Lipid Eye drops Containing Cyclodextrin and a Polymer

Formulation, Characteristics and Stability

Hróðmar Jónsson

M. Sc. Thesis in Pharmacy

University of Iceland

Faculty of Pharmaceutical Sciences

School of Health Sciences

Lipid Eye drops Containing Cyclodextrin and a Polymer

Formulation, Characteristics and Stability.

Hróðmar Jónsson

M.Sc. thesis in Pharmacy

Supervisor: Þorsteinn Loftsson

Faculty of Pharmaceutical Sciences

School of Health Sciences at the University of Iceland

June 2013

Augndropar úr Sýklódextríni, Fitu og Fjölliðu

Formúlering, Eiginleikar og Stöðugleiki

Hróðmar Jónsson

Meistararitgerð í lyfjafræði

Umsjónarkennari: Þorsteinn Loftsson

Lyfjafræðideild

Heilbrigðisvísindasvið Háskóla Íslands

Júní 2013

v

This thesis is for a M.Sc. degree in pharmacy and may not be reproduced in any way without

the permission of the author

© Hróðmar Jónsson 2013

Printed by Háskólaprent

Reykjavík, Iceland 2013

vi

Author Hróðmar Jónsson

Supervisor/ Þorsteinn Loftsson, Ph. D.

Professor

Faculty of Pharmaceutical Sciences, University of

Iceland

Instructors Sergey Kurkov, Ph.D.

Post doctoral fellow

Faculty of Pharmaceutical Sciences, University of

Iceland

vii

ABSTRACT

Lipid Eye drops Containing Cyclodextrin and a Polymer.

Formulation, Characteristics and Stability

The eye is a sensory organ that makes everyday life of more convenience. Even though eyes

are considerably protected from nature’s way, eye disease and discomfort are considerably

common, especially dry eye disease. Today the most common treatment of dry eye is

palliative instead of correcting the inflammatory state associated with dry eye disease. It has

been demonstrated that ω-3 fatty acids, which can be found in large quantities in cod liver oil,

are anti-inflammatory. The intake of these fatty acids has been linked to reduced risk of dry

eye disease and, when topically applied, to reduce various indicators used to assess dry eye

disease and inflammation. Long, polyunsaturated free fatty acids have also demonstrated anti-

bacterial and -viral effect.

Cyclodextrins are cyclic oligosaccharides that have the ability to form water soluble

complexes with lipophilic drugs and at the same time improve their stability. Cyclodextrins

have been shown to form complexes with the above mentioned lipids and improve both their

solubility and stability.

The purpose of this project was to formulate eye drops that contained cod liver oil as well as

free fatty acids, stabilized with cyclodextrin. Previously it has been demonstrated that only the

parent α-, β- and γ-cyclodextrin are able to form complexes with the lipids and that a

cyclodextrin concentration of 10% w/v and 10% v/v of the lipids were optimal. Of the

cyclodextrins tested, γ-cyclodextrin gave the best results. The effect of different polymers,

with and without preservatives, on flocculation, redispersion, surface tension, viscosity and

osmolality were studied. Formulation containing preservatives with 2.5% w/v poloxamer 407

and one with 5% poloxamer were superior to others. The particle size distribution complied

with the European Pharmacopeia on particle size in eye drops and the peroxide value

indicated that the CD protected the lipids from oxidation.

viii

ÁGRIP

Augndropar úr Sýklódextríni, Fitu og Fjölliðu

Formúlering Eiginleikar og Stöðugleiki

Augun eru eitt af skynfærum líkamans sem gera daglegt líf auðveldara. Þrátt fyrir að vera

talsvert vernduð frá náttúrunnar hendi eru augnsjúkdómar og aðrir kvillar algengir meðal

einstaklinga og er augnþurrkur einna algengastur. Flest öll lyfjameðferð í dag gegn augnþurrki

felur í sér skyndilausnir í stað þess að leiðrétta það bólguástand sem tengist honum. Sýnt

hefur verið fram á að ω-3 fitusýrur, sem meðal annars er að finna í miklum magni í

þorskalýsi, eru bólguhemjandi. Inntaka á þessum fitusýrum hefur meðal annars verið tengd

við minni hættu á augnþurrki auk þess sem staðbundin notkun í augu músa dró úr

mælikvörðum sem eru notaðir eru til að meta hann. Langar, fjölómettaðar fitusýrur á fríu

formi hafa auk þess sýnt fram á bakteríu- og veiruhemjandi verkun.

Sýklódextrín eru hringlaga fásykrur sem hafa þann eiginlega að mynda vatnsleysanlegur

fléttur með torleysanlegum lyfjaefnum og um leið auka stöðugleika þeirra. Sýnt hefur verið

fram á að sýklodextrín geta myndað fléttur með þessum fituefnum og aukið bæði leysanleika

og stöðugleika þeirra.

Markmið þessa verkefnis var að þróa augndropa sem innihéldu þorskalýsi sem og fríar

fitusýrur, stöðgaðar með sýklodextríni. Áður hefur verið sýnt fram á að aðeins náttúrulegu α-,

β- og γ-sýklodextrín gætu myndað fléttur með fituefnunum og að hlutföllin 10% w/v

sýklodextrín og 10% v/v fituefni væru ákjósanlegust. Af þessum sýklodextrínum kom γ-

sýklodextrín best út. Áhrif ýmissa fjölliða, með og án rotvarnarefna, á setmyndun,

upphristanleika, yfirborðsvirkni, seigjustig og osmólastyrk var rannsakaður og var það lausn

sem innihélt rotvarnarefni og 2.5% w/v poloxamer 407 ásamt lausn sem innihélt 5%

poloxamer 407 sem kom best út. Kornastærðin uppfyllti kröfur Evrópsku lyfjskrárinnar um

kornastærð augndropa og peroxíð gildi gáfu til kynna að sýklódexdrínin vernduðu fituefnin að

hluta fyrir oxun.

ix

LIST OF ABBREVIATIONS

ALA α linolenic acid

AV Anisidine value

ARA Arachidonic acid

BAK Benzalkonium chloride

CFS Corneal fluorescein staining

CLA Conjugated linolenic acid

CMC-Na Sodium salt of carboxymethyl cellulose

cP centipoise

DHA Docosahexaenoic acid

EPA Eicosapentaenoic acid

EDTA Ethylenediaminetetraacetic acid

FA Fatty acid

HPMC Hydroxypropyl-methylcellulose

HSV Herpes simplex virus

IL Interleukin

LA Linolenic acid

N neutons

Osm osmol

PGE Prostaglandins

PUFA Polyunsaturated fatty acid

PV Peroxide value

PVA Polivinyl alcohol

PVP Polivinyl pyrrolidone

SD standard deviation

TNF – α Tumor necrosis factor α

x

TABLE OF CONTENTS

1. INTRODUCTION .............................................................................................................. 2

1.1 THE EYE ............................................................................................................................ 2

1.1.1 Anatomy of the eye ................................................................ 2

1.1.2 The meibum ................................................................................... 4

1.1.3 Dry eye .......................................................................................... 4

1.1.4 Etiology of dry eyes ....................................................................... 6

1.1.5 Prevalence and treatment of dry eye ............................................. 7

1.1.6 Drug delivery to the eye ................................................................ 7

1.1.7 Formulation standards for eye drops ............................................ 8

1.2 LIPIDS ............................................................................................................................. 10

1.2.1 Fatty acids ........................................................................... 10

1.2.2 Essential Fatty Acids ........................................................... 11

1.2.3 ω-3 and diseases ................................................................. 13

1.2.4 Fish oil ................................................................................ 14

1.2.5 Antibacterial and antiviral effects of PUFAs ..................... 16

1.3 CYCLODEXTRINS ............................................................................................................ 17

1.3.1 Structure of cyclodextrins ................................................... 17

1.3.2 Cyclodextrin complexation ................................................. 18

1.3.3 Cyclodextrin toxicology ...................................................... 20

1.3.3.1 Ophthalmic toxicology .............................................................................................. 20

1.3.4 Cyclodextrins in ophthalmic drug delivery ......................... 21

1.3.5 Cyclodextrins and lipids...................................................... 21

1.4 POLYMERS ...................................................................................................................... 23

2. AIM OF THE STUDY ..................................................................................................... 25

3. EQUIPMENTS, MATERIALS AND METHODS ........................................................ 26

3.1 EQUIPMENTS ................................................................................................................... 26

3.2 MATERIALS .................................................................................................................... 26

xi

3.3 METHODS ....................................................................................................................... 27

