omeprazole would be inactivated by breastfed infants’ stomach acid. As for ranitidine intake through breast milk, the dose consumed is considered very low compared to known therapeutic doses for neonates. Therefore, it is not expected to cause significant adverse events in the infant.

Chapter 4. Methods of Assessing Infant Exposure to Drugs in Breast Milk

In addition to predicting drugs’ entry into breast milk using their physicochemical parameters (see Section 2.1.6.1), there are also empirical methods of assessing infants’ exposure to drugs in breast milk.

4.1 Milk/Plasma Ratio

Milk/plasma (M/P) ratio is the ratio of the average concentration of a drug in breast milk divided by the average concentration in maternal plasma.133 A ratio of > 1 indicates that the concentration is higher in breast milk; while a ratio of < 1 indicates that the concentration is higher in maternal plasma.134

Infant exposure through breast milk can be estimated using the following equation:133

퐴푣푒푟푎푔푒 𝑖푛푓푎푛푡 푑표푠푒 = 푀/푃 푟푎푡𝑖표 ∗ 퐶푎푣 ∗ 푉푚𝑖푙푘

Cav = average maternal plasma concentration; Vmilk = average volume of daily milk intake

Based on this equation, if the volume of milk intake or the maternal plasma concentration is low, infant exposure will be low regardless of the M/P ratio.

According to the works by Atkinson, Begg, and Duffull,135-137 M/P ratio may be estimated from known pharmacokinetic/physicochemical parameters using the following equations:

푀 1 + 10(푝퐾푎 − 7.2) 푀 푀 푢 = ( 푢 = 푟푎푡𝑖표 표푓 푢푛푏표푢푛푑 푑푟푢푔) 푃푢 1 + 10(푝퐾푎 − 7.4) 푃푢 푃

푓푢,푝 = 1 − 푃퐵 (푓푢,푝 = 푓푟푎푐푡𝑖표푛 표푓 푢푛푏표푢푛푑 푑푟푢푔 𝑖푛 푝푙푎푠푚푎 푃퐵 = 푝푟표푡푒𝑖푛 푏𝑖푛푑𝑖푛푔)

0.448 푓푢,푝 푓푢,푚 = −4 0.448 0.448 (푓푢,푚 = 푓푟푎푐푡𝑖표푛 표푓 푢푛푏표푢푛푑 푑푟푢푔 𝑖푛 푏푟푒푎푠푡 푚𝑖푙푘) (6.94 ∗ 10 ) + 푓푢,푝

푀𝑖푙푘: 퐿𝑖푝𝑖푑푃 = 101.29∗log 퐷푝퐻7.2−0.88

0.955 퐾 = + 0.045 ∗ 푀𝑖푙푘: 퐿𝑖푝𝑖푑푃 푓푢,푚 40

푀푢 푀 −0.09+2.54∗ln +0.8∗ln 푓푢,푝+0.46∗ln 퐾 = 푒 푃푢 푃

푀 푝푟푒푑𝑖푐푡푒푑 𝑖푛푓푎푛푡 푑표푠푒 푝푒푟 푑푎푦 = ∗ 퐶 ∗ 푉 푃 푠푠,푚푎푡푒푟푛푎푙 푚𝑖푙푘

(퐶푠푠,푚푎푡푒푟푛푎푙 = 푚푎푡푒푟푛푎푙 푠푡푒푎푑푦 푠푡푎푡푒 푐표푛푐푒푛푡푟푎푡𝑖표푛, 푉푚𝑖푙푘 = 𝑖푛푓푎푛푡 푑푎𝑖푙푦 푚𝑖푙푘 𝑖푛푡푎푘푒)

138,139 Note that logDpH7.2 is also known as logPapp.

For doxylamine:

푝퐾푎ℎ𝑖푔ℎ푒푠푡 = 8.87 푃퐵푤ℎ표푙푒 푝푙푎푠푚푎 = 0.38 log 퐷푝퐻7.2 = 1.43

휇푔 퐶 = 90.4 푉 = 1 퐿 푚푎푥 퐿 푚𝑖푙푘

As Css,maternal is unavailable, the maximum plasma concentration (Cmax) after a single dose of 2 27 Diclectin® tablets was used instead. 1 L is an arbitrary high estimate of Vmilk based on a summary of studies on breast milk consumptions by infants of 0-36 months old.140

35 Readers should be aware that the pKa, and logDpH7.2 values were predicted by computations , while fraction unbound (fu,p) is defined as follows: fu,p = 1 – fraction bound to whole plasma 141 proteins (PBwhole plasma = 0.38, measured experimentally ).

Below are the calculations:

푀 1 + 10(8.87 − 7.2) 푢 = = 1.12739 푃푢 1 + 10(8.87 − 7.4)

푓푢,푝 = 1 − 0.38 = 0.62

0.620.448 푓 = = 0.95453 푢,푚 (6.94 ∗ 10−4)0.448 + 0.620.448

푀𝑖푙푘: 퐿𝑖푝𝑖푑푃 = 101.29∗1.43−0.88 = 9.21934

0.955 퐾 = + 0.045 ∗ 9.21934 = 1.41536 0.95453 41

푀 = 푒−0.09+2.54∗ln 1.12739+0.8∗ln 0.62+0.46∗ln 1.41536 = 0.99196 푃

휇푔 푝푟푒푑𝑖푐푡푒푑 𝑖푛푓푎푛푡 푑표푠푒 푝푒푟 푑푎푦 = 0.99196 ∗ 90.4 ∗ 1 퐿 = 89.67318 휇푔 퐿

푀 = 0.99 푝푟푒푑𝑖푐푡푒푑 𝑖푛푓푎푛푡 푑표푠푒 푝푒푟 푑푎푦 = 89.67 휇푔 ≈ 0.09 푚푔 푃

Based on the predicted infant dose per day (0.09 mg/day), one would not expect doxylamine exposure through breast milk to be of clinical concern. However, as pKa, and logDpH7.2 values are not derived experimentally, they do not necessarily represent the actual value and hence this M/P ratio is not necessarily reliable.

4.2 Relative Infant Dose

Relative infant dose (RID) is the weight-adjusted dose received by infants through breast milk relative to the weight-adjusted maternal dose. Generally, when choosing drugs to use during lactation, we would prefer drugs with RID < 10%.133

Relative infant dose can be calculated using the following equation:

𝑖푛푓푎푛푡 푑표푠푒 [ ] 𝑖푛푓푎푛푡 푤푒𝑖푔ℎ푡 푅퐼퐷 = ∗ 100% 푤ℎ푒푟푒 𝑖푛푓푎푛푡 푑표푠푒 = 퐶 ∗ 푉 푚푎푡푒푟푛푎푙 푑표푠푒 푚𝑖푙푘 푚𝑖푙푘 [ ] 푚푎푡푒푟푛푎푙 푤푒𝑖푔ℎ푡

Cmilk = drug concentration in breast milk; Vmilk = volume of milk intake

Volume of milk intake may be measured or estimated as 150 mL/kg of infant weight.

