investigational medicinal product dossier omega-3 …

PURPOSE IMPD, version 1, February 5th 2016 1 of 46

INVESTIGATIONAL MEDICINAL PRODUCT DOSSIER

Omega-3 fatty acids and placebo

Version 1, dated February 5th 2016

Authors:

Matthijs Bossong

Gregor Berger

Conrad Vissink

Hugo Schurgast

Inge Winter


TABLE OF CONTENTS

LIST OF TABLES .................................................................................................................. 4

1. INTRODUCTION……….. ................................................................................................... 5

2. CHEMICAL PHARMACEUTICAL DATA… ....................................................................... 6

2.1.P MEDICINAL PRODUCT… ............................................................................................ 6

2.1.P.1 Description and Composition of the Medicinal Product .......................................... 6

2.1.P.2 Pharmaceutical Development ................................................................................ 6

2.1.P.2.1 Components of the Medicinal Product ......................................................... 6

2.1.P.2.2 Medicinal Product ........................................................................................ 7

2.1.P.2.3 Manufacturing Process Development .......................................................... 7

2.1.P.2.4 Container Closure System ........................................................................... 7

2.1.P.2.5 Microbiological Attributes ............................................................................. 7

2.1.P.2.6 Compatibility ................................................................................................ 7

2.1.P.3 Manufacture .......................................................................................................... 7

2.1.P.3.1 Manufacturer(s) ........................................................................................... 7

2.1.P.3.2 Batch Formula ............................................................................................. 8

2.1.P.3.3 Description of Manufacturing Process and Process Controls ....................... 8

2.1.P.3.4 Controls of Critical Steps and Intermediates ................................................ 8

2.1.P.3.5 Process Validation and/or Evaluation .......................................................... 8

2.1.P.4 Control of Excipients ............................................................................................. 8

2.1.P.4.1 Specifications .............................................................................................. 8

2.1.P.4.2 Analytical Procedures .................................................................................. 9

2.1.P.4.3 Validation of Analytical Procedures ............................................................. 9

2.1.P.4.4 Justification of Specifications ....................................................................... 9

2.1.P.4.5 Excipients of Human or Animal Origin ......................................................... 9

2.1.P.4.6 Novel Excipients .......................................................................................... 9

2.1.P.5 Control of Medicinal Product ................................................................................. 9

2.1.P.5.1 Specifications .............................................................................................. 9

2.1.P.5.2 Analytical Procedures ................................................................................ 10

2.1.P.5.3 Validation of Analytical Procedures ........................................................... 10

2.1.P.5.4 Batch Analyses .......................................................................................... 10

2.1.P.5.5 Characterization on impurities ................................................................... 10

2.1.P.5.6 Justification of Specification(s) .................................................................. 10

2.1.P.6 Reference Standards .......................................................................................... 10

2.1.P.7 Container Closure System................................................................................... 11


2.1.P.8 Stability.... ........................................................................................................... 11

2.1.P.8.1 Stability testing parameters ....................................................................... 11

2.1.P.8.2 Stability testing storage ............................................................................. 11

2.1.P.8.3 Stability testing scope ................................................................................ 12

2.2 NON-CLINICAL PHARMACOLOGY, PHARMACOKINETICS AND TOXICOLOGY ..... 13

2.2.1 Acute single dose toxicity ....................................................................................... 13

2.2.2 Repeated dose toxicity ........................................................................................... 13

2.2.3 Genotoxicity / Carcinogenicity ................................................................................. 15

2.2.4 Reproductive and developmental toxicity ................................................................ 17

2.2.5 List of studies Conducted & References ................................................................. 18

2.2.6 References ............................................................................................................. 19

2.3 CLINICAL DATA ........................................................................................................... 20

2.3.1 Clinical pharmacology............................................................................................. 20

2.3.2 Clinical pharmacokinetics ....................................................................................... 22

2.3.3 Human exposure .................................................................................................... 23

2.3.4 Overall risk and benefit assessment ....................................................................... 23

2.3.5 Conclusion .............................................................................................................. 23

2.4 OVERALL RISK AND BENEFIT ASSESSMENT .......................................................... 25

2.4.1 PUFAs and potential adverse effects in UHR ......................................................... 25

2.4.2 PUFAs and potential adverse effects in schizophrenia ........................................... 25

2.4.3 Specific adverse effects of PUFAs .......................................................................... 25

2.4.4 Risks versus benefits of PUFAs in emerging psychosis .......................................... 27

2.5 CONCLUSION ............................................................................................................... 27

APPENDIX 1 ....................................................................................................................... 32

APPENDIX 2 ....................................................................................................................... 33

APPENDIX 3 ....................................................................................................................... 34

APPENDIX 4 ....................................................................................................................... 35

APPENDIX 5 ....................................................................................................................... 36

APPENDIX 6 ....................................................................................................................... 39

APPENDIX 7 ....................................................................................................................... 42

APPENDIX 8 ....................................................................................................................... 45


LIST OF TABLES

Table 1 Qualitative composition of omega-3 capsules ........................................................... 6

Table 2 Qualitative composition of placebo capsules ............................................................. 6

Table 3 Release and Shelf-life Specification Product with active substance .......................... 9

Table 4 Release and Shelf-life Specification Placebo .......................................................... 10

Table 5 Stability testing parameters and methods ............................................................... 11

Table 6 Stability testing schedule of withdrawals ................................................................. 12

Table 7 Studies examining EPA/DHA repeated dose toxicity ............................................... 14

Table 8 Studies examining EPA/DHA genotoxicity / carcinogenicity .................................... 15

Table 9 Studies examining EPA/DHA reproductive and developmental toxicity ................... 17

Table 10 List of referenced non-clinical studies on omega-3 fatty acids ............................... 18


1. INTRODUCTION

This Clinical Trial Application presents information relating to omega-3 capsules containing 180

mg of EPA, 120 mg of DHA and 1.9 mg mixed tocopherols (antioxidant), as well as placebo

capsules. Omega-3 is being investigated in subjects at ultra-high risk for developing psychosis,

for the prevention of clinical transition to psychosis.

Omega-3 fatty acids are used worldwide as nutritional supplements with important physical and

psychosocial health benefits and has been studied elaborately in (the prevention of) various

disease states, ranging from cardiovascular disease to psychosis. Pre-clinical data demonstrate

very low level of oral toxicity of omega-3 fatty acids.