3.3.1 Water content determination ............................................... 27

3.3.2 Cyclodextrin solutions preparation .................................... 27

3.3.3 Deoxygenation of the CD solutions .................................... 28

3.3.4 Cyclodextrin-polymer solutions preparation ...................... 28

3.3.5 Preparation of the system of cyclodextrin, polymer and

lipids 29

3.3.6 Flocculation Test ................................................................. 29

3.3.7 Dispersion and smell ........................................................... 29

3.3.8 Surface tension .................................................................... 30

3.3.9 Viscosity .............................................................................. 30

3.3.10 Osmolality ........................................................................... 31

3.3.11 Particle size distribution ..................................................... 32

3.3.12 Peroxide value test .............................................................. 32

3.3.12.1 Lipid extraction. ........................................................................................................ 33

3.3.12.2 Titration..................................................................................................................... 34

3.3.12.3 Blending the solutions used in the titration ............................................................... 35

3.3.12.4 Statistics .................................................................................................................... 35

4. RESULTS .......................................................................................................................... 36

4.1 FORMULATIONS 1 - 29 .................................................................................................... 36

4.1.1 Water content of the CDs .................................................... 36

4.1.2 CD – lipid formulations ...................................................... 36

4.1.3 CD – polymer – lipid formulation ....................................... 37

4.1.4 Testing the formulations...................................................... 38

4.1.4.1 Flocculation test, redispersion and smell .................................................................. 38

4.1.4.2 Viscosity ................................................................................................................... 40

4.1.4.3 Surface tension .......................................................................................................... 41

4.1.4.4 Osmolality ................................................................................................................. 42

4.2 FORMULATIONS 30- 39 ................................................................................................... 42

4.2.1 CD – polymer – lipid formulation ....................................... 42

xii

4.2.2 Testing the formulations...................................................... 43

4.2.2.1 Flocculation test ........................................................................................................ 43

4.2.2.2 Surface tension .......................................................................................................... 43

4.2.2.3 Viscosity ................................................................................................................... 44

4.2.2.4 Osmolality ................................................................................................................. 44

4.3 FORMULATIONS 30 AND 33 ............................................................................................ 45

4.3.1 Particle size distribution ..................................................... 45

4.3.2 Peroxide value ..................................................................... 46

4.3.3 40-days flocculation ............................................................ 47

5. DISCUSSION .................................................................................................................... 48

5.1 CD-POLYMER-LIPID FORMULATIONS ............................................................................. 48

5.2 FORMULATIONS 6-29. ..................................................................................................... 48

5.3 FORMULATIONS 30-39. ................................................................................................... 51

5.4 FORMULATIONS 30 AND 33 ............................................................................................ 52

6. CONCLUSION ................................................................................................................. 54

7. ACKNOWLEDGEMENT ............................................................................................... 55

8. REFERENCES ................................................................................................................. 56

9. APPENDICES..................................................................................................................... B

9.1 APPENDIX A ........................................................................................................................ B

9.2 APPENDIX B ........................................................................................................................ C

9.3 APPENDIX C ....................................................................................................................... D

9.4APPENDIX D ......................................................................................................................... E

9.5 APPENDIX E ......................................................................................................................... F

9.6 APPENDIX F ........................................................................................................................ G

9.7 APPENDIX G ....................................................................................................................... H

9.8 APPENDIX H ....................................................................................................................... K

9.9 APPENDIX I ......................................................................................................................... N

xiii

TABLE OF TABLES

Table 1 A list of polymers used in combination with CD .............................................. 24

Table 2 The water content of the CDs used.................................................................... 36

Table 3 Results from the formulation of different cyclodextrins with cod liver oil and

free fatty acids ................................................................................................................ 36

Table 4 Concentrations and results from the CD – polymer – lipid formulations ......... 37

Table 5 Flocculation, redispersion and smell of the formulations ................................. 38

Table 6 Composition of re-prepared monophasic formulations ..................................... 42

xiv

TABLE OF FIGURES

Figure 1 Schematic view of the eye ................................................................................. 3

Figure 2 The cycle of dry eye ........................................................................................... 5

Figure 3 Schematic picture of the origin and fate of ω-3 and ω-6 FAs .......................... 12

Figure 4 Time dependence of Peroxide value, Anisidine Value and Totox numbers .... 15

Figure 5 Characteristics of the natural CDs.................................................................... 19

Figure 6 Illustration of how the particles were assessed. ............................................... 32

Figure 7 Stirring of the filtered polymer mud after filtering for 12 hours...................... 33

Figure 8 Appearance of formulations after being in the flocculation test for 8 days.. ... 39

Figure 9 Viscosity of the formulation in centipoises. ..................................................... 40

Figure 10 Surface tension of the formulations that were eligible for measurements ..... 41

Figure 11 Surface tension of the new formulations. ....................................................... 43

Figure 12 Viscosity of the new formulations in centipoises .......................................... 44

Figure 13 Osmolality, in milliosmoles/kg solute of the new formulations. ................... 45

Figure 14 Peroxide value of formulations 30, 33 and “pure cod liver oil plus free fatty

acids” at different conditions .......................................................................................... 46

Figure 15 40-days flocculation test ................................................................................ 47

2

1. INTRODUCTION

1.1 The eye

The eye is a complex organ that gives us the ability to see. Whether it is a simpler eye

of microorganism that can only distinguish between light and dark or a more complex

eye like that of hawk, it is usually paramount to life. As for human, although vision is

not a survival factor any more it still remains essential providing ca. 90% of all

information from surrounding environment.

1.1.1 Anatomy of the eye

The eye is one of five sensory organs of the human body and is responsible for vision.

The eyeball is about 2.5 cm in diameter with a slight projection in the front. The

eyeball sits protected in a bony hollow cavity where it is surrounded by fibrous tissue,

muscles and soft layer of fat. The lacrimal gland, which is located behind the upper

eyelid, produces tears that lubricate the eye with the help of the eyelid, as well as

nourishing and cleaning it off foreign substances (Harvard Medical School, 2010). As

well as helping to lubricate the eye, the eyelids protect the vulnerable ocular surface

from physical injury (Benitez-Del-Castillo, 2012). The normal mean tear volume is

6,5µl (Scherz, Doane, & Dohlman, 1974). The wall surrounding the eye is made up of

three distinct layers. The first layer, called the surface layer, is made up of tough

collagen. It can be seen in front of the eye as both the sclera and the cornea. The middle

layer, called the uveal tract, contains the iris, ciliary body and choroid. The iris can be

seen as a pigmented segment around the pupil. Essentially a circular muscle fiber, the

iris regulates how much light enters the eye. Depending on the brightness, the

involuntary muscles relax or stretch allowing more light into the eye when it is dusk or

less light when it is bright. The choroid membrane is crowded with blood vessels

carrying oxygen and other nutrients to the nearby outer portion of the retina. The

crystalline lens, located behind the pupils and iris, focuses light rays on the thin, light

sensitive retina which is referred to as the third layer. Muscles located in the ciliary

body enable the retina to alter its shape for focusing on objects at varying distances

(Harvard Medical School, 2010; Silverthorn, 2009). Located within one of the ten layers

3

of the retina (Herzlich A A., Patel M., Sauer T C., & Chan, 2010) are cones and rods,

specialized cells that, with the help of visual pigment molecules, enable us to see. Cones

are responsible for color vision and work best in relatively bright light. About 7 million

cones are located within each eye where they are densely packed in the fovea but

quickly reduce in numbers toward the periphery of the retina. Rods can function in less

light than cones and are mainly used in peripheral vision. About 150 million rods are

located within each eye where they are evenly distributed throughout the retina

(Harvard Medical School, 2010). The cornea consists of three membranes, the

epithelium which is in contact with the tears, the inner stroma and the endothelium. The

lipophilic layered epithelium acts as a barrier to ion transport. Tight junctions located at

the epithelium prevent the diffusion of large molecules via the paracellular route but

selectively allow some smaller molecules to be absorbed. The stroma is a highly

hydrophilic layer and makes up 90% of the cornea. The endothelium maintains corneal

hydration (Le Bourlais et al., 1998).

The ciliary epithelium generates

aqueous humor generally found

between the iris and the cornea

providing water-dissolved nutrients

to the lens and carrying waste

products away from the lens,

draining into the Schlemm’s canal

(Davies, 2000; Harvard Medical

School, 2010; Silverthorn, 2009).

The clear and gel-like vitreous

humor, located behind the lens,

supports and fills the rear two-thirds

of the eyeball with a volume of about

4ml in adults. Made up, almost

entirely, of water with glucose, hyaluronic acid, collagen fibers, inorganic salts and

ascorbic acid it serves as a pathway for light coming through the lens and maintains the

shape of the eyeball (Bochot & Fattal, 2012; Silverthorn, 2009). Figure 1 shows a

schematic diagram of the most important parts of the eye.