4.3 Oral Bioavailability

Regardless of the M/P ratio or the RID, if a drug has low oral bioavailability, it is unlikely to affect the infant. For instance, in the case of omeprazole (Section 3.3), it is inactivated by stomach acid. Therefore, regardless of the amount consumed by the infant, unprotected omeprazole will unlikely reach concentrations that can cause adverse/therapeutic effects in the breastfed infant. 42

4.4 Infant Plasma Concentrations

Measuring an infant’s plasma drug concentration is the most direct measure of whether the intake warrants clinical concern, as it accounts for the infant’s pharmacokinetics as well. However, there are several limits to this approach as suggested by Begg et al.133:

1. Ethical concerns

2. Requires parental cooperation, usually can only collect one sample

3. Needs to be drawn by an experienced pediatric phlebotomist to minimize parental anxiety and trauma to the infant

a. Cost of hiring the phlebotomist

4. Small sample volume may raise the limit of detection

Chapter 5. Methods 5.1 Setting

Subjects were enrolled through the Motherisk Program, which provides evidence-based information to women, their partners, and their health care providers on the safety and risks associated with exposures, such as drugs, chemicals, radiations, and infectious diseases during pregnancy and lactation. Upon receiving a call, Motherisk counsellors would document medical and obstetrical history as well as details about exposures, concomitant medications, medical conditions and use of drugs of abuse on a standard Motherisk intake form (written or digital) for clinical purposes.

The Motherisk NVP Disease Management Helpline, part of the Motherisk information services, is the only such service worldwide, where women with NVP are counselled about symptom management based on guidelines from the Society of Obstetricians and Gynaecologists of Canada.

5.2 Study Design

This is an analytical retrospective-prospective cohort study of women who used Diclectin® during breastfeeding and their breastfed infants. I call this study retrospective-prospective as participants were recruited retrospectively and prospectively

5.3 Study Group Subject Recruitment

Mothers who had breastfed and used Diclectin® concurrently, or pregnant and asked for the safety of using Diclectin® during breastfeeding were contacted by the study coordinator, who would provide them study information and request for consent to participate. Mothers were recruited when they agreed to participate. I had intended to recruit 100 mothers who called the Motherisk helplines during 2012-2015.

5.3.1 Prospective Recruitment

At the end of a call, Motherisk counsellors informed eligible mothers about this study and asked if they would like to be contacted by the study coordinator (myself). Once verbal consent was given, I would call the mother to provide further information about the study and request for 44 verbal consent to conduct the follow-up interview. Interviews were conducted at least a week after the mother initiated Diclectin® therapy.

5.3.2 Retrospective Recruitment

I had called eligible mothers who had contacted Motherisk regarding use of Diclectin® while breastfeeding before the study initiated. During the call, I would inform the mother about the study and request for verbal consent to conduct the follow-up interview. Corresponding consent procedures were performed depending on their consent.

5.3.3 Interview

After a mother was recruited, I would then arrange a time for the interview. Each interview took approximately 30-40 minutes.

5.3.4 Inclusion and Exclusion Criteria

Inclusion criteria:

 Age of mother when she consulted Motherisk on using Diclectin® while breastfeeding: 18-45

 Taking/took Diclectin® while breastfeeding at the same time

Exclusion criteria:

 Mothers who used other sedating medications for > 3 times throughout the period of interest (concurrently taking Diclectin® and breastfeeding), including but not limited to the following types/classes: opioids, barbiturates, benzodiazepines, non-benzodiazepines, or other sedating antihistamines.

 Mothers who had used:

o Alcohol > 1 time per week, and > 2 drinks each time

o > 2 cigarettes per day

o I believe that such low frequency of use would unlikely cause AEs in infants142,143 45

 Infants with medical conditions that may affect their mental state (e.g. CNS anomalies)

 Mother unable to communicate in English

 Mothers who had refused to participate or provide oral consent

5.3.5 Recruitment Outcome

After initial screening by applying inclusion/exclusion criteria on Motherisk records, 237 callers were selected. Below listed the cases removed due to various reasons:

 Wrong number

 Excluded prior to interview base on inclusion/exclusion criteria (not including did not breastfeed or did not take Diclectin®

 Did not breastfeed or did not take Diclectin®

 Could not communicate in English

 Refused to participate

 Incomplete interview

5.4 Data Collection and Target Endpoints

For all participants, demographics and medical information of both the mother and their breastfed infant were collected. Adverse events in mothers and infants were recorded––with a primary focus on CNS depression due to the sedative property of doxylamine.

CNS depression may manifest as difficulty in latching to the breast, sleepiness, limpness, amongst other signs.144 To capture these adverse events, I had created a questionnaire adapted from questionnaires used in previous Motherisk studies on the use of opioids and benzodiazepines during breastfeeding, which effectively captured symptoms of CNS depression.145,146

Secondary endpoints included: 46

 Non-CNS depression adverse events: unusual excitement, irritability/fussiness, irregular breathing, vomiting, constipation, change in skin colour, etc.

 Whether any observed adverse events in the breastfed infant necessitated medical attention

 Whether observed adverse events in the breastfed infants correlated with maternal adverse events

 Whether weight-adjusted maternal dose of doxylamine (in Diclectin®) correlated with the occurrence of adverse events in either the mother or the infant

 Efficacy of Diclectin® in treating NVP as rate by the mothers on a scale of 1 (no effect) to 10 (totally alleviated NVP)

Near the end of an interview, mothers were asked to provide verbal consent for us to send a questionnaire to the infant’s healthcare provider requesting for the infant’s medical history. This was to ensure that information collected in the interview was accurate. However, due to unforeseeable circumstances, this part could not be performed for some consented mothers. Therefore, results presented in this thesis are based on information collected in the interview only.

Mothers were also asked if they would like to receive a summary of the results. If they answered yes, their e-mail addresses were recorded.

5.5 Analysis

Of the 237 mothers contacted, 67 had completed the full questionnaire and 1 had partially completed the questionnaire. The partially completed case was excluded for there was insufficient information for analysis. 1 case was excluded as the mother confused information regarding her 2 pregnancies/lactation during the interview. An additional 11 cases had to be excluded due to protocol violations. Although I had interviewed 8 mothers who consulted Motherisk during 2006-2011, these mothers had to be excluded from the analysis due to concerns of recall bias. One mother was excluded for she was older than 45 years old when calling Motherisk. One mother had to be excluded as her dimenhydrinate use overlapped with Diclectin® for a period. In the end, data from 41 mother-infant pairs were eligible for analysis.

5.5.1 Statistical Analysis

Categorical data (e.g. rates of adverse events) were compared using Fisher’s exact test due to small sample size (which disallows χ2 test). Fisher’s exact test calculations were performed using this website: http://graphpad.com/quickcalcs/contingency1/. 95% confidence interval (CI) of the rates of individual AEs in the current study was calculated using the Agresti and Coull method on this website: http://epitools.ausvet.com.au/content.php?page=CIProportion. The Agresti and Coull method was chosen over the traditional Wald method as per the recommendation by Brown et al.147

Continuous data are expressed as mean ± stand deviation unless otherwise stated. Statistical significance was defined as p < 0.05.