Due to the generally high level of overlap between IMPDs and Investigator Brochures, and the

resulting cross referencing between the documents, it has been decided to combine both

documents in the current IMPD.


2. CHEMICAL PHARMACEUTICAL DATA

2.1.P MEDICINAL PRODUCT

2.1.P.1 Description and Composition of the Medicinal Product

Omega-3 capsules of 600 mg, containing 180 mg of EPA, 120 mg of DHA, 60 mg of other

omega-3 fatty acids and 1.9 mg mixed tocopherols (antioxidant), as well as placebo capsules.

The qualitative composition of the verum capsules is listed in Table 1. Please also see Appendix

1.

Table 1 Qualitative composition of omega-3 capsules

Component Reference to

standards Function

Fish oil 33/22 TG In house Active ingredient

Mixed tocopherol In house Active ingredient

The qualitative composition of the placebo capsules is listed in Table 2. Please also see

Appendix 2.

Table 2 Qualitative composition of placebo capsules

Component Reference to

standards Function

Fish oil 33/22 TG In house Active ingredient

Mixed tocopherol In house Active ingredient

Coconut oil Ph. Eur. Active ingredient

Medium-chain triglyceride Ph. Eur. Active ingredient

2.1.P.2 Pharmaceutical Development

The gel capsules with verum and placebo which are used for the present study have been used

in a clinical trial conducted by Amminger et al. (2010) in a similar subject population.

2.1.P.2.1 Components of the Medicinal Product

The mixed tocopherol (vitamin E) is a common addition to omega-3 fatty acid capsules, and

has been added to the placebo as well, in order to align the content of the capsules on

factors which may influence pharmacokinetics/-dynamics and/or therapeutic effect. Fish oil

is added to the placebo capsule in order to prevent unblinding due to differences in smell


and taste compared to the verum capsule. Placebo capsules will contain a 1:1 combination

of coconut oil and medium chain triglycerides because it does not contain polyunsaturated

fatty acids and has no impact on omega-3 fatty acid metabolism.

2.1.P.2.2 Medicinal product

No additional relevant information applicable.

2.1.P.2.3 Manufacturing Process Development

The manufacturing process development is described in detail in section 2.1.P.3.3.

2.1.P.2.4 Container Closure System

The capsules will be packed in customized polyethylene bags or in tubular film made

of Polyethylene for the transport to ACE Pharmaceuticals in the Netherlands, where

the capsules will be bottled using 400 ml (dark brown) glass bottles with a pressure

seal lid.

2.1.P.2.5 Microbiological Attributes

Not applicable.

2.1.P.2.6 Compatibility

Not applicable.

2.1.P.3 Manufacture

2.1.P.3.1 Manufacturer(s)

Manufacturing and QP release:

AYANDA GmbH & Co. KG

Am Huenengrab 20

16928 Pritzwalk

Germany

QP release:

Antistress / Burgerstein

Fluhstrasse 30

8640 Rapperswil-Jona

Switzerland


Packaging, blinding, labeling and QP release:

ACE Pharmaceuticals BV

Schepenveld 41

3891 ZK Zeewolde

The Netherlands

Phone +31 36 5227201

Fax +31 36 5229096

2.1.P.3.2 Batch Formula

Please see Appendix 3 and 4 for the batch formula of omega-3 and placebo capsules,

respectively.

2.1.P.3.3 Description of Manufacturing Process and Process Controls

Please see Appendix 5 and 6 for a general description of the manufacturing process as well

as a flow chart mentioning the in-process controls for omega-3 fatty acids and placebo,

respectively.

2.1.P.3.4 Controls of Critical Steps and Intermediates

Please see Appendix 5 and 6 for a general description of the manufacturing process as well

as a flow chart mentioning the in-process controls for omega-3 fatty acids and placebo,

respectively.

2.1.P.3.5 Process Validation and/or Evaluation

The currently manufactured product Omega-3 Purpose (item code: 5007242; batch

number: 30016995) has not been validated. Production of soft gelatin capsules with an oil

mixture filling is a well-established procedure at Ayanda GmbH & Co. KG. The used

equipment is qualified and the staff is well trained. Additionally, the manufacturing site in

Germany is certified according to EU-GMP rules, International Featured Standard and

Global Standard for Food Safety. The parameters on the certificate of analysis are within

the specification.

2.1.P.4 Control of Excipients

2.1.P.4.1 Specifications

Please see Appendix 7 and 8 for the Ph. Eur. monographies of the excipients medium

chain triglycerides and coconut oil (version 8.3) used in our products. All raw materials are


Ph. Eur. Grade and are tested according to Ph. Eur. methods or with validated methods, if

applicable.

2.1.P.4.2 Analytical Procedures

The analytical procedures are available on request.

2.1.P.4.3 Validation of Analytical procedures

All Ph. Eur. methods used for our standard analytical regime are per se regarded as

validated.

2.1.P.4.4 Justification of Specifications

Not applicable.

2.1.P.4.5 Excipients of Human or Animal Origin

Not applicable.

2.1.P.4.6 Novel Excipients

Not applicable.

2.1.P.5 Control of Medicinal Product

2.1.P.5.1 Specifications

Table 3 Release and Shelf-life Specification Product with active substance

Test item Method Acceptance Criteria

Description Visual observation 12 oval clear, slightly yellow soft gel capsule

ID GC, Ph.Eur. 2.2.28 Retention times of EPA/DHA complies with CRS

Uniformity of filling mass Ph.Eur. Complies

Assay GC, Ph.Eur. 2.2.28 Min. 180 mg EPA (TG) Min. 120 mg DHA (TG) Min. 360 mg Omega3 (TG) Shelf life spec.: Min. -10%

Peroxide Value PH.Eur. 2.5.5 Max. 5 Shelf-life spec.: for Information

Anisidine Value PH.Eur. 2.5.36 Max. 20 Shelf-life spec.: for Information

Disintegration Ph.Eur. 2.9.1 max. 30 min


Table 4 Release and Shelf-life Specification Placebo

Test Item Method Acceptance Criteria

Description Visual observation 12 oval clear, slightly yellow soft gel capsule

ID GC, Ph.Eur. 2.2.28

No signals for EPA/DHA compared to CRS

Uniformity of filling mass Ph.Eur. complies

Disintegration Ph.Eur. 2.9.1 Max. 30 min

2.1.P.5.2 Analytical Procedures

The analytical procedures are available on request.