Figure 1 Schematic view of the eye

(Davies, 2000; Rhcastilhos, 2007)

4

1.1.2 The meibum

The tear film is a highly important layer between the eye surface from one side and

either environment or the eyelid from another side. The tear film is mostly aqueous in

nature, formed from secretions of the lacrimal glands. However, although minor in

quantity, the lipids secreted from the meibomian glands are crucial to its function. The

thickness of the tear film is approximately 10µm where approximately 99% of the

thickness is occupied by an aqueous layer containing inorganic salts, small molecular

weight organic substances and biopolymers. Over the aqueous tear film is a much

thinner film containing the meibomian lipids (Tiffany, 1985) sometimes referred to as

meibum. The meibum is squeezed out of the meibomian glands during the course of an

eye blink due to contractions of nearby muscles. During sleep and periods of reduced

blinking the meibum accumulates in the ducts of the glands and can be expressed in

quantity by forced blinking (Benitez-Del-Castillo, 2012; Perry, 2008). The meibum

consists of polar and nonpolar lipids. The polar lipids act as a surfactant to help spread

the nonpolar lipids over the aqueous part of the tear film. Healthy meibum is vital for

healthy ocular surface, as the lipids in the meibum help to spread and stabilize the tear

film, prevent the contamination of the tear film by sebum, help seal the apposed lid

margins during sleep, lubricate the eye during blinking and last but not least, slow down

the evaporation of the aqueous tear component (Goto, 2003; Tiffany, 1985). Production

of meibum is regulated by a number of endogenous substances, including androgens,

progestin, estrogen, corticotrophin-releasing hormone, substance P and the autonomic

nervous system (Perry, 2008). Goblet cells and conjunctival epithelial cells also aid in

the protection of the ocular surface by their ability to secrete mucin (Stern, Gao,

Siemasko, Beuerman, & Pflugfelder, 2004).

1.1.3 Dry eye

As we proceed into life we are more susceptible to various disorder of the body. With

increased age the appearance and function of the eye change. Eyelid muscles weaken

and the skin becomes thinner. Eyelashes and eyebrows become thinner and tear

production slows down. Meibum secretion decreases which could result in increased

evaporation of the tear film (Harvard Medical School, 2010).

5

The ocular surface is one of the most fragile ones on the human body, yet it is

challenged by many factors such as different air currents, low humidity, foreign bodies

and attacks by microorganisms. To cope with these challenges the ocular surface and its

individual components, especially the tear film, are in a highly dynamic state and share

feedback mechanisms which results in simultaneous reaction to cope with these

challenges. Tear production is essential for sustaining the health of the ocular surface as

they help to cleanse, lubricate and nourish the eye as well as providing physical

protection against infection (Rolando & Zierhut, 2001). Ocular surface disorders, dry

eye disease in particular, are the leading reason for visits to eye care professionals. The

cause of dry eye, sometimes referred to as keratoconjunctivitis sicca meaning “dry

inflammation of the cornea and conjunctiva” in Latin , can be diverse (Fig. 2) and can

rely upon both underlying conditions and external assaults (Baudouin, 2001). Not

shown on the picture is the hypothesis that some medication, and poly pharmacy in

particular, can be the cause of dry eye (Fraunfelder, Sciubba, & Mathers, 2012).

Compared to normal individuals with a mean tear volume of about 6,5µl, dry eye

patients had a moderate decrease or a mean of 4,8µl (Scherz et al., 1974).

Figure 2 The cycle of dry eye (Baudouin, 2001)

6

1.1.4 Etiology of dry eyes

Dry eye disease can be distinguished into two forms, aqueous or tear deficient dry eye

in which the primary etiology is reduction in the amount of tears produced and

evaporative dry eye in which tear production is sufficient but evaporation due to

deficiency of the lipid layer reduces the effectiveness of the tear film. Aqueous deficient

dry eye can further be classified to Sjögren’s syndrome dry eye, autoimmune disorder

affecting both the lacrimal and salivary glands, and non-Sjögren’s syndrome which

include other causes of tear deficiency (Baudouin, 2001). The usual cause of

evaporative dry eye is obstruction of the meibomian glands, resulting in deficiency of

the lipid layer that allows faster evaporation of moisture than the lacrimal glands can

compensate for, from the eye surface. This is referred to as meibomian gland

dysfunction (Foulks et al., 2012; Rolando & Zierhut, 2001). A lack of tear production or

reduced amount of tears due to increased evaporation exposes the ocular surface to the

risk of damage caused by environmental factors. Due to the desiccating environment, a

chronic inflammatory state arises at both the ocular surface and at the lacrimal glands.

This chronic inflammatory state leads to secretion of inflammatory cytokines from the

lacrimal gland and the ocular surface (Stern et al., 2004). Epithelial cells may

independently produce interleukin (IL)-1α, IL-6, IL-8 and tumor necrosis factor-α

(TNF-α), all of which are inflammatory cytokines or chemokine, and might participate

in or amplify immune-based inflammation (Baudouin, 2001; Stern et al., 2004). T-cells

are activated, resulting in the release of inflammatory mediators, causing further

inflammation and damage to the ocular surface. As the disease progresses, tear

production diminishes due to infiltration of lymphocytes which amplify the immune

response with the secretion of more inflammatory cytokines, impairment of the

conjunctival epithelium, dysfunction or destruction of the lacrimal glands and loss of a

reflex tear response to sensory nerve impulses (Stern et al., 2004). In addition, these

inflammatory mediators might inhibit neural signals of the lacrimal glands, depriving

the gland of the stimulation needed for its maintenance, progressing its destruction

(Zoukhri, Hodges, & Dartt, 1998).

7

1.1.5 Prevalence and treatment of dry eye

The prevalence of dry eye increases with age. Epidemiological studies have reported

more than 6% of the population over 40 to suffer from dry eye with the prevalence

increasing to 15% of the population over the age of 65 (McCarty, Bansal, Livingston,

Stanislavsky, & Taylor, 1998; Schein, Munoz, Tielsch, Bandeen-Roche, & West, 1997).

A recent online study conducted in the U-S-A by Allergan on 9034 individuals who

were listed on the Harris Interactive Online dry eye panel, found out that 48% of the

individuals regularly experienced dry eye symptoms (Patel, Watanabe, Strauss, &

Dubey, 2011).

The most common treatment of dry eye is the use of artificial tears or eye drops.

Traditional therapies for dry eye are palliative, their purpose is not to correct the

underlying disease but only replace or conserve the patient’s tears. Artificial tears have

diverse composition that may include cellulose ethers, carbomers, polyvinyl alcohol

(PVA), sodium hyaluronate, polivinil pyrrolidone (PVP) and a number of different

lipids. Applying artificial tears up to 4 times per day can successfully manage mild

cases of dry eye. In the case of moderate dry eye, applying unpreserved artificial tears

up to 12 times per day as well as unpreserved lubricating ointment at bedtime might

manage the symptoms. In the case of severe dry eye, additional therapy will be needed

such as tear-conserving therapies (Calonge, 2001). Preservatives in eye drops,

especially the most commonly used benzalkonium chloride (BAK), have been linked to

toxic effects in laboratory experiments and clinical studies. BAK has been shown to

cause tear film instability, loss of mucin producing goblet cells and disruption of the

corneal epithelium barrier (Harvard Medical School, 2010). In the same Allergen dry

eye study mentioned above, 63% stated that over-the-counter artificial tears are only

just or not at all successful in managing their symptoms (Patel et al., 2011).

1.1.6 Drug delivery to the eye

Drug delivery to the eye is a bothersome task due to various barriers. Topical

application in the form of eye drops is the most common method used to treat both the

outside of the eye, such as dry eyes, and to provide intraocular treatment with

absorption through the cornea, such as glaucoma (Gipson, 2004). Precorneal factors

8

such as short contact time of the drug (1-2 min) due to lacrimal fluid production

followed by drainage into either the nasolacrimal ducts or conjunctiva, and blinking,

induce a rapid elimination of the topically applied drug solution (Ahmed & Patton,

1985). The three layered cornea also limits the absorption with the epithelium limiting

the absorption of hydrophilic drugs and the stroma limiting the absorption of lipophilic

drugs. Mucins secreted to protect the ocular surface also forms a hydrophilic layer over

the tears (Le Bourlais et al., 1998). Conjunctival drug absorption into the eye is limited

due to rich blood flow and large surface area, which can cause a significant systemic

absorption (Ahmed & Patton, 1985). Blood-retinal barriers and blood-aqueous barriers

express tight junctions which limit drug penetration from the systemic bloodstream into

the intraocular environment (Barar, Javadzadeh, & Omidi, 2008). Systemic

administration of drugs will thus, in most cases, not be able to reach therapeutic levels

in the eye and orally administered drugs will not reach therapeutic levels in the eye

unless given in very high dose. These high doses could result in systemic side effects

(Gaudana, Ananthula, Parenky, & Mitra, 2010). Periocular and intravitreal

administration have become increasingly more common since they partly overcome the

inefficient drug delivery related to topical and systemic dosing to the posterior segment

of the eye. However these routes are not very patient compliant and may result in

tedious side effects (Gaudana et al., 2010).

1.1.7 Formulation standards for eye drops

Physiological conditions of the eye, physico-chemical properties of the drug and the eye

preparation formulation all have an impact on the effectiveness, tolerance and stability

of the eye drops. (Kråmer, 2002).