Calculation of means and standard deviations of continuous data (e.g. age of mother during pregnancy) were performed using Microsoft Excel 2013. Means and standard deviations of the current study data and others were compared using Student’s t-test on this website: http://graphpad.com/quickcalcs/ttest1/?Format=SD. When compared with data without standard deviation, one-sample t-test was used instead: http://graphpad.com/quickcalcs/oneSampleT1/?Format=SD.

I had also investigated if there were any correlations between weight-adjusted maternal highest dose per pregnancy per day (maternal dose) and maternal adverse events, infant adverse events, relieving infant eczema, or total infant sleeping time per day. Data for one mother had to be excluded from correlation analyses, as I could not calculate her weight-adjusted dose (due to uncertain weight data). Microsoft Excel 2013 was used to calculate Pearson’s r for each sets of comparisons. Calculation of p-values for Pearson’s r values were performed using this website: http://www.danielsoper.com/statcalc3/calc.aspx?id=44.

Doses were expressed as mg/kg/day and were calculated using this equation:

푚푔 ℎ𝑖푔ℎ푒푠푡 푡표푡푎푙 푑푎𝑖푙푦 푑표푠푒 ( ) 푑푎푦

푚푎푡푒푟푛푎푙 푤푒𝑖푔ℎ푡 (푘푔)

5.5.2 Post-Hoc Comparisons

After the statistical analysis was completed, rates of adverse events with ≥ 5 occurrences would be statistically compared to rates reported by other studies. Only maternal sedation and infant sedation met this requirement. The rate of maternal sedation was compared to that in Atanackovic et al.19, which reported the highest rate of sedation amongst studies on Diclectin®. Whereas the rate of infant sedation was compared to previous Motherisk studies on other medications, including other antihistamines117, benzodiazepines146, acetaminophen145, oxycodone145, and codeine145.

Figure 4 – Flow Chart of Recruitment and Data Collection

Chapter 6. Results 6.1 Study Population

The time period between Motherisk initial consult date and the interview date was on average 11.0 ± 8.3 months, with the range being approximately 1 week-2.2 years. Maternal age at the time of consult (for using Diclectin® while breastfeeding) was 33.6 ± 4.7 years old (range: 25.8- 43.0 years old).

In general, most mothers had 2-4 pregnancies (gravidity), 1-3 living children (parity), 0-1 miscarriage, and 0-1 termination. None of the mothers had other pregnancy losses. Maternal body-mass index (BMI) at the time of Diclectin® consult was 24.7 ± 5.3 kg/m2, ranging from 17.0 to 39.6 kg/m2. BMI could not be calculated for one mother due to uncertain maternal weight. Based on World Health Organization’s BMI classification, of the 40 mothers, 1 was underweight, 23 were within normal range, 10 were overweight, and 6 were obese.148

Regarding maternal race, 82.9% of the mothers were White, 7.3% were Oriental Asian, 4.9% were mixed, 2.4% were Indo-Asian, and 2.4% were Middle Eastern.

Male-to-female ratio of the breastfed infants was 1.16 (22 males and 19 females).

The gestational age at birth for the breastfed infants was 39.3 ± 1.6 weeks, with a minimum of 35 weeks and maximum of 42 weeks. Regarding breastfed infants’ birth weight, the average was 3.5 ± 0.5 kg, with a range of 2.4-4.7 kg. Under ICD-10 definitions,149 1 was classified as Low Birth Weight (1000-2499 g) and 2 were classified as Exceptionally Large Baby (> 4500 g).

Note: demographics of prospectively recruited participants were similar to those of retrospectively recruited participants, except: duration between Motherisk consult and follow-up interview, maternal race, and breastfed infant sex.

Table 4 – Demographics Part 1

[Values are presented as: mean ± Overall (n=41) Prospective Retrospective SD (range)] group (n=11) group (n=30)

Duration between Motherisk consult 11.0 ± 8.3 0.9 ± 0.4 14.7 ± 6.5 and follow-up interview (months) (0.2-26.5) (0.2-1.8) (3.8-26.5)

Maternal age at time of Motherisk 33.6 ± 4.3 33.9 ± 4.6 34.7 ± 4.2 consult (years) (25.8-43.0) (26.5-41.5) (27.9-44.5)

Maternal body-mass index at time of 24.9 ± 5.3 23.4 ± 4.4 25.4 ± 5.5 Motherisk consult (kg/m2) (17.0-39.6) (18.8-33.3) (16.9-39.6) (n=40) (n=29)

Breastfed infant’s birth weight (kg) 3.5 ± 0.5 3.5 ± 0.3 3.5 ± 0.6 (2.4-4.7) (3.0-3.9) (2.4-4.7)

Breastfed infant’s gestational age at 39.6 ± 1.5 39.7 ± 1.3 39.5 ±1.6 birth (weeks) (35-42) (37.29-42) (35-41.5)

Maternal pregnancy history*

Gravidity 2.7 ± 0.9 (1-5) 2.3 ± 0.6 (1-3) 2.8 ± 0.9 (2-5)

Parity 1.8 ± 0.7 (1-4) 1.1 ± 0.3 (1-2) 2.1 ± 0.6 (1-4)

Spontaneous abortion 0.4 ± 0.7 (0-2) 0.3 ± 0.5 (0-1) 0.5 ± 0.8 (0-2)

Medical termination 0.1 ± 0.2 (0-1) 0 0.1 ± 0.3 (0-2)

Others 0 0 0

* At time of follow-up interview

Table 5 – Demographics Part 2

[Values are presented as: incidence Overall (n=41) Prospective Retrospective (%)] group (n=11) group (n=30)

Maternal Race:

White 34 (83) 10 (91) 24 (60)

Oriental Asian 3 (7) 1 (9) 2 (7)

Mixed 2 (5) 0 2 (7)

Indo-Asian 1 (2) 0 1 (3)

Middle Eastern 1 (2) 0 1 (3)

Breastfed infant’s sex:

Male 22 (54) 8 (73) 14 (47)

Female 19 (46) 3 (27) 16 (53)

Maternal BMI: (n=40) (n=29)

Underweight (<18.5) 1 (3) 0 1 (3)

Normal range (18.5-24.99) 23 (58) 8 (73) 15 (50)

Overweight (25-29.99) 10 (25) 2 (18) 8 (27)

Obese (≥30.00) 6 (15) 1 (9) 5 (17)

Breastfed infant’s birth weight:

Low birth weight* 1 (2) 0 1 (3)