2.1.P.5.3 Validation of Analytical Procedures

All Ph. Eur. methods are per se regarded as validated. Our method for the assay testing of

omega-3 fatty acids is validated according to the directive.

2.1.P.5.4 Batch Analyses

All Ph. Eur. methods are per se regarded as validated. Our method for the assay testing of

omega-3 fatty acids is validated according to the directive.

2.1.P.5.5 Characterization of Impurities

No impurities have been defined for the finished capsule; hence, no limits were set either.

However, the active principle is a multi-component fish oil concentrate. Related substances

for fish oils are not defined, only oxidation products (POV, AV) are summarily specified. As

acceptance criteria only minimum values can be characterized since no upper limits are

defined for fish oils.

2.1.P.5.6 Justification of Specification(s)

Medication is considered safe for testing in human subjects. Please see paragraphs 2.2

‘Non-clinical pharmacology, pharmacokinetics and toxicology’, 2.3 ‘Clinical data’, and 2.4

‘Overall risk and benefit assessment’ for an elaborate description of previous studies with

omega-3 fatty acids.

2.1.P.6 Reference standards

Our reference standards for the determination of omega-3 content are commercial standards

with lot numbers and CoAs. However, no analytical testing of the omega-3 content was


performed on the delivered batch. Please note that this will be part of the stability program

(paragraph 2.1.P.8).

2.1.P.7 Container Closure System

The gel capsules used in the clinical trial will be packed in dark brown glass bottles, which are

closed with a pressure seal lid in order to maximize stability of the capsules as well as possible

(i.e. minimize exposure to light and air). The bottles will be packed in cardboard boxes to further

protect the capsules from light.

2.1.P.8 Stability

All commercially available omega-3 fatty acid products manufactured by Antistress / Burgerstein

have been proven stable for a period of 36 months, assessed with the stability testing methods

as described below. They never experienced any problems in guaranteeing the stability of their

capsules for a period of 36 months.

Stability testing needs to be done for the current batch of capsules, and will be performed by

SGS Institut Fresenius GmbH, Berlin, Germany. By accelerated stability testing of the verum

capsules stored at 40°C/75%RH, a 24-month stability of samples can be assessed within six

months. All tests will be carried out at SGS’ laboratories accredited according to

DIN/EN/ISO/IEC 17025. Climatic chambers for controlled stability storage are referring to GMP

conditions. Please note that 1) stability studies on verum capsules will be initiated prior to the

start of the clinical trial to ensure stability of capsules, and 2) there are no reasons to expect that

the placebo product will undergo changes in physical characteristics or degradation.

2.1.P.8.1 Stability testing parameters

One batch of omega-3 capsules have to be tested on the content of nutritional, sensory and

microbial parameters (Table 5).

Table 5 Stability testing parameters and methods

Parameter Methods

Fatty acid spectrum (including DHA, EPA) DGF C-VI 10a, 11a mod., GC/FID

Organoleptic (appearance, consistency, taste) ASU L 00.90-6 mod.

Microbiology

TAMC, TYMC, molds, yeasts Ph.Eur. 8, Kap. 2.6.12

Escherichia coli Ph.Eur.8, Kap. 2.6.13

Salmonella (absent in 10 g)


Staphylococcus aureus (absent in 1 g)

Bile-tolerant gram-negative bacteria

2.1.P.8.2 Stability testing storage

Samples are checked upon arrival at SGS on its completeness and compliance with the

documentation provided by the client and on its sound condition. Each single sample

including packaging is labelled. Samples are stored under controlled conditions regarding

room temperature, stress conditions and protected from light. Stability storage referrers to

GMP conditions with continuous 24/7 control of temperature and documentation. Under the

assumption of a shelf life of 24 months the stability study is set up for a speeded period of

time (6 month). Samples for analysis are taken from the chamber at distinct points of time

(removal at t=X months).

2.1.P.8.3 Stability testing scope

Full testing (X*) and reduced testing (X) will be done at certain points of the stability study

as indicated (Table 6). All parameters as listed in Table 5 will be assessed for full testing.

Fatty acid spectrum and organoleptic will be assessed for reduced testing.

Table 6 Stability testing schedule of withdrawals

Climatic Zone

°C / % RH

Withdrawal from climatic chamber [months]

0 1 2 3 6 9 12 18 24

40 / 75 X* X X X X*


2.2 NON-CLINICAL PHARMACOLOGY, PHARMACOKINETICS AND TOXICOLOGY

In addition to the non-clinical and clinical pharmacology, pharmacokinetics and toxicology as

described in paragraphs 2.2 and 2.3 of this IMPD, we would like to emphasize that additional

information is publically available in the Summary of Product Characteristics of registered

products containing EPA/DHA such as for example Omacor ®

(https://www.medicines.org.uk/emc/medicine/10312/SPC/Omacor/).

2.2.1 Acute single dose toxicity

No acute single dose toxicity studies for eicosapentaenoic acid (EPA) and docosahexaenoic

acid (DHA) were identified in the literature.

2.2.2 Repeated dose toxicity

Several animal studies were identified in which the safety of EPA and DHA consumption was

examined (Table 7). In one study, 5-week-old male Otsuka Long-Evans Tokushima Fatty

(OLETF) rats (which are used to model Type 2 diabetes) were provided with diets containing 1.0

g EPA/kg body weight(bw)/day, 1.0 g oleic acid/kg bw/day or a non-supplemented rat chow diet

until they were 30 weeks of age (Minami et al., 2002). No adverse events or signs of toxicity

were reported in OLETF rats consuming a diet providing 1.0 g EPA/kg bw/day. Moreover,

consumption of the EPA-containing diet had beneficial effects on various lipid and biochemical

parameters, causing significant reductions in plasma TG levels and abdominal fat accumulation,

and significantly improving insulin resistance. Thus, EPA was found to have a potentially

beneficial role in improving insulin sensitivity in Type 2 diabetes.