Viscosity is an important factor. If the eye drops are too liquid the contact time and

therefore bioavailability is too low. However, if they are too viscous they can reduce the

patients’ vision, and therefore are only suitable for application before night. A viscosity

of about 20 centipoises (cP) has been considered optimum viscosity for contact time,

where higher viscosity offers no advantage with respect to drug contact and usually

leaves a residue on the margin of the eyelid. Numerous polymers can be used to

increase the viscosity of solutions, for example methylcellulose, hydroxyethylcellulose,

9

hydroxypropylmethylcellulose, PVA and PVP (Kråmer, 2002). pH is an important

factor since it determines the rate of drug dissociation and penetration into the cornea as

well as bioavailability. Ideally, ophthalmic solutions should have the same pH as the

lacrimal fluid (7.4), but pH values from 7 to 9 are tolerated by the eye without marked

irritation. The buffer capacity of the lacrimal fluid (0.01 ml) should not be exceeded due

to increased tear production and eye movement, resulting in increased eye drop

clearance (Kråmer, 2002). The lacrimal fluid is isotonic (i.e. has the same tonicity) with

blood with 287 mOsm/l (Kråmer, 2002). Ideally, an ophthalmic solution should have

the same tonicity values as the lacrimal fluid but the eye can tolerate a rather broad

range of tonicity from ~205-683 mOsm/l (USP, 1995). It should be noted that this

information is relatively old and may have changed. The surface tension of the lacrimal

fluid ranges from 40 to 50 mN/m. Low surface tension provides good spreading effect

on the cornea possibly improving the contact between the drug and corneal epithelium

(Ammar, Salama, Ghorab, & Mahmoud, 2009). The particle size distribution in eye

drops has to meet defined standards according to the European Pharmacopeia. No more

than 20 particles may have a maximum dimension greater than 25 µm, not more than

two of these may have a maximum dimension greater than 50 µm and none above 90

µm (Council of Europe, 2013).

10

1.2 Lipids

Lipids are biomolecules made up of hydrogen, carbon and oxygen and defined as fatty

acids (FA) and their derivatives. They can be characterized by the fact that they are

nonpolar and consequently poorly soluble in water. Lipids are technically called fats if

they are solid at room temperature and oils if they are liquid at room temperature

(Silverthorn, 2009). Lipids have diverse function in the human body. While most of

them end up serving as a source of energy in the form of triglycerides, some of the

lipids represent as FA, fat-soluble vitamins or are even converted into prostaglandins

(PGE) or steroids (Harvey, 2011).

1.2.1 Fatty acids

FAs are carboxylic acids possessing a massive hydrocarbon skeleton. The carboxyl

group is known to be ionized at physiological pH.

FAs are called saturated if there are no double bonds between carbons,

monounsaturated if there is one double bond and polyunsaturated (PUFA) if there are

two or more double bonds. For each double bond, the molecule has two fewer hydrogen

atoms attached to the carbon chain. The more saturated the FA is, the more likely it is to

be a solid at room temperature (Silverthorn, 2009). The double bounds of PUFAs are

rarely conjugated and almost always in the cis-configuration. The cis-configuration

restricts rotation and introduces a rigid bend in the hydrocarbon chain, resulting in

interference with the tight packing in organic membranes (Lehninger, Nelson, & Cox,

2008). There are two ways of naming FAs. The standard one designates the carboxyl

carbon the number 1. The number of carbon atoms is written followed by the number of

double bonds, denoted X:Y. The position of double bonds is designated with delta (∆)

followed by a superscript of the lower numbered carbon in the double bond. Linoleic

acid (LA), for example, is 18:2(∆9,12

). The other one, which is only used when naming

PUFAs, assigns the methyl carbon the number 1 as well as omega (ω). The position(s)

of the double bond(s) are indicated relative to the ω carbon. Therefore, a PUFA

containing a double bond between carbons 3-4 is referred to as ω-3 FA (Lehninger et

al., 2008).

11

1.2.2 Essential Fatty Acids

Humans require certain FAs, namely ω-3 and ω-6, but do not have the enzymatic

capacity to synthesize them, thus they have to be obtained from the diet. α-linolenic acid

(ALA 18:3(∆9,12,15

)), an ω-3 FA, is a precursor for eicosapentaenoic acid (EPA

20:5(∆5,8,11,14,17

)) and docosahexaenoic acid (DHA 22:6(∆4,7,10,13,16,19

) (Lehninger et al.,

2008) whereas LA, an ω-6 acid, is the precursor for, most notably, arachidonic acid

(ARA 20:4(∆5,8,11,14

)) (Macsai, 2008). The conversion from the shorter parent FAs to

the longer downstream FAs involve a series of elongation enzymes that add two carbon

units to the backbone and desaturation enzymes that insert double bonds into the

molecule (Arterburn, Hall, & Oken, 2006).

Increased dietary intake of ω-3 PUFAs results in increased incorporation of those FAs

into inflammatory cell phospholipids. The incorporation occurs in a dose-respondent

fashion and is partly at the expense of ARA. ARA acts as a substrate for

cyclooxygenase and 5-lipoxygenase in the synthesis of thromboxanes, prostaglandins

and leukotrienes, collectively known as eicosanoids. Eicosanoids are known to be

involved in modulating the intensity and duration of inflammatory response. The ω-3

PUFA EPA competes with ARA as a substrate for cyclooxygenase and 5-lipoxygenase,

decreasing the production of pro-inflammatory prostaglandins and leukotrienes (Calder,

2006; Funk, 2001; Surette, 2008). ω-3 PUFAs have also shown to be effective at

reducing the generation of TNF-α, IL-6, IL-8 and expression of various adhesion

molecules through decreased activation of nuclear factor κ-light-chain-enhancer of

activated B cells, sometimes only referred to NFκB (Calder, 2006). Additionally, ω-3

PUFAs especially DHA have been linked to the production of resolvins, anti-

inflammatory mediators that moderate the time course and magnitude of inflammatory

processes (Fig. 3). It should be noted however that the precursor ALA does not appear

to exert anti-inflammatory effect at accomplishable intakes (Calder, 2006; Rand &

Asbell, 2011).

12

Figure 3 Schematic picture of the origin and fate of ω-3 and ω-6 FAs. Resolvins are also derived from EPA

(Roncone, Bartlett, & Eperjesi, 2010).

It has been demonstrated that the high ratio of ω-6 to ω-3 in the diet leads to

overproduction of pro-inflammatory PGE2 and underproduction of PGE1 and PGE3

(Calder, 2003). The optimal dietary ratio should be between 1:1 and 4:1, seen in the

Mediterranean diet rich in cold-water fish and natural oils. The typical dietary ratio of

Americans and Northern Europeans is from 10:1 and 30:1 (Lehninger et al., 2008;

Macsai, 2008). This high ratio in the Western diet has been blamed by some to be the

cause of the high prevalence of cardiovascular diseases, autoimmune diseases and

cancer. Dietary intake of the ω-3 FAs may shift the body to more anti-inflammatory

state resulting in decreased prevalence of these diseases (Simopoulos, 1991).

Food rich in ω-3 FAs include oils from certain plants such as flaxseed or canola oil, in

fish such as halibut, herring, mackerel, salmon and tuna, and fish oils such as cod liver

oil. The composition of ω-3 FAs is different between plant oils and fish where plant oils

mostly contain ALA but fish and fish oil a bigger proportion of EPA and DHA. Food

rich in ω-6 include grains, meat and the seeds of most plants. ALA and LA compete for

the same enzymes to be converted into the longer chained EPA and ARA. Because of

this competition, and the fact that the ratio between ALA and ARA is undesirable,

conversion of ALA to EPA (and then to DHA) is low, with exact figures in the range of

1-15% (Covington, 2004; Emken, Adlof, & Gulley, 1994). Since ALA does not appear

13

to exert any anti-inflammatory effects and the rate of conversion is low, consumption of

fish or fish oils is a better source of EPA and DHA than plant oils.

1.2.3 ω-3 and diseases

The effects of ω-3 supplementation on both meibomian gland dysfunction and/or

evaporative dry eye have been carried out although only one was found. In that study

patients were randomly assigned to either the placebo group, which received olive oil,

or the study group, which received 6 grams (g) of flaxseed oil. This is equivalent to 3.3

g of ω-3 FAs since only 55% of the oil is ω-3. The study was carried out over the course

of 1 year to minimize seasonal changes. 30 individuals reached the primary endpoint, 7

were lost to follow up and 1 was removed from the study due to the diagnosis of

Sjögren’s syndrome. At the end of one year, improvements in dry eye symptoms and

overall ocular health were observed. The flaxseed group had a decrease in saturated FAs

in the meibum and significant improvement in the Ocular Surface Disease Index

compared to the placebo group. (Macsai, 2008). Perhaps if fish oil, which has high

levels of EPA and DHA, would have been used instead of flaxseed oil, which has low

levels of EPA and DHA, better results could have been obtained. To maximize patient

compliance, flaxseed oil was used instead of fish oil which sometimes has a “fishy”

aftertaste.