Exceptionally large baby** 2 (4) 0 2 (7) 53

Hospitalized for NVP after Motherisk 1 (2) 0 1 (3) call

# values are presented as: frequency (percentage)

* ICD-10 definition: 1-2.49 kg

** ICD-10 definition: ≥ 4.5 kg

6.2 Diclectin® and NVP-Related Data

Overall, NVP had started at 5.9 ± 1.7 gestational weeks, ranging from 4 to 11 weeks (n = 39). In 18 participants where exact NVP stop time could be documented, their NVP had stopped at 24.9 ± 9.4 gestational weeks, ranging from 13 to 40 weeks. One mother reported hospitalization after her last consultation by Motherisk. Mothers were also asked to rate Diclectin®’s efficacy on a scale of 1 (did not alleviate NVP) to 10 (totally alleviated NVP). The highest score given for Diclectin® per pregnancy was 6.9 ± 1.8 and ranged from 2 to 10. There were 23 mothers where the start and stop dates for Diclectin® use could be ascertained/estimated. On average, these mothers used Diclectin® for 15.2 ± 10.2 weeks, with a range of 2-34.4 weeks. Dose of Diclectin® for the mothers varied day-to-day. The highest daily dose per mother was on average 4.4 tablets (each tablet contains 10 mg doxylamine/10 mg pyridoxine), ranging from 1 to 8 tablets (10 mg doxylamine/10 mg pyridoxine to 80 mg doxylamine/80 mg pyridoxine). After weight-correction, the highest daily dose per mother was 0.7 ± 0.3 mg/kg of doxylamine or pyridoxine, with a range of 0.1-1.3 mg/kg. The average age of the breastfed infants at initial Diclectin® exposure was 16.9 ± 6.7 months old, with a range of 4.5-37.2 months.

Table 6 – Diclectin and NVP related data

[Values are presented as: mean Overall (n=41) Prospective Retrospective ± SD (range)] group (n=11) group (n=30)

Gestational age* when NVP 5.9 ± 1.7 5 ± 1.2 6.2 ± 1.7 started or was first recorded by (4-11) (4-8) (4-11) Motherisk (weeks) (n=39) (n=10) (n=29)

Gestational age* when NVP 24.9 ± 9.4 16.5 25.4 ± 9.5 ended (weeks) (13-40) (n=1) (13-40) (n=18) (n=17)

Subjective score for how 6.9 ± 1.8 7.4 ± 1.3 6.8-1.9 efficacious was Diclectin® in (2-10) (5-9) (2-10) alleviating NVP (1 = did not (n=36) (n=9) (n=27) alleviate NVP at all, 10 = total alleviation)

Time length of Diclectin® use 15.2 ± 10.2 No data 15.2 ± 10.2 (weeks) (2.0-34.4) (2.0-34.4) (n = 23) (n = 23)

Highest daily dose of each mother 4.4 ± 1.7 4.1 ± 1.0 4.6 ± 1.9 (tablets) (1-8) (2-6) (1-8)

Highest daily dose of each mother 0.7 ± 0.3 0.7 ± 0.2 0.7 ± 0.3 (weight-adjusted) (mg/kg of (0.1-1.3) (0.2-1.1) (0.1-1.3) doxylamine or pyridoxine) (n=40) (n=29)

Breastfed infant’s age when 16.9 ± 6.7 19.2 ± 8.0 16.1 ± 6.1 mother started using Diclectin® (4.5-37.2) (10.0-37.2) (4.5-32.8) (months old)

* Gestational age of the pregnancy during which the mother used Diclectin® while breastfeeding a previously born child 55

6.3 Maternal Other Exposures

Amongst the mothers, there was a high rate of prenatal/multivitamins supplementation (85%). Some mothers also used folic acid supplements (29%). However, 5 mothers (12%) did not use prenatal/multivitamin or folic acid supplements. In addition, 3 mothers (7%) had drunk alcohol and 1 mother had smoked cigarette (2%) while they were pregnant, taking Diclectin®, and breastfeeding. For these four mothers:

 Mother 1 had two glasses of wine for one time only.

 Mother 2 would have 1-2 glasses of wine every weekend but this stopped some time during her pregnancy.

 Mother 3 had drunk alcohol on a few occasions: beer – 1 time, 1/2 glass; wine – 3 times, 1/4 glass each; champagne – 1 time, 1/3 glass.

 Mother 4 would smoke two cigarettes per day from the beginning of her pregnancy until at least the time of interview (pregnancy was ongoing).

Table 7 – Other maternal exposures while breastfeeding and using Diclectin®

(N = 41) Number of cases (%) CI (%)

Prenatal/multivitamin 35 (85) 71-94

Folic acid 12 (29) 18-45

No prenatal/multivitamin or folic 5 (12) 5-26 acid supplementation

Alcohol 3 (7) 2-20

Cigarette smoking 1 (2) –0.7-14

6.4 Dietary Patterns and Other Infant Characteristics

Concerning the dietary patterns of the breastfed infants, majority of them would have solid food for 3-6 times per day. The frequency of breastfeeding ranged from 1-11 times per day, and averaged at about 4 times per day. The time length of each breastfeeding was on average about 10 minutes but could range from 5 to 15 minutes each time. The number of hours of sleep every 24-hours was on average 11-14 hours, with a range of 9.5-16.5. The number of bowel movements per day was on average 1-3 times, and ranged from 1 to 5.5 times per day (one mother reported her infant would have 5-6 bowel movements per day, hence the non-integer value).

Note: dietary patterns and other infant characteristics were not different between prospective and retrospective groups.

Table 8 - Breastfeeding and infant-related data

[Values are presented Overall (n=41) Prospective group Retrospective group as: mean ± SD (range)] (n=11) (n=30)

Number of solid food 4.2 ± 1.3 4.9 ± 0.9 3.7 ± 1.3 feeding per day (1-6) (n=26) (3-6) (1-5.5) (n=15)

Number of breastfeeding 3.7 ± 2.2 2.2 ± 1.3 4.2 ± 2.2 per day (1-11) (n=40) (1-4) (n=10) (1-11)

Time length of each 9.9 ± 3.2 8.7 ± 3.4 11 ± 2.7 breastfeeding (minutes) (5-15) (n=19) (5-15) (n=9) (7.5-15) (n=10)

Hours of sleep per day

All age groups 12.6 ± 1.4 13.3 ± 1.2 12.3 ± 1.4 (9.5-16.5) (n=37) (12-16.5) (9.5-15) (n=26)

3-6.99 months 12.0 ± 3.5 No data 12 ± 3.5 (9.5-14.5) (n=2) (n=0) (9.5-14.5) (n=2)

7-12.99 months 12.9 ± 1.4 13.8 ± 1.1 12.5 ± 1.4 (10.5-14.5) (n=7) (13-14.5) (n=2) (10.5-14) (n=5)

13-18.99 months 13.0 ± 1.4 13.8 ± 1.6 12.7 ± 1.3 (11-16.5) (n=15) (12.5-16.5) (n=5) (11-15) (n=10)

19-24.99 months 12.2 ± 0.9 12.8 ± 0.4 12 ± 1 (10-13) (n=9) (12.5-13) (n=2) (10-13) (n=7)

≥ 25 months 11.9 ± 1.0 12.5 ± 0.7 11.3 ± 1.1 (10.5-13) (n=4) (12-13) (n=2) (10.5-12) (n=2)

Number of bowel 1.9 ± 1.1 2.2 ± 1.4 1.7 ± 1.0 movements per day (1-5.5) (n=30) (1-5.5) (n=10) (1-4) (n=20) 58

6.5 Maternal Adverse Events

The most common maternal adverse event reported was sedation (71%), followed by weakness (10%), dizziness (5%), constipation (5%), reduced milk supply (5%), abdominal pain (2%), increased appetite (2%). Of all the mothers, 24% reported that they did not experience any adverse event at all. 3 mothers reported that sedation had gradually faded over time, within 1-2 weeks since initiation of Diclectin® therapy.