In another animal study, diets containing corn oil, corn oil and 0.1 g EPA/kg bw/day, or corn oil

and 1.0 g EPA/kg bw/day were fed to ovariectomised rats for 9 weeks (Poulsen and Kruger,

2006). No significant differences were noted between the bone breaking strength and serum

type-1 collagen concentrations of the treatment and control rats. Ovariectomised rats

consuming 1.0 g EPA/kg bw/day were reported to have a significant lower femur bone density,

which was attributed to a decrease in bone density, possibly caused by an increase in bone

resorption. In contrast to these findings, the administration of EPA to older women (mean age

79.6 years) had beneficial effects on bone density (Kruger et al., 1998).

The administration of a diet containing 2% EPA or DHA ethyl esters, representing a dose of

approximately 1,000 mg/kg bw/day, to normal Sprague-Dawley rats for 3 weeks did not result in

any adverse events (Hung et al., 2000). In contrast, consumption of EPA and DHA had

beneficial effects on lipid metabolism and leukotriene synthesis, with the effects elicited by EPA


consumption being stronger than those elicited by consumption of DHA. In another study the

administration, by gavage, of diets containing 0, 0.5, or 1.25 g/kg bw/day of an oil containing 40

to 50% DHA for 90 days to normal CD rats did not result in significant differences in any of the

parameters examined (i.e., hematology, clinical chemistry, pathology, ophthalmology,

neurobehavioral and neuropathological indices) (Arterburn et al., 2000). The No-Observed-

Adverse-Effect Level (NOAEL) for normal CD rats was calculated from the highest dose of DHA

administered, and corresponded to a dose of approximately 0.5 to 0.625 g DHA/kg bw/day.

One study was identified in which the safety of DHA, purified form tuna oil, was examined in

pigs (Merritt et al., 2003). Newborn piglets were administered formula providing approximately

387 or 653 DHA/kg bw/day for male piglets and 412 or 691 mg DHA/kg bw/day for female

piglets. The consumption of either dose of DHA did not result in any significant effects on bw,

clinical signs, food consumption, clinical chemistry, hematology, organ weight, or gross or

histopathology in the male and female piglets. These study findings further corroborate the

safety of DHA supplementation.

Table 7 Studies examining EPA/DHA repeated dose toxicity

Reference Animals Sample Size Investigational product + duration

Dose Results

(Minami et al., 2002)

Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats

3 groups (n=8 per group)

Powered chow, EPA, oleic acid (OA) for 25 weeks

Group 1: powered chow Group 2: powered chow + 10 g EPA/kg bw/day Group 3: powered chow + 10 g oleic acid/kg bw/day

No adverse events or signs of toxicity were reported in OLETF rats consuming a diet providing 1.0 g EPA/kg /day.

(Poulsen and Kruger, 2006)

Ovariectomised Sprague–Dawley rats

4 groups (n=14 to 16 per group)

0.1 g (LOW) or 1.0 g (HIGH) of EPA/kg bw/day for 9 weeks

Group 1: LOW diet Group 2: HIGH diet Group 3: control with diet devoid of n-3 fatty acids Group 4: sham-operated control with diet devoid of n-3 fatty acids

Ovariectomised rats consuming 1.0 g EPA/kg bw/day were reported to have a significant lower femur bone density, which was attributed to a decrease in bone density, possibly caused by an


increase in bone resorption.

(Hung et al., 2000)

Sprague-Dawley rats


High linoleic safflower oil (SAF), EPA, DHA for 3 weeks

Group 1: 8% SAF + 2% EPA Group 2: 8% SAF + 2% DHA Group 3: control, 10% SAF

No reported adverse events.

(Arterburn et al., 2000)

CD rats 4 groups (n=40 per group)

DHASCO oil (containing 40 to 50% DHA) for 90 days

Group 1: 0.5 g/kg bw/day Group 2: 1.25 g/kg bw/day Group 3: Vehicle control Group 4: Control, untreated

The No-Observed-Adverse-Effect Level (NOAEL) for normal CD rats was calculated from the highest dose of DHA administered, and corresponded to a dose of approximately 0.5 to 0.625 g DHA/kg bw/day.

2.2.3 Genotoxicity / carcinogenicity

The results of studies examining the mutagenic and genotoxic potential of EPA and DHA are

summarized in Table 8. EPA and DHA were neither mutagenic nor genotoxic in any study. On

the contrary, EPA and DHA showed anti-mutagenic activity in a study with V79 lung cells of

Chinese hamsters (Kuroda et al., 2001).

Table 8 Studies examining EPA/DHA genotoxicity / carcinogenicity


Dose Results

(Fedorova-Dahms et al., 2011)

Sprague-Dawley rats


Algal oil (43% DHA and 8% EPA) or fish oil (60% DHA and 8% EPA) for 90 days

Group 1: 0.5% algal oil diet (340-907 mg/kg bw/day) Group 2: 1.5% algal oil diet (1022-2782 mg/kg bw/ day)

No mutagenic properties of DHA-rich oil were observed. Also no other treatment related effects were


Group 3: 5% algal oil diet (3466-8322 mg/kg bw/day) Group 4: Control, fish oil diet (3511-7155 mg/kg bw/day) Group 5: Control, basal diet

observed. Therefore, the highest Algal oil dose was considered NOAEL.

(Blum et al., 2007)

Sprague-Dawley rats


Combination of fish oil (containing 26.7% DHA and 11.4% EPA) and DHA-algal (containing 45% DHA) for 90 days or 90 days + an additional 4-week recovery period (no treatment).

Group 1: 2000 mg/kg fish oil Group 2: 1500 mg/kg fish oil + 500 mg/kg DHA-algal oil Group 3: 1000 mg/kg fish oil + 1000 mg/kg DHA-algal oil Group 4: 2000 mg/kg DHA-algal oil Group 5: Control Group 6: 2000 mg/kg fish oil + 4 week recovery Group 7: 2000 mg/kg DHA-algal oil + 4 week recovery Group 8: Control + 4 week recovery

In the 90-day toxicity study no toxicologically significant side effects were observed in genotoxicity- and chromosomal aberration studies, following oral administration of DHA oil, both fish and algal derived. The highest dose of 2000 mg/kg bw/day was considered NOAEL.

(Abril et al., 2003)

Growing pigs (mixed breed of Landrace and Large White)


Dried DHA-rich microalgae, DRM, (containing 22.3% DHA) added to the diet for 120 days.