The Woman’s Health Study is a randomized, double blind, placebo controlled trial

examining the use of low-dose aspirin and vitamin E for primary prevention of

cardiovascular disease and cancer. At their 4 year follow up questionnaire they were

asked if they had been diagnosed by a clinician to have a dry eye, where about 4.7% of

the study population had. The authors discovered that woman with higher ω-3 FA intake

had decreased risk of dry eye as compared to those who had lower intakes, and the ω-6

to ω-3 ratio was associated with significantly greater risk for dry eye (Miljanovic et al.,

2005). The effects of topically applied ALA (ω-3), LA (ω-6) and an ALA/LA

combination on a murine model have been studied. For control they used the vehicle or

a placebo. The total daily dosage was 1µl of 0.2% concentration in a solution with

compatible surfactants given for 10 days. Corneal fluorescein staining (CFS) was

evaluated, the number and maturation of CD11b, a trans membrane protein expressed in

inflammation found on numerous cells that are part of the immune system, were

14

determined both at the center of the eye and the periphery as an indicator of

inflammation, and a real time polymerase chain reaction was used to quantify the

expression of various inflammatory cytokines at days 5 and 10. Treatment with ALA

alone resulted in a significant decrease in CFS compared with the vehicle and untreated

control. CFS stains dead or degenerated corneal epithelial cells and therefore is a good

indicator of corneal epithelial damage. The number of CD11b+ cells in the center of the

eye was found to be significantly decreased in the ALA group compared to all other

groups. ALA treatment also decreased corneal IL-1α and TNF-α and conjunctival TNF-

α. The authors speculate why the combined treatment with ALA and LA had no positive

effect, neither clinically nor cellularly, and wonder if the ratio was not high enough to

offset the pro-inflammatory status already present in the eye (Rashid, 2008)

ω-3 FAs may also be important in preventing or treating a number of cardiovascular

diseases (Kris-Etherton, Harris, & Appel, 2002; Lecerf, 2009; Riediger, Othman, Suh,

& Moghadasian, 2009), asthma (Villani, Comazzi, De Maria, & Galimberti, 1998),

rheumatoid arthritis (Calder & Zurier, 2001; Cleland, James, & Proudman, 2003; James

& Cleland, 1997) and depression (Freeman et al., 2006). The motivation on the research

into the ω-3 FAs has resulted in the approval of Omacor® used to lower very high

triglyceride levels. The drug contains both ethyl esters of EPA and DHA (FDA, 2004).

1.2.4 Fish oil

Fish oil, produced either from the meat of the fish or its liver, is an important source of

EPA and DHA and other long-chain ω-3 PUFAs. Fish oils are characterized by their

high degree of unsaturation, the long-chain ω-3 type PUFAs and the great number and

variety of FAs present in the triacylglycerols (Haraldsson & Hjaltason, 2001). More

than 50 different FAs are present in a typical fish oil which include C14-C24, saturated,

monounsaturated, polyunsaturated, ω-3, ω-6, branched, odd-numbered and so forth. The

origin of the important ω-3 PUFAs is in the lipids of photosynthetic microalgae which

are eaten by the fish (Sargent, McEvoy, & Bell, 1997). FAs in the form of

triacylglycerol are in most abundance in the fish oil with small amounts of mono- and

diacyl-glycerol and other minor nontriacylglycerol substances. Refinement of the oil

aims at reducing these small and minor amounts since they may influence the flavor and

odor qualities of the oil as well as its stability (Haraldsson & Hjaltason, 2001). When

15

producing fish oil from fish meat or fish liver, a variety of impurities can be found.

Protein, dirt and rust are considered insoluble impurities and tend to precipitate out of

the oil during storage which can affect the stability of the oil. Saponification in the oil

removes moisture, which could lead to deterioration in storage. Unsaponifiable

materials include free cholesterol and vitamins A and D. While the vitamins are usually

not removed, free cholesterol is removed by vacuum stripping of the oil. Heavy metals

are removed by refining, trace metals by degumming and refining and chlorinated

hydrocarbons must remain within regulatory limits (Haraldsson & Hjaltason, 2001). A

number of analyses indicate the

quality of commercial fish oil.

First of all, dark colored oils

indicate the oil might contain

impurities or have been

overheated during refining. Acid

values, also known as

neutralization number or acid

number, indicate the quantity of

carboxylic acid groups in a

chemical compound, in this case free FAs. High acid values indicate poor quality of the

oil. Peroxide value (PV) reflects recent oxidation and anisidine value (AV) reflects

oxidation that has taken place in the past. However, these two values do not represent

overall rancidity; PVs follow an (inverse) parabola curve and AVs are delinquent to rise

until considerable oxidation has occurred, see Figure 4. Totox value expresses a

relationship between the PV and AV (Haraldsson & Hjaltason, 2001) (PV × 2 + AV)

and is used more commonly for indicating overall oxidation of the oil (Miller, 2012).

The double bonds in unsaturated fats play a role in autoxidation, where PUFAs are more

susceptible to oxidation than monounsaturated FAs. The autoxidation occurs

preferentially adjacent to a double bond in unsaturated FAs (Simic, 1981) and is

initiated by hydrogen abstraction from allylic or bis-allylic position, leading to

oxygenation and the formation of peroxyl radicals. In the presence of more PUFAs, the

peroxyl radical abstracts hydrogen to generate PUFA hydroperoxides which are prone

to further transformations by free radical routes (Gardner, 1989). Crude fish oil always

Figure 4 Time dependence of Peroxide value (PV), Anisidine Value

(AV) and Totox numbers (Miller, 2012)

16

contains some natural antioxidant such as vitamin E and astaxanthin. During processing

these natural antioxidants are removed which results in less stability due to oxidation.

Thus, antioxidants are usually added to the oil after processing where blends of various

forms of tocopherols are commonly used. Lipid oxidation and rancidity is usually

caused by light, heat, oxygen or enzymatic activity (Haraldsson & Hjaltason, 2001).

1.2.5 Antibacterial and antiviral effects of PUFAs

The antibacterial effects of PUFAs longer than 15 carbons in length have been reported.

The antibacterial effects are primarily effective against Gram-positive bacteria, but

activity against Gram-negative bacteria has also been reported (Kenny et al., 2009;

Shin, Bajpai, Kim, & Kang, 2007). The exact anti-bacterial mechanism has not been

determined but numerous possible mechanisms have been proposed. They include for

example specific inhibition of FabI, a catalyzer in the final and rate limiting step in FA

biosynthesis, inhibition of glucosyltransferases, interference with energy metabolism or

that the PUFAs inhibit all major bacterial biosynthetic pathways (Kenny et al., 2009).

The degree of unsaturation and oxidation has been directly linked to the antibacterial

effects of PUFAs (Rybin et al., 2000). The antiviral effects of free FAs have been

reported. In a study comparing the antiviral effects of PUFA’s, a 1% FA extract from

cod liver oil resulted in a 4.7 log reduction in HSV-1 concentration (Loftsson et al.,

1998). Another study, comparing the monounsaturated FAs oleic- and palmitoleic-acid,

as well as monoglycerides, medium and long chained monounsaturated FAs and fatty

alcohols, showed a significant reduction (2-4.5 log scale) in herpes simplex virus (HSV)

type 1 and 2 compared to control groups (Hilmarsson, Kristmundsdóttir, & Thormar,

2005).

17

1.3 Cyclodextrins

Cyclodextrins (CD) are manufactured by bacterial fermentation of starch followed by

product purification. First believed to be discovered in 1891 by a French scientist

named A. Villiers, the different CDs were not isolated until years later. The isolation

step was tiresome which resulted in high prices. With the biotechnological advances in

the early 1970s came new ways to produce CD and high-grade CDs were available at

affordable prices (Loftsson & Brewster, 2010).

1.3.1 Structure of cyclodextrins

Cyclodextrins are cyclic oligosaccharides consisting of six (α-CD), seven (β-CD), eight

(γ-CD) or more D-glucopyranose units linked with α-(1,4) bonds. CDs consisting of

more than eight glucopyranose units are of relatively little importance in pharmaceutical

industry and will not be discussed here. Due to chair structure of the glucopyranose

units the CD molecule is shaped like a truncated cone with the primary hydroxyl (-OH)

groups extending from the narrow edge and the secondary hydroxyl groups from the

wider edge. The hydroxyl groups extending from the edges of the molecule give the CD

a hydrophilic outer surface while the inner cavity, lined with carbons and ethereal

oxygen of the glucose residue, is rather lipophilic. However, due to high crystal lattice

energy and intra-molecular hydrogen bonding between C-2 and C-3 hydroxyl groups,

the aqueous solubility of parent CDs and their complexes is limited, especially for βCD

(Loftsson & Brewster, 1996; Jozsef Szejtli, 1989; J. Szejtli, 1998). The low solubility

has been overcome by creating CD derivatives, which are of pharmaceutical interest and

include hydroxypropylated-βCD and -γCD (HPβCD and HPγCD), randomly

methylated-βCD (RMβCD) and sulfobutyl ether βCD sodium salt (SBEβCD) (Loftsson

& Brewster, 2011). The physicochemical properties of the derivatives depend on the

structure, location and number of the substituents (Loftsson & Brewster, 2010). The CD

derivatives also have different hydrophobic cavity volume compared to the parent

molecules (Del Valle, 2004).