Table 9 – Maternal adverse events

(N = 41) Number of cases (%) CI (%)

Sedation (drowsiness, tiredness) 29 (71) 55-83

Weakness 4 (10) 3-23

Dizziness 2 (5) 0.5-17

Constipation 2 (5) 0.5-17

Reduced milk supply 2 (5) 0.5-17

Abdominal pain 1 (2) –0.7-14

Increased appetite 1 (2) –0.7-14

No adverse event 10 (24) 14-40

6.6 Adverse Events amongst Breastfed Infants

Majority of the breastfed infants (80%) were not reported to have any adverse events. The most common adverse event amongst breastfed infants was sedation (12%), followed by constipation (2%), loose bowel (2%), slept less (2%), and problem with latching onto mother’s breast (2%). Irritability, irregular breathing, vomiting, or change in skin colour were not reported in any breastfed infant. For the cases where the infant had problem with latching or constipation, both mothers suggested that the adverse event was unrelated to Diclectin®. One mother had also reported that the eczema of her breastfed infant became less severe after she started taking Diclectin®.

Table 10 - Breastfed infant adverse events

(N = 41) Number of cases (%) CI (%)

Sedation (drowsiness, tiredness) 5 (12) 5-26

Constipation 1 (2)* –0.7-14

Problem with latching 1 (2)* –0.7-14

Loose bowel 1 (2) –0.7-14

Slept less 1 (2) –0.7-14

Irritability, irregular breathing, vomiting, 0 –2-10 change in skin colour, or other adverse events

No adverse event (including 1 case of relieving 33 (80) 66-90 eczema)

* Mother suggested that this was unrelated to Diclectin® use.

Presented below are details of the sedation cases (demographics of these cases can be seen in Table 11):

 Case 1: Mother reported that her infant used to be a bad sleeper. However, the infant had slept better when the mother used Diclectin®. This had reverted gradually over time after the mother stopped using Diclectin®.

 Case 2: Mother reported that her infant was sleepy for 8-10 days until she stopped using Diclectin®. She did not consult the physician and no change was made.

 Case 3: Mother reported that her infant had slept more when she took 2 tablets daily (slept for 10-12 hours at night, napped for 2-4 hours during the day). This occurred when the infant was approximately 9 months old and had lasted for a couple of weeks. The mother did not consult her doctor but lowered the dose to 1 tablet daily.

 Case 4: Mother reported that normally the infant would sleep for 10.5 hours at night and nap for 2 hours during the day. However, during the period she used Diclectin®, the infant slept for 12 hours at night, and had two naps (3 hours and 1.5 hours) during the day. Mother noticed the significant change in sleep pattern 2 days after she started Diclectin®. Hence, she stopped taking Diclectin® and breastfeeding. However, she could not tolerate the NVP and had to re-initiate Diclectin®. Hence she stopped breastfeeding altogether. Infant sedation had subsided within a week.

 Case 5: Mother reported that the infant would nap for 1 hour before she started taking Diclectin®. After she started taking Diclectin®, the infant napped for 1-2 hours, and the mother would wake him up if he napped for more than 2 hours. The mother suggested that she might have noticed this within the first week of use, even at 2 tablets/day. She did not consult the physician and no change was made.

Table 11 – Demographics of sedated infants

Case (n=5) 1 2 3 4 5

Prospective (P)/ Retrospective (R) R R R P P

Infant age (years)* 1.2 1.4 0.9 1.3 1.9

Maternal age (years)* 39.9 32.1 41.1 30.1 32.5

Time between Motherisk call and interview 18.0 12.5 8.5 1.0 0.7 (months)

Maternal highest weight-adjusted dose 0.6 0.9 0.4 0.7 0.6 (mg/kg/day)

Maternal highest dose (tablets) 4 6 2 5 4

Score on Diclectin® efficacy 9 8 8 9 n/d

Mother used sedating drugs/drugs of abuse 0 0 0 0 0

Average number of breastfeedings per day 6 6 4.5 1 3

Duration of each breastfeeding session (min) n/d n/d n/d 10 10

* When the mother started using Diclectin® and breastfed concurrently

Please refer to Table 12 for a summary of the demographics of infants who experienced non- sedation AEs.

1 infant was reported to have slept less after the mother started taking Diclectin®. The infant used to sleep for 16 hours per day before her mother started taking Diclectin®. When the mother took Diclectin® and breastfed the infant, the infant would sleep for 13-14 hours per day. The mother then stopped breastfeeding the infant and switched her to goat milk and the change in sleep pattern was reverted. 62

There was also one case where an infant had loose bowel one week after the mother initiated Diclectin®. Before the mother started and after the mother stopped taking Diclectin®, the infant had 1-2 bowel movements per day. When the mother took Diclectin®, the infant had 5-6 bowel movements per day. The mother reported that this had lasted for 7 weeks and she had consulted her physician. Bacterial and blood tests were negative. She did not make any changes but the boy weaned off breastfeeding himself.

Table 12 – Demographics of infants who experienced non-sedation AEs

Case (n=5) Loose bowel Slept less

Prospective (P)/ Retrospective (R) P R

Infant age (years)* 1.0 0.8

Maternal age (years)* 29.7 30.6

Time between Motherisk call and interview (months) 1.8 6.7

Maternal highest weight-adjusted dose (mg/kg/day) 0.7 1.1

Maternal highest dose (tablets) 4 7

Score on Diclectin® efficacy 8 7

Mother used sedating drugs/drugs of abuse 0 0

Average number of breastfeedings per day n/d 3.5

Duration of each breastfeeding session (min) n/d n/d

6.7 Correlations

Significant moderate positive correlations were found between weight-adjusted maternal dose and maternal constipation or maternal increased appetite (Table 13). No significant correlations were found for the remaining correlation tests.

Table 13 – Correlations between weight-adjusted maternal dose and maternal adverse events, infant adverse events, and total infant sleeping time per day

(N = 40*) Pearson’s r Directional/One- tailed p-value

Maternal sedation –0.21 0.10

Maternal dizziness –0.04 0.40

Maternal weakness –0.17 0.15

Maternal constipation 0.29 0.03**

Maternal abdominal pain –0.07 0.34

Maternal increased appetite 0.31 0.03**

Reduced milk supply 0.16 0.16

Infant sedation –0.03 0.43

Infant constipation 0.02 0.45

Infant problem latching –0.12 0.24

Infant loose bowel –0.02 0.45

Infant slept less 0.25 0.06

Relieved infant eczema 0.07 0.34

Infant hours of sleep per day 0.13 0.21

* Weight-adjusted maternal dose could not be calculated for one case, this case was removed from the correlation analysis.