Group 1: 1.10% DRM Group 2: 1.10% DRM on day 79-106 and 0.39% DRM on day 107-120 Group 3: 3.30% DRM on day 79-106 and 1.17% DRM on day 107-120 Group 4: 5.51% DRM on day 79-106

No treatment-related adverse effects were observed in these commercial strains of swine.


and 1.94% DRM on day 107-120 Group 5: Control

2.2.4 Reproductive and developmental toxicity

Table 9 summarizes available literature on reproductive and developmental toxicity. Overall no

effect on developing animals were observed related to EPA and DHA consumption.

Table 9 Studies examining EPA/DHA reproductive and developmental toxicity


Dose Results

(Schmitt et al., 2012)

Sprague-Dawley rats


DHA rich oil (42% DHA, total omega-3 fatty acid concentration of 44%) or fish oil (DHA 26-27% and EPA content of 6-7%). Treated for 89-91 days in males en 75-77 days in females. Male and female offspring were exposed for 106-107 and 110-111 consecutive days, respectively.

Group 1: Fish oil Group 2: Fish oil Group 3: DHA rich oil Group 4: DHA rich oil Group 5: Control, basal diet

There was an absence of maternal and developmental toxicity. Also postnatal survival and development were not different with the control group. The highest dose, 2000 mg/kg bw/day, was considered the NOAEL.

(Blum et al., 2007)

Sprague-Dawley rats


Combination of fish oil (containing 26.7% DHA and 11.4% EPA) and DHA-algal (containing 45% DHA) for 90 days or 90 days + an additional 4-week recovery period (no treatment).

Group 1: 2000 mg/kg fish oil Group 2: 1500 mg/kg fish oil + 500 mg/kg DHA-algal oil Group 3: 1000 mg/kg fish oil + 1000 mg/kg DHA-algal oil Group 4: 2000 mg/kg DHA-algal oil Group 5: Control Group 6: 2000

There were no adverse effects reported related to DHA-containing oil consumption.


mg/kg fish oil + 4 week recovery Group 7: 2000 mg/kg DHA-algal oil + 4 week recovery Group 8: Control + 4 week recovery

(Kroes et al., 2003)


Diet including an oil containing 45% DHA (DHA45) for 10 weeks prior to mating (for both sexes) and 14 days during gestation and lactation (only females)

Group 1: 1.5% DHA45 (360-1215 mg/kg bw/day) Group 2: 3.0% DHA45 (675-2385 mg/kg bw/day) Group 3: 7.5% DHA45 (1530-5040 mg/kg/day) Group 4: Control (7.5% corn oil)

No effect on reproductive parameters (e.g. percoital time, mating index, fertility indices, fecundity, gestation index and duration, numbers of stillborn pups and post-implantation losses.

2.2.5 List of studies conducted & references

Table 10 List of referenced non-clinical studies on omega-3 fatty acids

Study Number Author(s) Title of report

1 Minami et al, 2002 Effect of eicosapentaenoic acid ethyl ester v. oleic acid-rich safflower oil on insulin resistance in type 2 diabetic model rats with hypertriacylglycerolaemia.

2 Poulsen and Kruger, 2006

Detrimental effect of eicosapentaenoic acid supplementation on bone following ovariectomy in rats.

3 Hung et al., 2000 Dietary effects of eicosapentaenoic and docosahexaenoic acid esters on lipid metabolism and immune parameters in Sprague-Dawley rats

4 Arterburn et al., 2000 A combined subchronic (90-day) toxicity and neurotoxicity study of a single-cell source of docosahexaenoic acid triglyceride

5 Fedorova-Dahms et al., 2011

Safety evaluation of DHA-rich Algal Oil from Schizochytrium sp

6 Blum et al., 2007 Genotoxicity and subchronic toxicity studies of DHA-rich oil in rats

7 Abril et al., 2003 Safety assessment of DHA-rich microalgae from Schizochytrium sp. Part V: target animal safety/toxicity study in growing swine.

8 Schmitt et al., 2012 Toxicologic evaluations of DHA-rich algal oil in rats: developmental toxicity study and 3-month dietary toxicity study with an in utero exposure phase.

9 Kroes et al., 2003 A review of the safety of DHA45-oil.


2.2.6 References

Abril R., Garrett J., et al. (2003) Safety assessment of DHA-rich microalgae from Schizochytrium sp. Part V: target animal safety/toxicity study in growing swine. Regul Toxicol Pharmacol 37(1), 73-82.

Arterburn L.M., Boswell K.D., et al. (2000) A combined subchronic (90-day) toxicity and neurotoxicity study of a single-cell source of docosahexaenoic acid triglyceride (DHASCO oil). Food Chem Toxicol 38(1), 35-49.

Blum R., Kiy T., et al. (2007) Genotoxicity and subchronic toxicity studies of DHA-rich oil in rats. Regul Toxicol Pharmacol 49(3), 271-284.

Fedorova-Dahms I., Marone P.A., et al. (2011) Safety evaluation of DHA-rich Algal Oil from Schizochytrium sp. Food Chem Toxicol 49(12), 3310-3318.

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2.3 CLINICAL DATA

2.3.1 Clinical pharmacology

The role of polyunsaturated fatty acids (PUFAs) and their metabolites for the cause and

treatment of psychotic disorders are widely discussed. Controlled studies are usually small and

still seem to yield conflicting results (1,2). The efficacy as an augmenting agent in chronic

schizophrenia seems to be small or not present (3), however epidemiological data, as well as

some recent controlled studies in emerging psychosis point towards possible preventive effects

of PUFAs in early and very early stages of psychotic disorders (4, 5) and some potential

secondary or tertiary beneficial long-term effects in later, more chronic stages, in particular for

metabolic or extra-pyramidal side effects.

The clinical staging model for psychotic and severe mood disorders proposed by Patrick

McGorry (6) reflects that very notion of the differentiation between earlier and milder clinical

phenomena and progressing, more chronic manifestations of an illness. This model, which is

considered for inclusion in the DSM-V (7), suggests three risk stages before onset of full

threshold first episode psychosis. Evolving clinical and neurobiological knowledge on high-risk

stages generally seems to support the clinical staging model. The potential role of PUFAs for

brain maturational processes, the regulation of synaptic plasticity and myelination have not yet

been explored in these early stages of emerging psychotic disorders.