Natural CDs are more resistant towards starch hydrolyzing enzymes and non-enzymatic

hydrolysis than the linear oligosaccharides. In aqueous solutions, non-enzymatic

hydrolysis of the α-acetal linkages produces glucose maltose and linear

18

oligosaccharides. The derivatives are degraded at similar speed with ring opening the

dominant pathway. CDs are slowly hydrolyzed by α-amylase found in human saliva and

pancreatic juice. The rate depends on ring size and fraction of free CD, with bigger

rings being more susceptible to hydrolysis. After ingestion, α- and β-CD are digested by

bacteria in the colon whereas γ-CD is almost completely digested in the digestive tract

(Loftsson & Brewster, 2010)

1.3.2 Cyclodextrin complexation

The lipophilic microenvironment in the cavity of CD gives it the ability to form

inclusion complex with lipophilic nonpolar structure or substructures of a guest

molecule. The ability of CD to form an inclusion complex is determined by two factors.

The first one is steric and relates to relative size of the CD molecule and that of the

guest molecule or a substructure of it. As can be seen in Figure 5, height of the three

parent CD molecules is identical but the number of glucose units determines the internal

diameter and thus its volume. Based on the dimensions, α-CD can normally complex

low molecular weight molecules or compounds with aliphatic side chains, β-CD can

typically complex aromatics and heterocycles while γ-CD can form stable complexes

with larger molecules such as steroids or macrocycles. The second one relates to

thermodynamic interactions. For a complex to form a net driving force must be able to

pull the guest molecule into the CD cavity. These driving forces relate to both the CD

and the guest as well as the solvent (Del Valle, 2004). During complex formation no

covalent bonds are formed or broken and complexes are easily dissociated in aqueous

solution since equilibrium exists between free guest molecules and the ones bound

within the CD cavity (Loftsson & Brewster, 1996). Water molecules located within the

CD cavity do not satisfy their hydrogen-bonding potential and have higher enthalpy

than bulk water molecules in the aqueous environment. The main driving force in

complex formation was believed to be the replacement of these high enthalpy water

molecules with more hydrophobic guest molecules (Loftsson & Brewster, 1996; J.

Szejtli, 1998). This replacement attains apolar-apolar association between CD and the

guest molecule, and decreases the ring strain of the CD molecule resulting in a more

stable, lower energy state (J. Szejtli, 1998). However, recently it was reported that the

replacement of high enthalpy water molecules with guest molecules is not the driving

19

force for CD complexation, but forces such as van der Waals interaction, hydrogen

bonding, hydrophobic and charge-transfer interactions were to be thanked (Liu & Guo,

2002). Whatever the main driving force is, it appears that multiple driving forces are

behind CD complexation which may be exerted simultaneously (Brewster & Loftsson,

2007).

Linear oligosaccharides and polysaccharides are able to form complexes with lipophilic

molecules by intermolecular hydrogen bonding between hydroxyl groups. Similarly,

CDs can form non-inclusion complexes where the guest molecule is not localized in the

inner cavity but bound with hydrogen bonds to the CD’s exterior (Loftsson, Vogensen,

Desbos, & Jansook, 2008). Most frequently, one CD molecule entraps one guest

molecule within its cavity creating a 1:1 guest/CD complex. More complicated

guest/CD complexes do exist such as 2:1, when two or even more guests of small size

may fit into the cavity or, 1:2, when two or more CD molecules may bind to bulky

molecules (J. Szejtli, 1998).

Figure 5 Characteristics of the natural CDs (Loftsson & Brewster, 2010)

20

1.3.3 Cyclodextrin toxicology

Lipinski’s rule of five states that any molecule with less than 5 hydrogen bond donors,

less than 10 hydrogen bond acceptors, a molecular mass over 500 and an octanol-water

partition coefficient lower than 5 is not readily absorbed (Lipinski, Lombardo, Dominy,

& Feeney, 2001). CDs violate three of these criteria: they contain a significant number

of both hydrogen bond acceptors and donors and have molecular weight in the range of

973-2163 Dalton. Thus their oral bioavailability is generally below 4%. CDs that are

absorbed intact are furthermore rapidly excreted in the urine (Loftsson & Brewster,

2010). Toxicological studies have demonstrated that orally administered CDs are

practically non-toxic due to the fact that they are unable to permeate lipophilic

membranes such as gastrointestinal mucosa and skin, with the exception of RMβCD

which has higher bioavailability due to increased lipophilicity (Del Valle, 2004;

Loftsson & Brewster, 2010). All of the CDs and CD derivatives except βCD and

RMβCD can be used in parenteral formulations but only two, HPβCD and SBEβCD, are

approved by the Food and Drug Administration for intravenous injection (Stella & He,

2008). βCD and RMβCD have low aqueous solubility and adverse effects (Loftsson &

Brewster, 2010). When tested in animals, γ-CD was found to be virtually nontoxic when

given intravenously (Loftsson & Duchêne, 2007).

1.3.3.1 Ophthalmic toxicology

Loftsson proposed three possible mechanism in which CDs could cause irritation or

damage to the ocular surface after topical application: First, it is possible that small

fraction of the more lipophilic CDs may penetrate into the cornea, conjunctiva, sclera or

other eye surface tissue. However, the more hydrophilic CDs, such as HPβCD, have

been found to be non-irritating. Secondly, the CDs may be able to extract components

from the cornea or other ocular membranes. Nevertheless, after a complex formation

between the CD and the guest molecule, their ability to interact with biological

membranes is greatly reduced and usually only seen in vivo at relatively high

concentrations. At last, aqueous eye drops containing large proportion of CD (12-25%)

given to dry eye patient results in the formation of crust in the eyelids with consequent

irritation. When given to patients with normal tear production no irritation was

21

observed. HPβCD, the most commonly used CD in ophthalmic drug delivery, has been

shown to be well tolerated at high concentrations (Loftsson. & Jarvinen, 1999).

1.3.4 Cyclodextrins in ophthalmic drug delivery

The fact that higher concentration of free CD in solution leads to a lower flux over

biological membranes and increased concentration of CDs does not increase the

absorption of water soluble drug molecules in solution has led to the conclusion that the

main mechanism of enhanced transcorneal drug delivery is not the disruption of cell

membrane. In eye drop solutions, CDs act as true carriers by hiding the hydrophobic

substructure of the guest molecule inside their cavity from the aqueous environment.

CDs carry the guest molecule through the aqueous-mucin layer to the surface of the

ocular barrier (the cornea or conjunctiva), where the guest molecule can partition into

the lipophilic membrane (Loftsson. & Jarvinen, 1999). Irritation is a common drawback

of many commercially sold ophthalmic eye drops due to high drug concentrations or

irritating additives. By forming inclusion complexes with the irritating guest molecules,

CDs might be able to mask these irritating characteristics (Loftsson. & Jarvinen, 1999).

Like mentioned before, drug delivery to the eye with eye drop solutions is the most

common and preferred route. Usually, eye drop solutions contain drug molecules

dissolved in water. In aqueous environment, drug molecules are subject to chemical

degradation which reduces potency and formation of possibly harmful degradation

products. Stability can be increased with adjustment of pH and optimization of storage

conditions. Additionally, CDs can be used to increase stability in aqueous eye drop

solutions. With inclusion of the labile drug substructure, the CDs can shield it from

reactive molecules and thus decrease the rate of hydrolysis, oxidation, steric re-

arrangement, racemization and, possibly, enzymatic degradation (Loftsson. & Jarvinen,

1999).

1.3.5 Cyclodextrins and lipids

Lipids are extremely sensitive to oxidation and have a very limited solubility in water -

factors that limit their uses considerably. Antioxidants are well known agents that can

be used to slow down this oxidation. CDs are another agent that has gained considerable

attention to slow down the oxidation by forming inclusion complexes. The

22

characteristic of CD complex depends on both the nature of the CDs (α-, β-, γ-CD or a

derivative) and that of the FA (number of double bonds and chain length). When testing

the oxidative protecting effects of CD, an experiment carried out in 2011 showed a

significant difference when comparing the oxidation of conjugated linolenic acid (CLA)

to CLA/β-CD complex. With no antioxidants present, about 97% of the CLA was

oxidized after 40 minutes compared to less than 1% in the CLA/β-CD complex. And

still after 250 hours, about 35% of the CLA still remained (Ying, Ming-Li, Yan-Hua, &

Hua-Jie, 2011). When comparing different CDs, a study in 2000 showed that

microencapsulation of CLA with CDs completely protected the FA against oxidation.