** Significant (p < 0.05) 64

Chapter 7. Discussion 7.1 Study Population

Evaluation of maternal age at the time of consult for using Diclectin® while breastfeeding suggests that these pregnant mothers are on average slightly (and significantly) older than the national average of maternal age at time of delivery in 2011 (33.6 versus 30.2 years old) (p < 0.0001, one-sample t-test).150 This is expected given the fact that there has been an upward trend of maternal age at time of delivery since 1991. Moreover, mothers in the current study had at least 1 living child, whereas the national average included mothers having their first child.

Maternal race composition of this study is partially dissimilar to the findings of the 2011 National Household Survey conducted by Statistics Canada.151 The percentage of White mothers (83%) in the current study is significantly higher than the percentage of people with European origins (61%) in the Survey (p = 0.0168, Fisher’s exact test). While the percentages of Oriental Asian (7%), Indo-Asian (2%), and Middle Eastern (2%) mothers in the current study were not significantly different from the percentages of people with East and Southeast Asian (8%), South Asian (5%), West Central Asian and Middle Eastern (2%) origins in the Survey respectively. However, no participants of African (2% on the Survey), Caribbean (2% on the Survey), and Latin, Central and South American (2% on the Survey) origins were included in the analysis of this study. The difference in maternal race composition between prospectively and retrospectively recruited mothers could be attributed to the small sample size.

The male-to-female ratio of breastfed infants for this study (1.16) was slightly higher than that of newborns discharged from hospitals in Canada in 2011-2012 (1.05).152 Since it is unclear if infant sex would affect drug response at this age range, it is uncertain whether this would affect the results significantly. The difference in breastfed infant gender composition between prospectively and retrospectively recruited mothers could be attributed to the small sample size.

The percentages of breastfed infants classified as Low Birth Weight (2.44%) or Exceptionally Large Baby (4.88%) at birth are both below 10%. This is expected as mothers who consult Motherisk are thought to be more health conscious and more careful with their pregnancy. 65

7.2 Diclectin® and NVP-Related Data

In most participants, NVP started at 5.91 ± 1.65 weeks (1st trimester). This range corroborates with the ranges reported by previous studies.153,154 However, the range of time when NVP ended (24.86 ± 9.41 weeks) is wider and later than those reported by previous studies.153,154

In 1993, Gadsby et al. reported a prospective study in 363 pregnant women, 292 of which had NVP.153 The mean day of onset for NVP was 39 days (5.6 weeks, since last menstrual period), with 72.2% of women having NVP onset during 29-49 days (4.1-11.3 weeks). NVP ceased in 60% of the women by 84 days (12 weeks) and in 90.8% by 112 days (16 weeks).

In 2000, Lacroix et al. reported a similar prospective study.154 This study was completed in 160 participants. 118 women reported having nausea, with the mean time of onset being 5.7 weeks (gestational age). Nausea lasted for a mean of 34.6 days (4.9 weeks) with a 95% CI of 0-90 days (0-12.9 weeks). 60 women also reported having vomiting, which occurred for a mean of 5.6 days, with a 95% CI of 0-28 days (0-4 weeks)). NVP had resolved in about 50% of women by 14 weeks, and in 90% of women by 22 weeks.

The discrepant findings between the current study and previous studies may be because the current study included mothers calling the NVP helpline, who were likely to have suffered more severe NVP than the general population. One may also speculate that Diclectin® delayed the end time of NVP, which will require further investigation.

7.3 Maternal Other Exposures

The rate of prenatal/multivitamin/folic acid supplementation in the current study (87.8%) is similar to the rate reported by the Canadian Maternity Experiences Survey (89.7% in the first three months of pregnancy).155 The Survey also reported that 22.4% of women did not know taking folic acid prior to pregnancy could help prevent some birth defects. Although the rate of supplementation is high in both the current study and the survey, it can still be improved and there still exists a need to educate women about the benefits of starting folic acid supplementation prior to conception.

Although statistical significance was not reached, the rates of alcohol use and cigarette smoking in the current study were lower (alcohol: 7.3%, smoking: 2.4%) than the rates estimated from the 66

National Longitudinal Survey of Children and Youth conducted during 2005-2008 (alcohol: 10.7%, smoking: 12.3%).156

7.4 Maternal Adverse Events

Compared to studies mentioned in section 2.1.2, mothers of this study reported similar symptoms (weakness, dizziness, constipation, abdominal pain) at similar rates. Meanwhile, a comparison using Fisher’s exact test (Table 14) showed that the rate of sedation in this study is significantly greater than that reported by Atanackovic et al.19

Table 14 – Comparison of the rate of maternal sedation between this study and that of Atanakovic et al.19 with Fisher’s exact test

Sedation No sedation CI (%)

This study 29 12 55-83

Atanckovic et al.19 42 80 27-43

Two-tailed p-value < 0.0001.

I suspect that mothers in this study were more prone to the sedative effects of the drug because they had to take care of both their pregnancy and their breastfed infants, tiring them more than just having a pregnancy (as in Atanakovic et al.’s study19).

Study participants had also reported some symptoms not reported in the literature, including lower milk production and increased appetite. Although increased appetite/increased appetite has not been reported for the ethanolamine class of antihistamines such as doxylamine, it has been reported for cyproheptadine (piperidine class), possibly due to serotonin antagonism.157,158

Reduced milk production reported by 2 women in the current study warrants further discussion.

One study had associated pregnancy with reduced lactation in some mothers.159 In this study, 30% of the mothers reported no change in breast milk production, while 34% reported decline in first trimester, 18% reported decline in second trimester, and 18% reported complete cessation. Indeed, one of the mother who experienced lactation suppression suggested that this had started even before she started taking Diclectin®. 67

Another explanation may be that antihistamines could suppress serum PRL, one of the hormones involved in maintaining lactation. In 1985, Messinis et al.160 reported that administration of 100 mg promethazine intramuscularly or 20 mg d-chlorpheniramine (both first generation H1 antihistamines) intravenously to non-lactating puerperal mothers suppressed basal PRL release. However, it is unclear whether this would affect lactation or not.

7.5 Adverse Events amongst Breastfed Infants

There were 4 infants in this study who experienced either of the following symptoms: “constipation”, “problem with latching”, “loose bowel”, or “sleeping less”. Mother of the infant with problematic latching had suggested that this was unrelated to Diclectin. The infant who had slept less may have had the paradoxic excitation effect of antihistamines.161 While the case with constipation may be attributed to the anticholinergic effect of doxylamine;162,163 it is unclear if the case of diarrhea is related to Diclectin®/doxylamine. However, given the low incidence of these 4 adverse events, no statistical comparison may be performed and no conclusion can be drawn.