Physiological role of PUFAs

Neuronal membrane bilayers are largely made up of phospholipids and cholesterol.

Phospholipids consist of a glycerol or sphingosine backbone with 3 carbon molecules. A

phosphate molecule and a hydrophilic head group, such as choline, ethanolamine, inositol or

serine are attached to the 3-position. Hydrocarbon tails ranging from 16 to 24 carbon molecules

are attached to the 1- and 2 positions. These hydrocarbon tails are called fatty acids. Fatty acids

can be either saturated or unsaturated if they contain one (monounsaturated) or more

(polyunsaturated) double bonds. Typically, fatty acids attached to the 1-position are saturated

and the ones attached to the 2-position are unsaturated. The number of double bonds, i.e. the

degree of unsaturation influences membrane fluidity.

Polyunsaturated fatty acids (PUFAs) can be classified into omega-3 and omega-6 fatty acids.

The terms omega-3 (n-3) and omega-6 (n-6) specify the location of the first double bond from

the methyl end of the molecule. Mammals have lost the ability to introduce a double bond

beyond carbon 9, thus humans cannot synthesize these fatty acids de novo. Therefore, the

parent molecules of n-3 and n-6 PUFAs, alpha-linolenic acid (ALA, 18:3 n-3) and linoleic acid


(LA, 18:2 n-6), are termed essential fatty acids (EFAs); they have to be obtained through dietary

intake and hold important physiological functions. In the liver, EFAs serve as substrates for

further conversion to long-chain PUFAs (LC-PUFAs) with higher number of carbon atoms and

double bonds. The synthesis of LC-PUFAs from the parent substrates ALA and LA is a multi-

stage process involving desaturases and elongases, where n-3 PUFAs and n-6 PUFAs

compete for the same enzymes.

The conversion rates from ALA and LA are limited in humans, wherefore some LC-PUFAs, such

as arachidonic acid (AA, 20:5, n-6) and eicosapentaenoic acid (EPA, 20:5, n-3) need to be

mainly ingested with AA and EPA-rich foods. Dietary sources of ALA are nuts and seeds, such

as walnuts, flaxseed and rapeseed oil; sources of the LC-PUFAs such as EPA and DHA include

fatty fish (e.g. salmon), certain white fish, shellfish and other seafood, as well as some animal

products depending on the animals’ diet (8, 9). In early life, breast milk also constitutes a source

of intake (10). LA and all other n-6 PUFAs are ingested with plant and vegetable seeds and oils,

such as margarines as well as those included in processed foods.

PUFAs and inflammatory mediators

AA and EPA act as precursors for eicosanoids such as prostaglandins (PGs), thromboxane (Tx)

and leukotriens (LTs). Prostaglandins can be synthesized after the release of fatty acids from

membrane phospholipids. Eicosanoids regulate a number of cellular processes.

Phospholipases

Phospholipids are broken down by different types of phospholipases (A1, A2, B, C, D). In the

brain, phospholipases A2 (PLA2) and C play the most important role. PLA2 are key enzymes in

regulating the AA content in the cell membrane, as well as the release and production of

bioactive lipids in general. In short, omega-3 LC-PUFAs are potentially anti-inflammatory agents

and influence the PLA2 catalyzed eicosanoid metabolism.

PUFAs and the brain

AA and DHA are concentrated in the grey matter of the human brain and account for 20% of the

membrane fatty acids. EPA and DHA exert different physiological effects on neuronal cell

membranes. DHA has been shown to alter densities of dopamine, serotonin and muscarinic

receptors, whereas EPA affects the brain through its function as a precursor for eicosanoids.

These PUFAs are particularly important for the specific properties and functions of neuronal

cells since their availability determines the phospholipid structure and structure in turn impacts

function. Other processes regulated by PUFAs (11) include brain development (12), synaptic


functioning (13), regulation of corticotrophin-releasing hormone (14), prevention of neuronal

apoptosis (15, 16), neurite growth (17), gene expression (18), as well as neuronal migration,

pruning and synaptic plasticity (13). The balance of specific type of PUFAs also affects

inflammatory processes: EPA as well as ALA supplementation (19) has been shown to reduce

the production of pro-inflammatory cytokines (20, 21).

Direct effects of omega-3 fatty acids on the brain can be measured in vivo with magnetic

resonance imaging (MRI) by measuring brain water proton transverse relaxation times (T2),

which is an index of the water in neuronal tissues. Reduced neuronal health is thought to be

associated with T2 increases (22). Indeed, preclinical findings on neuroprotective properties of

EFAs are supported by studies in patients with bipolar disorders (23) and first episode

psychosis (FEP) using T2 relaxation time (22).

Fish intake, cognition and early brain development

Regarding the importance of PUFAs for brain development, maternal fish and seafood

consumption of less than 340g per week during pregnancy has been reported to increase the

risk for a low IQ and deficient neurodevelopmental outcomes in childhood (24). Some (25, 26)

but not all (27-29) studies have yielded findings of protective effects of EFAs in pregnancy and

breastfeeding against schizophrenia and related disorders. Some of the negative findings were

population-based studies, whereas the positive ones rather investigated high-risk populations

with family histories of schizophrenia, very premature birth, birth complications, viral infections

or extreme malnutrition. Possibly, the protective role of EFAs in breastmilk might be relevant

only for risk populations.

2.3.2 Clinical pharmacokinetics

In vivo comparison of three dosages (3 g, 6 g, 12 g) of two different fish oil preparations in terms

of plasma concentrations of their major active components eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA) has been quoted most often when describing the

pharmacokinetics of omega-3 fatty acids. The plasma accumulation was measured during 28

days of ingestion and an equally long wash out period. Data were scrutinized for bioavailability

in order to distinguish between the efficiency of the two preparations. Rapid increases in EPA

and DHA plasma concentrations can be demonstrated at all dosages during a 28-day ingestion

period. EPA accumulated more during ingestion of high than of low dosages of fish oil. DHA

revealed almost identical increases and peak values in plasma concentrations in all subgroups.