The protective oxidative effects were in the order α-CD > β-CD > γ-CD (Kim et al.,

2000). In concordance with that, a study in 2002 came to the conclusion that the

oxidative protecting effects of CDs were inversely proportional to their size (Park et al.,

2002). On the contrary, a study in 1993 showed no significant differences between the

stabilizing effects of α- and β-CD when studying the oxidation of LA/CD complex

(Szente, Szejtli, Szemán, & Kató, 1993). Inclusion of FAs into CDs increases their

water solubility. The increase is proportionally higher for longer FAs (C12) than for

those who are shorter (C6), even though their water solubility is still very low compared

to their shorter comrades. When forming inclusion complexes with FAs, it has been

shown that the CD with the narrowest cavity, α-CD, has the highest affinity for both

short (≤C8) and long (≥C12) chain FAs. This can be explained by the fact that a shorter

distance between atoms of host and guest molecules results in stronger interaction.

However, this affinity does not translate directly to an increase in solubility because β-

CD has been shown to be superior in increasing water solubility of C10-C11 FAs and at

least equally successful to α-CD for C12 FAs. Supposedly, this can be attributed to the

fact that the C10-C11 FAs are slightly twisted inside the β-CD cavity leading to better

molecular interactions. Due to the narrow cavity in α-CD there isn’t enough room for

that twist. Starting with C12, part of the chain is outside the cavity for both α- and βCD.

Unfortunately, the authors did not include γ-CD to their review (Duchêne, Bochot, Yu,

Pépin, & Seiller, 2003). For fatty acids C16-C18 in length, γ-CD was superior to the other

two parent CDs in complexation and provided the best stabilization against auto-

oxidation (Regiert, Wimmer, & Moldenhauer, 1996). The solubility enhancement data

of several C18-C22 FAs with methyl and hydroxypropyl βCDs derivatives showed that

23

increasing number of double bonds within a FA molecule results in a more stable

inclusion complex. This might be due to the more compact geometry and non-linear

structure of the FA which provides a better fitting into the CD cavity (Szente et al.,

1993).

The effect of pH of the solution and temperature has been tested. The equilibrium

constant between free and complexed FA (K1) was used to assess the effects, defined as

K1

where [FA-CD] is the concentration of the fatty acid-cyclodextrin complex, [FA]f is the

concentration of free fatty acid and [CD]f is the concentration of cyclodextrin. The

effects of pH on K1 were obvious. When the pH was increased from around 7.5 to 9.5

the equilibrium constant K1 decreased from a steady 11,000 M-1

to 1,000M-1

. The

authors conclude that this is due to titration of the FAs carboxy group. As it turns out,

the mean value of K1 is very close to the pKa 7.9 of the LA. A likely explanation is that

the protonated carboxyl group of the FA forms hydrogen bond with one of the

hydrophilic groups of the CD at pH below the pKa. Based on nuclear magnetic

resonance imaging, it is probably the hydroxyl group at position 6. Increase of

temperature resulted in an increase in K1, which is normally not seen due to the fact that

hydrogen bonds are usually weakened by heat. A likely explanation is that higher

temperature resulted in an increase in protonated species rather than stronger hydrogen

bonds (Lopez-Nicolas, Bru, Sanchez-Ferrer, & Garcia-Carmona, 1995)

1.4 Polymers

Polymers are additives used widely in pharmaceutical systems for numerous objectives,

i.e. as suspending, emulsifying and flocculating agents, adhesives, and for packaging

and coating materials. Polymers are made up of repeating monomer units with a high

molecular weight. Their chemical reactivity depends on the chemistry of their monomer

units, but their properties depend mainly on how the monomers are assembled together.

Nearly all polymers exist with a range of molecular weight and for convenience the

reported molecular weight of a polymer is the average molecular weight. Water soluble

polymers have the capability to increase the viscosity of solvents at low concentrations,

24

to swell in solutions and sometimes even to adsorb at surfaces (Florence & Attwood,

2003). However they are primarily used to stabilize the system they are intended to be

used in. A list of the polymers used in this project in combination with CD can be found

in Table 1.

Table 1 A list of polymers used in combination with CD. CMC-Na = Carboxymethylcellulose Sodium, HPMC = Hydroxypropylmethylcellulose, PVA – Polyvinyl Alcohol, PVP

= Polyvinyl Pyrrolidone (Aldrich, 2012, 2013; BASF, 2010; LMS, 2006; LubrizolCorporation, 2013; ScienceLab, 2005)

Polymer Monomer Structure

Poloxamer 407

Ethylene oxide and propylene oxide

Carbomer 974 P Acrylic Acid

CMC-Na Cellulose-O-CH2-COONa

HPMC C6O5-R3

PVA CH2CHOH

PVP C6H9NO

25

2. AIM OF THE STUDY

The aim of this study is to formulate stable, monophasic aqueous eye drops from CD,

lipids, and polymer. The characteristics relevant to eye drops are assessed and the

stability as well as protective effects of these formulations are evaluated.

26

3. EQUIPMENTS, MATERIALS AND METHODS

3.1 Equipments

Equipment Model Manufacturer

Moisture analyzer MX50 A&D

Autoclave - Astell

Light microscope BH2 Olympus

Water purification system Q Gard Millipore

Purelab Option - Elga

Rotavapor REII Buchi

Scale AG 285 & PJJ60 Mettler Toledo

Shaker GmbH KS-15 kontrol Edmund Bühler

Sonicator 8892 Cole-Parmer

Vapor Pressure Osmometer K7000 Knauer

Viscosity meter DV1 Prime Brookfield

Water bath Polystat Cole-Parmer

3.2 Materials

Chemical Batch number Manufacturer

Acetic acid - isooctane solution* - Lýsi

-cyclodextrin - Cavamax® W6 Pharma 60P304 Wacker Chemie AG

Bensalkonium Chloride S32836-516 Sigma Aldrich

β-cyclodextrin - Cavamax® W7 Pharma 70P093 Wacker Chemie AG

Carbopol 974 CC61NAB896 Noveon

Carboxymethylcellulose-Sodium 6356A ICN

Chloroform SZBC073MV Sigma Aldrich

Cod liver oil PC0079102 Lýsi

Cod liver oil CPC2121202 Lýsi

Distilled water - Elga

Free fatty acids from hydrolysis 15.1.2010 Lýsi

γ-cyclodextrin Cavamax® W8 Pharma 80P241 Wacker Chemie AG

Hydroxypropylmethylcellulose 87F0148 Sigma Aldrich

Lutrol F-127 47-0646 BASF

Methanol SZBC1903V Sigma Aldrich

Nitrogen 1066 Ísaga

Poly(vinyl)alcohol 124K0052 Sigma

Polyvinylpyrrolidone 65H0040 Sigma

Potassium Iodide 50620 Riedel De Haën Potassium Iodide SZBC0500V Sigma Aldrich Purified water - Millipore

Sodium Chloride 80650 Sigma Aldrich

27

Sodium Thiosulphate solution* - Lýsi

Starch solution* - Lýsi

Titriplex III (EDTA) 630 KD2158618

Merck

*Solutions were donated by Lýsi hf.

3.3 Methods

3.3.1 Water content determination

MX-50 moisture analyzer was used to measure the water content of the parent CDs.

About 1 gram of CD was spread evenly onto the plate to ensure reproducible results, the

analyzer was closed and the halogen lamp turned on at a pre-defined temperature of

130°C to ensure water evaporation. The analyzer simultaneously computes the weight

of the water that evaporates and expresses it in percentages of the original weight. The

measurement automatically stops when the change is less than 0.1% per minute. Water

content of each CD was performed at least in triplicate

3.3.2 Cyclodextrin solutions preparation

Earlier research performed at the Faculty of Pharmaceutical Sciences, University of

Iceland suggests that the parent CDs, compared to their derivatives, are more suitable

for forming inclusion complexes with the lipids. To get a, for example, 10% w/v

solution, one must take into account the water content of the CDs as well as the

proportion of CD and lipids. The proportion that has shown the best result is 9:1

CD:lipid (Geirsson, 2008). For a 50 ml α-CD solution with an original water content of

9.8%, the amount in grams (g) is calculated

Amount of α-CD =

( ) (

) = 6.16 g

The amount of CD was then weighed exactly and dissolved in water. To help with the

dissolving process, the CD solutions were put into a sonicator until fully dissolved.