The only adverse event with more than 1 case amongst infants is sedation, with 5 cases (12%). Fisher’s exact test was again used to compare the rate of sedation in this study with that of other medications in others studies (Table 15).

Table 15 – Comparisons of the rate of infant sedation between Diclectin® and other medications with Fisher’s exact test

Sedation No sedation p-value* CI (%)

This study: Diclectin® 5 36 n/a 5-26

Ito et al.117: Multiple antihistamines 1 58 0.0409 –0.6-10 – Terfenadine, Astemizole, Dimenhydrinate, Diphenhydramine, Chlorpheniramine

Kelly et al.146: Multiple 2 122 0.0108 0.1-6 benzodiazepines – Alprazolam, Bromazepam, Clonazepam, Diazepam, Flurazepam, Lorazepam, Midazolam, Oxazepam, Temazepam, Triazolam

Lam et al.145: acetaminophen 1 183 0.0008 –0.2-3

Lam et al.145: oxycodone 28 111 0.3581 14-28

Lam et al.145: codeine 35 175 0.6415 12-22

* When compared with the sedation rate in this study using Fisher’s exact test.

These comparisons suggested that the rate of sedation in infants breastfed by mothers taking Diclectin® is significantly higher than the rates in infants whose mothers took benzodiazepines (sedating), acetaminophen (non-sedating), or other antihistamines (sedating); but not significantly different from the rates in infants whose mother took oxycodone (sedating) or codeine (sedating).

Although severe CNS depression has been reported for infants exposed to opioids through breast milk,145,164 the cases of sedation presented in the current study are mild and did not require medical attention. Therefore, one can conclude that while using Diclectin®/doxylamine during 69 breastfeeding may sedate the breastfed infant, it is highly unlikely to be as severe as those cases for opioids.

7.6 Correlations

As mentioned before, cyproheptadine is a piperidine antihistamine with antiserotonergic activity that may cause increased appetite in a patient. While doxylamine or other ethanolamine antihistamines are not known to be antiserotonergic, the positive correlation between maternal Diclectin® dose and maternal increased appetite suggests that this may be possible at high dose, which warrants further investigation.

At high doses, the anticholingeric effects of doxylamine is expected to become more pronounced, as reflected in the toxidrome of overdoses. Since constipation could be an anticholinergic effect, this correlation has a pharmacological basis.

While the correlations I found may have pharmacological bases, sample size of the current study and the low incidence of increased appetite/constipation limits the generalizability of these correlations. Hence, these correlations should be tested in future studies with larger sample sizes.

Although no correlation has been found between maternal dose and any infant side effects, mothers should still be cautious in monitoring their breastfed child for the occurrence of sedation. This is because the current study’s small sample size disallows the conclusion that there is no dose-effect relation.

7.7 Limitations of the Study

One limitation of the current study is that data for only some mother-infant pairs could be collected within 6 months after Motherisk consultation. Hence, recall bias could not be eliminated.

Since mothers calling Motherisk is a self-selected population, their demographics might differ from that of the general population (e.g. education level). Unfortunately, my questionnaire did not include sufficient questions to permit full analysis of differences in demographics. Future studies should include questions like education level, occupation, marriage status, family income, etc. 70

The small sample size of this study also limits the power and generalizability of study results. For this reason, study results are prone to interference by random effects. For instance, the significant correlations between maternal dose and maternal constipation/increased appetite could well be chance findings.

Some mothers who had refused to participate due to time constraints had asked for an online questionnaire. This was not possible due to concerns of patient confidentiality. However, I do believe that it is feasible, as exemplified by Health Canada’s MedEffect online reporting system: http://www.hc-sc.gc.ca/dhp-mps/medeff/report-declaration/index-eng.php.

In the current study, AE assessments were based on mothers’ assessments and recall. Future studies should assess the causality of AEs using quantitative methods, such as the Naranjo scale or the WHO-UMC assessment.165 By applying these scales to the cases, in spite of some missing data points, most of the AEs were classified as “possible” on the Naranjo scale and “probable” or “possible” on the WHO-UMC assessment.

In addition, although I have attempted to collect information on infants’ sleeping patterns, I did not collect specifically the infant sleeping patterns before, during, and after the period the mother used Diclectin®, hence I could not perform any meaningful analysis. Future studies should ask mothers about infant sleeping patterns before, during, and after the period she used Diclectin®, to account for any changes. Collecting sleeping pattern at only one point would be useless, as sleeping pattern also depends on the infant’s age and habits.

7.7.1 Limitations of Comparison with Previous Studies on Using Other Medications While Breastfeeding

One should be aware that settings of these studies are very different from that of the current study.

1. Study design of the comparator studies are in general similar to the current study. However, the questions, though based on similar template(s), may be modified and/or asked differently, which may/may not affect how mothers would answer the questions or reporting AEs.

2. There are population/maternal differences between the current study and the compared studies.

a. Participants of the current study were pregnant and breastfeeding at the same time, whereas mothers in the comparator studies were breastfeeding only.

b. Drugs in the comparator studies are indicated for different illnesses. Different drugs and illnesses may affect mothers’ perception of AEs. For instance, mothers who use opioids may experience significant sedation and may hence be less observant of any AEs that may occur in their infants

i. This study – Diclectin® – nausea and vomiting of pregnancy

ii. Ito et al.117 – other antihistamines (amongst multiple drugs) – indication not specified

iii. Kelly et al.146 – benzodiazepines – indication not specified

iv. Lam et al.145 – codeine and oxycodone – mainly dental/minor surgery, Caesarean delivery

v. Lam et al.145 – acetaminophen – mainly headache/migraine, cold/cold- related pain

3. Since the infant age at time of exposure in the current study is on average higher than the comparator studies, participating infants would likely be breastfeeding less and eating 72

more solid food than younger infants. Therefore, their doxylamine intake through breast milk would likely be lower than young infants solely breastfed by mothers taking Diclectin®. Hence, one may question the biological plausibility of such low dose doxylamine in breast milk for causing the reported adverse effects.

a. This study – 17.06 ± 6.71 months

b. Ito et al.117 – 4.0 ± 4.9 months (not just antihistamines)

c. Kelly et al.146 – average 11 months at time of follow-up, range 2-24 months

d. Lam et al.145 oxycodone cohort – 53.6 ± 32.9 weeks (12.3 ± 7.6 months)

e. Lam et al.145 codeine cohort – 53.9 ± 16.4 weeks (12.4 ± 3.8 months)

f. Lam et al.145 acetaminophen cohort – 53.9 ± 13.3 weeks (12.4 ± 3.1 months)

4. The duration between Motherisk call and follow-up interview is significantly longer in this study when compared to Ito et al.’s study117 (p < 0.0001, t-test), hence the current study is more prone to recall bias than Ito et al.’s study. Ito et al.117 found that mothers followed-up 5 months post-exposure reported much lower rates of adverse events, whereas mothers contacted within 4 months post-exposure reported similar rates of AEs. If the same principle applies to the current study, it is likely that there was also under- reporting of AEs.

a. This study – 47.7 ± 36.0 weeks (range 1-115.1 weeks)

b. Ito et al.117 – 11.7 ± 7.3 weeks (range 1-31 weeks) (not just antihistamines)

c. Kelly et al.146 – not reported

d. Lam et al.145 – unclear, acetaminophen and codeine group contacted Motherisk during January 2004-December 2008; oxycodone group contacted Motherisk recruited during January 2007-October 2010

5. Comparison of other cohort characteristics did not show any significant differences between maternal age at time of exposure. Therefore, maternal age is not expected to 73

influence the comparisons significantly. Parity was also similar between the current study and Ito et al.’s study117, but cannot be compared to Lam et al.’s study145 as Lam et al. only reported the number of nulliparous women.