The present data demonstrate dose dependent increases of EPA concentrations whereas DHA

plasma concentrations are comparable in all dosages investigated. Measurable EPA and DHA

plasma concentration levels are inappropriate means to explain clinical effectiveness. These


results were found in both commercially available fish oil preparations. Direct comparison of

both preparations revealed no differences in bioavailability (30). EPA and DHA are absorbed

largely via the lymph. It is apparent that EPA and DHA ethyl esters are hydrolyzed during

absorption from the small intestine (31) and re-esterified mainly into triglycerides for transport in

the chylomicrons in lymph (31, 32). Elaborate evidence for high oral bioavailability is available,

for instance, from an excretion study in which less than 10% of the dose of radioactive carbon

(14C)-labelled EPA ethyl ester or DHA ethyl ester was excreted in faeces over 7 days.

2.3.3 Human exposure

Polyunsatured omega-3 fatty acids (PUFA) are essential for normal brain development. Cell

biological and molecular studies suggest that omega-3 fatty acids modulate membrane fluidity

(33), dopaminergic (34), glutamatergic (35) and serotonergic (36) neurotransmission. PUFA can

also regulate gene expression (37), influencing synaptic plasticity and function (38). Preclinical

studies suggest that omega-3 fatty acids have neuroprotective properties (39, 40). Animal and

human studies show that omega-3 PUFA are crucial during the embryonic stage, early postnatal

life and childhood (41). PUFA are thus relevant to neurodevelopment, neurodegeneration and

behavioural neurobiology, all factors proposed as playing a pivotal role in the genesis of

schizophrenia (42).

The precursors of omega-3 fatty acids (alpha-Linolenic acid ALA) and omega-6 fatty acids

(Linoleic acid LA) are essential fatty acids (EFAs) meaning that humans cannot synthesize them

de novo. Humans have to supply EFAs through diet similar to vitamins. The source of the

omega-3 LC-PUFAs is mainly oily fish, e.g. salmon or sardines, whereas the precursor ALA is

also present in plant oils, such as canola oil, nut oils, or algae oils. A range of epidemiological

and experimental studies provide some evidence for a link between cardiovascular disorders,

metabolic syndrome or depressive disorders that are all proposed to be associated with an

increase in proinflammatory cytokines and a deficiency in omega-3 fatty acids. The latter may

have direct implications for prevention and treatment (43, 44).

Omega-3 fatty acids and psychotic disorders

The “membrane hypothesis “ of schizophrenia (45, 46) was originally based on early findings of

lipid alterations in the cerebrospinal fluid or peripheral cell membranes of patients with

schizophrenia (42) and of a prostaglandin–deficit syndrome (mainly metabolites of arachidonic

acid or eicosapentaenoic acid) (47). There is also evidence suggesting decreased omega-3

fatty acid levels during the early phases of schizophrenia, in blood cell membranes of

antipsychotic-naïve first episode patients (48). Imaging methods such as magnetic resonance

spectroscopy have allowed researchers to investigate membrane turnover in the living human


brain, confirming significant membrane abnormalities in schizophrenia (49, 50), independent of

treatment (51, 52).

Although there is only limited evidence supporting that EPA augmentation of antipsychotic

medication in established psychosis improves symptomatic outcome (53), they may exert

positive effects as a preventive agents, in particular during the pre-psychotic phases. A recent

study has confirmed a mixture of EPA and DHA significantly reduces transition rates in subjects

at ultra-high risk for psychosis (54) pointing to a potential neuroprotective effect of omega-3 fatty

acids supplementation in at risk mental states. There is growing evidence suggesting that the

emergence of psychosis is associated with subtle structural (for a review see (55)), functional

(for reviews see (56, 57)) and neurochemical (mostly involving dopamine and glutamate, for

reviews see (58, 59)) alterations which may ultimately lead to the onset of frank psychosis. The

potential underlying mechanism of the therapeutic action of omega-3 fatty acids during the pre-

psychotic phases may be neuroprotective, e.g. via modulation of the antioxidative intracellular

defense (35), antiapoptotic actions (60) or be the result of a direct interaction between EPA and

glutamatergic neurotransmission (for a review on omega-3 fatty acid interventions in early

psychosis see (42)).

Published omega-3 fatty acids RCTs in minors with psychiatric conditions

The available scientific literature concerning effects of omega-3 fatty acids in children with

psychiatric conditions is limited to childhood-onset depression and attention deficit and

hyperactivity disorders (ADHD). Studies in adolescents include an RCT first-episode psychosis

(5), as well as an RCT in adolescents at ultra-high risk for psychosis (4). The latter concerned

an RCT in 81 adolescents at ultra-high risk (UHR) for developing a psychotic disorder (mean

age 16.4) that used 1.2g of an EPA-enriched omega-3 fatty acids oil as a sole agent (4). 27.5%

in the placebo group progressed towards a first psychotic episode compared to only 4.9% in the

omega-3 fatty acids group. This pilot study has recently been replicated but study results are not

available yet (the NEURAPRO study (61)). The use of omega-3 fatty acids in prevention of

mental disorders is promising, yet an avenue to be further explored.


2.4 OVERALL RISK AND BENEFIT ASSESSMENT

2.4.1 PUFAs and potential adverse effects in UHR

Only one study has investigated the effects of supplementation with omega-3 PUFAs as

treatment in UHR individuals (4). Thus, the only data regarding the safety of omega 3 PUFAs

are derived from this trial. Applying the Udvalg for Kliniske Undersogelser scale (UKU) to detect

adverse effects, frequency and intensity of possible side effects in the omega-3 FA condition

were not different from placebo. Although not statistically different from the placebo group, the

following adverse effects were reported in the omega-3 group: diarrhoea, nausea/vomiting,

tension/inner unrest, depression, concentration difficulties, emotional indifference, tension

headache, reduced duration of sleep, increased fatigability. All but “increased fatigability”

occurred – nonsignificantly - less frequently in the omega-3 group Not listed are side effects

that occurred with a prevalence of less than 5% in the omega-3 or in the placebo group.

2.4.2 PUFAs and potential adverse effects in schizophrenia

Additional data regarding adverse effects of omega-3 PUFAs supplementation are available

from add-on treatment trials in patients suffering a first episode of psychosis or in chronic

schizophrenia patients. Generally, the assessments of adverse effects in these trials varied

between structured assessments (e.g. UKU) and spontaneous patient reports. In Fenton and

colleagues’ omega-3 PUFA supplementation RCT patients with schizophrenia/schizoaffective

disorder of the verum condition showed upper respiratory infection (8/43 patients, 19%) and

diarrhoea (8/43 patients 19 %) (62). A lower dose of omega-3 PUFAs (600mg EPA/DHA) in

Arvindakshan and colleagues’ open-label study was not associated with any gastrointestinal

problems, but 5 of 33 patients did drop out due to the fishy odour of the capsules (63). Taken

together, the following common but clinically rarely significant side effects were reported here:

mild nausea or diarrhoea, fishy eructation and loose stool (53, 64, 65).

2.4.3 Specific adverse effects of PUFAs

Further data regarding safety issues are available from trials performed in patients with

schizophrenia, other psychiatric and somatic illnesses and the general population.

PUFAs and bleeding time

There is evidence that the intake of omega-3 PUFAs is associated with a prolonged bleeding

time in healthy men (66) and in both healthy men and women (66-68). A literature review by

Knapp suggested that the prolongation of bleeding time is usually modest and there have been

no reports of clinically significant bleeding (69). A statement by the FDA (70) confirmed that fish

oils can significantly prolong bleeding time but a dose of up to 3g/d EPA/DHA is considered as


safe (71). According to a study by Emsley and colleagues (72) in chronic schizophrenia patients

a dose of 2g/day EPA caused a significant increase in bleeding time, but remained in the high

end of the normal range and was not associated with clinically relevant bleeding complications.

Since patients with schizophrenia and other psychoses have been shown to be at increased risk

for cardiovascular conditions and mortality (73), a slightly prolonged bleeding time within normal

range as well as other metabolic effects of PUFAs (outlined below) are discussed as being even

beneficial.

PUFAs and metabolic parameters

There is sound evidence from studies in healthy populations and in individuals with lipid

metabolism disorders that omega-3 LC-PUFAs are effective in lowering triglycerides (TG) levels

(74-76). Only one trial explicitly investigated metabolic effects of EPA in patients with chronic

schizophrenia (72). This study evaluated the effects of 2g/day EPA vs. placebo on weight gain,

total cholesterol, HDL, LDL and TG levels, on fasting blood glucose and additionally on tardive

dyskinesia (TD) over a period of 12 weeks with a 40-week open extension phase. The results

point to a significant, but clinically probably only slightly relevant increase in Body Mass Index

(BMI) in the EPA group (baseline 24.5 to end of treatment 25.2 kg/m2; p = 0.0001). With respect

to total cholesterol, HDL, LDL and TG levels, the findings depict a significant decrease in total

cholesterol levels as well as in HDL levels, but there were changes in neither LDL nor in TG

plasma levels (77). Fasting blood glucose increased at trend level. Meta-analytical data of

studies in individuals who were either healthy, had diabetes, hypertension or dyslipidemia, or

cardiovascular disease, show that omega-3 LC-PUFAs do not seem to have relevant effects on

blood glucose (78, 79). At the moment, no published data regarding PUFAs and metabolic

parameters are available in patients with UHR for psychosis.

PUFAs and cardiovascular factors

Epidemiological studies suggest benefits of omega-3 PUFAs on cardiovascular health (80, 81).

Indeed, the American Heart Association recommends 1g/d of EPA/DHA for secondary

cardiovascular disease prevention (76, 82). No valid data are yet available in patients with

schizophrenia. However, it is well known that patients with schizophrenia and other psychiatric

disorders are at increased risk for cardiovascular disease as a consequence of a higher

prevalence of the metabolic syndrome (83). Therefore, positive omega-3 PUFA

supplementation effects in patients with emerging psychosis might not only arise from protection

against transition to acute psychosis, but there may also be a gain from the potential to prevent

metabolic and cardiovascular detriments (84).


Other common but clinically rarely relevant side effects of n-3 fatty acid supplementation include

mild nausea, mild diarrhoea, fishy eructation and exacerbation of asthma in aspirin-sensitive

patients (85). Only two of available treatment trials in patients at UHR, with a FEP or chronic

schizophrenia report differences in adverse effects with n-3 LC-PUFAs supplementation (62,

64): they report gastrointestinal symptoms and upper respiratory infections.

2.4.3 Risks versus benefits of PUFAs in emerging psychosis

Taken together, one promising aspect of omega-3 PUFAs is that there seem to be no reports of

relevant deleterious side effects in humans, even at high doses. However, the differential effects

on lipid parameters described above are noteworthy and need further clarification. As

mentioned, the slight risk of prolonged bleeding time is outweighed by the benefits. Aside from

the benefit that treatment with omega-3 PUFAs may prevent or at least delay the onset of

psychotic disorders in UHR groups, a notable benefit is that treatment with omega-3 PUFAs

also significantly reduced positive, negative, and general symptoms on the positive and

negative syndrome scale (PANSS) and improved functioning (4) .

2.5 CONCLUSION

The current stage of knowledge based on the most recent trial of indicated prevention in UHR

groups holds promise that treatment with omega-3 PUFAs may prevent or at least delay the

onset of psychotic disorder. As replication studies are still underway, conclusions in terms of

clinical effectiveness are still preliminary. In contrast, in terms of safety issues evidence from the

RCT in UHR individuals and a variety of studies in schizophrenia patients, strongly suggests

PUFAs are safe and well-tolerated by young people at ultra-high risk of psychosis. Omega-3

PUFAs have been shown to be safe even when used in relatively high doses, and except for

mild gastrointestinal symptoms they are presumed to be free of clinically relevant adverse

events (86). Nevertheless, clinicians should be aware of possible increases in bleeding time.

Although a statement by the FDA (70) reported that fish oils can significantly prolong bleeding

time and a dose of up to EPA/DHA of 3g/d was considered as safe, clinical trial evidence does

not support relevant increases in bleeding time, not even in patients treated with anti-platelet or

antithrombotic agents (71). Generally, tolerability of n-3 LC-PUFAs can be assumed as being

very good. When balancing risks and benefits of omega-3 PUFA supplementation, it is

important to note that additionally to the common positive effects on the cardiovascular and

other systems omega-3 PUFAs might also unfold neuroprotective properties (87) which are of

particular interest in the field of indicated prevention and psychosis research.


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