Table 16 – Comparison of cohort maternal age and parity between the current study and comparator studies

N Maternal age (years)* Parity

This study (Diclectin®) 41 33.6 ± 4.3 1.8 ± 0.7

Ito et al.117 (include other 838 31.6 ± 4.7 1.8 ± 1.1 drugs)

Kelly et al.146 124 33.5 ± 5.0 (infant sedated group) Not reported (benzodiazepines) 33.6 ± 10.6 (infant non-sedated group)

Lam et al.145 (acetaminophen) 184 32.0 ± 5.0 109/179 nulliparous

Lam et al.145 (codeine) 210 32.7 ± 4.5 107/179 nulliparous

Lam et al.145 (oxycodone) 139 32.9 ± 4.6 51/139 nulliparous

* At exposure

7.7.2 Recall Bias

As suggested by Ito et al.117, recall bias may be an issue when participants are followed-up too long after an exposure. This issue has also been reported by a few other studies.

In 2013, van Gelder et al.166 reported a validation study of a questionnaire which assessed prescription medication use from 3 months before through pregnancy. The study used pharmacy records as a reference, as all dispensing records are computerized in the Netherlands. For most participants, 1 month or more after the questionnaire was sent and completed, a telephone interview would be performed where interviewers would ask whether the mother used the medication on the pharmacist’s list. The median time between delivery and administration of questionnaire was 1.2 years, with a range 0.1-15.3 years. The study found increasing disagreement between the reference standard and the questionnaire at the time between delivery and completion of questionnaire increased. At > 2 - ≤ 5 years, the level of disagreement was already (almost) significantly greater than the reference (≤ 6 months) for pregnancy-related medications (adjusted OR = 2.0, CI = 0.9-4.6) and any prescription medication (adjusted OR = 1.6, CI = 0.9-2.9). And the highest level of disagreement occurred when the questionnaire was taken > 5 years postpartum for pregnancy-related medications (OR = 3.3, CI = 1.5-7.4). The authors concluded that completing the questionnaire > 2 years postpartum led to increased disagreement, particularly for any prescription medication and pregnancy-related medication. Based on this conclusion, it is possible that some participants of the current study may have poor recall of their Diclectin® use (particularly ones where the time difference between Motherisk call and follow-up interview was > 2 years). While this was partly alleviated by using records from Motherisk to prompt the participants about their medication use, it was almost impossible to ask mothers what doses did they use at different periods, hence the use of “highest dose” in the analysis rather than the exact dose used during the period participants took Diclectin® and breastfed. There was a similar issue with recalling AE occurrence, where it was almost impossible to ask about when the AE occurred (and what dose it was associated with).

To reduce recall bias due to time length between counselling and follow-up interview, future studies should perform follow-up calls within 6 months after initial counselling. Alternatively, they may recruit participants prospectively and request them to record the occurrence of any AEs on an online standardized form for a certain period after counselling, so that the time of the 75 follow-up becomes less of an issue. Participants may be reminded monthly through email to record AEs.

In 1995, Taddio et al.167 evaluated if different counselling styles affected the rate of AE reporting, in this case infant diarrhea. Participants were callers who called Motherisk regarding use of antibiotics during breastfeeding. While all participants were informed that it is safe to use antibiotics while breastfeeding, only some were informed (primed) that infant diarrhea may be a potential AE. In the follow-up, all mothers were asked if there were any clinical events/changes in their breastfed infants while they were taking the antibiotic and breastfeeding concurrently. While more unprimed mothers reported occurrence of clinical events and AEs in their infants, the rates were not significantly different from those of primed mothers. Therefore, it was concluded that counselling about AEs did not affect the rates of reporting AEs in their infants. Based on this finding, it is probable that in the current study, the reported infant AE rates would not have been affected by whether Motherisk counsellors mentioned about potential AEs or not. However, it is also possible that mothers who were informed about potential AEs, particularly when they are mild, would be less worried about it and more likely to forget it. Future studies may investigate this by repeating Taddio et al.’s study design, but in addition to asking for any AEs, they should also ask for specific potential AEs related to the study drug. They may also include a control group with the same disease but used a different drug or did not use any drug.

Chapter 8. Conclusion and Future Directions

Results of this study suggests that using Diclectin®/doxylamine while breastfeeding may lead to infant sedation. Though the rate of infant sedation for Diclectin® is similar to that of oxycodone and codeine, it is not expected to be as severe as the CNS depression caused by opioids.

During my literature search, I had also found an information gap regarding the safety of using first generation antihistamines or other old drugs during breastfeeding. This may be attributed to the lack of incentive to the generic makers of these drugs. Since pharmaceuticals are constantly designing new drugs based on old drugs, if they would like to create a drug that can be used by breastfeeding women, it may be more cost-effective to study the breastfeeding safety of the old drug first. This would allow prediction of the breastfeeding safety of chemical leads related to the old drug, and allow chemists to improve them so that they are more likely to be safe for breastfeeding.

Future studies on the safety of using medications during breastfeeding should attempt to collect the data prospectively by following-up breastfeeding mothers, as this will greatly reduce recall bias. Recruitment could be improved by collaborating with multiple centres or databases. Recruitment may also be improved by utilizing online questionnaires. This would accommodate potential participants who are too busy for a telephone interview. Alternatively, mothers could be asked to fill in an online diary. Participants should be instructed to record the use of any medication and its dosing regimen, health status, and occurrence of AEs in both the mother and the infant; as well as the sleeping and feeding pattern of the infant. Once an AE ends, the participant should be directed to assess causality of the AE using either the Naranjo or the WHO- UMC assessments.

Future studies on the safety of using Diclectin® while breastfeeding should also include control groups. However, I cannot include a negative/placebo control group as it would be unethical to not treat NVP patients when treatments are available. Nevertheless, positive control groups may be used, in particular dimenhydrinate and diphenhydramine, since they are closely related to doxylamine and would be expected to cause similar AEs in the breastfed infant.

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Appendix 1 – Questionnaire

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Appendix 2 – Approval from Research Ethics Board of the Hospital for Sick Children