28

3.3.3 Deoxygenation of the CD solutions

To prevent the oxidation of both the cod liver oil and the free fatty acids, the aqueous

CD solution was deoxygenated. This was done by allowing nitrogen to bubble through

air steel stone that distributes the nitrogen out into the CD solution for at least 2 hours

and shielding it against further contact with oxygen using parafilm. This was done

before adding the polymers into the solution because deoxygenating each solution

independently would have been too time consuming and resulted in the formation of

considerable amount of foam that could have overflowed the glassware (Hákonarson,

2009). This overflow could have resulted in the loss of polymers as well as CD from the

solutions.

3.3.4 Cyclodextrin-polymer solutions preparation

The water soluble polymers (and preservatives in some cases) were weighed and placed

into a 50 ml volumetric flask. One to two different concentrations of each polymer were

used, both with and without preservatives. This was done to see the effect of the

preservatives on the formulation. For example, in the case of 50 ml of 2.5% w/v Lutrol

solution, the amount of polymer in grams is calculated

Amount of polymer =

(

)

= 1.39 g

Due to Lutrols physicochemical properties (Kojarunchitt, Hook, Rizwan, Rades, &

Baldursdottir, 2011), the polymer and the CD solution were refrigerated before

merging. This was done to solubilize Lutrol. Blending of the other polymers was

executed by simply mixing the CD solution with the polymers at room temperature.

Before the CD solution was added to the polymers, nitrogen was sprayed into the

volumetric flask to get rid of the oxygen. Then the flasks were put on the shaker for one

day to let the polymers solubilize in the CD solution. Each flask was filled to 80% of

the final volume because some of the polymers swell during solubilization and the lipids

still remained to be combined with the solution.

29

3.3.5 Preparation of the system of cyclodextrin, polymer and lipids

When the CD-polymer solutions were fully solubilized, lipids from the cod oil were

ready to be mixed into the solutions. The free fatty acids were kept in the freezer to

protect them from heat and light and needed to be heated to liquid in a water bath before

mixing. The proportion of CD-polymer solution, cod liver oil and free fatty acids was

always the same in every sample. The proportions were:

I- 1/100 Free fatty acids

II- 9/100 Cod liver oil

III - 90/100 CD-polymer solution

II was measured and poured into the volumetric flask containing III. Then I was

measured and poured into the same flask. I turns to a solid at room temperature which

could affect the final volume of the sample. To prevent that, I was heated occasionally

in a water bath throughout the mixing process. When I, II and III were merged in the

volumetric flask, CD solution was added until the volume was exactly 50 ml, then

nitrogen was sprayed over it and they were mixed together and poured into an oxygen-

free 100 ml jug made of light-protective brown glass. The jugs were shaken at rotation

speed 250 for 1 week to promote complexation. When the samples had to be opened for

a measurement, nitrogen was sprayed over before closing.

3.3.6 Flocculation Test

The flocculation test was performed using an in-house method. After having been in a

shaker for at least 1 day and shaken vigorously before insertion, 10 ml of each solution

were poured in a 10-ml measuring cylinder. Observations were made periodically for 8

days.

3.3.7 Dispersion and smell

Dispersion was determined by both intensity and length of shaking. The solution was

shaken until uniformity had been achieved. The intensity and time of shaking were

given a value between 1 and 5, where 1 represents a solution that is redispersed easily

and 5 is a solution which was difficult to redisperse. The smell was objective of the

30

smeller and was made by simply smelling the formulations and giving it a score of 1,

representing no cod liver oil, to 6, representing strong cod liver oil smell.

3.3.8 Surface tension

A digital Tensiometer K9 (Krüss) with a roughened platinum plate was used to measure

the surface tension of the samples. Before every measurement, the glass cell and the

platinum plate had to be rinsed, cleaned and then ignited in the flame of Bunsen burner

to destroy any surface-active substance left on their surface. Prior to use, the plate was

wetted with water to make the contact angle close to 0°. The surface tension is

calculated with the equation

σ =

where σ is surface tension, F is force acting on the balance, L is wetted length and θ is

contact angle. The contact angle is virtually 0°, and therefore the value cos θ is close to

zero, only the measured force and length of the plate need to be taken into consideration

(KRÜSS, 2013). The sample, about 20-40 mL, was poured into the sample vessel and

inserted into the sample support. The sample stage is then raised by means of the coarse

stage adjustment and the device reset to zero when the plate was just above the sample.

The sample stage is raised again and when the plate has just touched the sample a

reference mark is set. Then the sample stage is raised again about 5mm and then

lowered back to the reference mark. This is done so no buoyancy error occurs during

measurement. After the tensiometer had stopped fluctuating the value was read. Before

measuring the samples, water with a surface tension of approximately 72 mN/m

(Vargaftik, 1983) was used to calibrate the equipment.

3.3.9Viscosity

A Brookfield DV1 Prime visco meter supplied with a S40 or S52 spindle and a

thermostated cell was used to measure the viscosity of the solutions. The S40 spindle

was used for the less viscous solution while the S52 for the more viscous solutions. The

Brookfield viscometers employ the principle of rotational viscometry, where the torque

required to turn a spindle in a fluid indicates the viscosity of the fluid. In the Brookfield

31

viscometer, the spindle is moved through a calibrated spring and the deflection of the

spring measures the viscous drag of the fluid against the spindle, which is proportional

to viscosity (Brookfield, 2013). First, the gap between the solution and the spindle had

to be set at a defined distance with the sample cup attached. This was done my moving

the micrometer adjustment ring until the yellow light flickered, then move the sliding

reference marker to the nearest dash and then move the micrometer adjustment ring one

dash to the left. 1 ml was then applied at the center of the sample cup and attached to the

viscometer. A care had to be taken not to move the micrometer adjustment ring while

attaching the sample cup. The measurements were carried at 25 (± 0.2)°C with a

minimum torque of 10%. If the torque was considerably higher than 10%, the spindle

speed was decreased to give a torque value closest to 10%. The sample cup was cleaned

with a paper tissue between samples. The samples were stirred before measurement to

ensure uniformity. The viscometer was calibrated using a standard with appropriate

viscosity.

3.3.10 Osmolality

A K7000 Knauer Pressure Osmometer was used to determine the osmolality of the

samples. The vapor pressure osmometer is based on the principle that the vapor pressure

of any solution is lower than the vapor pressure of a pure solvent. The replacement of a

drop of pure solvent with one that contains solute leads to a vapor pressure difference

between the two droplets. To compensate for this, the pure solvent’s vapor that is

already saturated in the gas phase condenses on the solution droplet until the vapor

pressure is balanced. Condensation leads to an increase of droplets temperature. This

temperature difference between the reference and sample thermistors is always

proportional to the number of particles/number of moles dissolved in the solution

(GmBh, 2007). Before measurement, pure solvent must be placed into the glass beaker

located inside the cell. To promote vapor saturation, paper wetted with solvent, in this

case water, is also placed inside the beaker, the cell cover is attached and the cell closed,

the head thermostat closed and all the syringes installed to their ports to maintain

25.0°C inside the cell. To make sure the vapor saturation inside the cell was sufficient,

the osmometer was left running overnight before measurements began. An aqueous

NaCl solution with 400 milliosmoles per kilogram of solvent (mOsmol/kg) was used as

32

a standard. When measuring it is important to keep the pure solvent drop and the sample

drop similar in size and allow a couple of drops to flow over the thermistor probe before

measuring a new sample.

3.3.11 Particle size distribution

The particle size was measured using an Olympus BH2 light microscope with DPlan

100X lens from Olympus. The eyepiece power was 10X, therefore giving total

magnification of 1000X. The eyepiece

scale was calibrated with 1mm stage

micrometer from Olympus with intervals

equal to 0.01mm. Single interval of

eyepiece scale corresponded to 0.1

intervals of the stage micrometer, equal

to 1.0 μm. Each sample was prepared in

the following way: small droplet of the

formulation was introduced onto an

object plate followed by application of

a cover glass to spread the sample

evenly. The sample was then inserted

into the micrometer and the particle size assessed, counting each particle as the plate

was moved from left to right, down few millimeters and then right to left to ensure no

particle was counted twice, see Figure 6. The particle size distribution was determined

from observations of more than 200 particles per sample.

3.3.12 Peroxide value test

To assess the PV, formulations were prepared as discussed before and shaken for 1

week. The formulations and lipids without any formulation were kept at different

conditions for a period of 4 weeks. The conditions were:

Figure 6 Illustration of how the particles were assessed to

ensure none were counted twice.

33

A0 – Only formulated.

A1 – Kept under nitrogen and protected from light.

A2 – Kept protected from light, opened and left open for a period of ~2 hours

every day.

A3 – Kept under nitrogen, not protected from light

Note that condition A1, A2 and A3 were all formulated, shaken for a week and then

kept at the defined conditions.

3.3.12.1 Lipid extraction.

To carry out the peroxide test, the lipids had to be extracted out of the CD-polymer

solution using a scaled down Bligh and Dyer method (Bligh & Dyer, 1959). This was

achieved by first diluting the formulation with 75ml of methanol:chloroform solution

with the ratio of 2:1 and stirred until monophasic, then adding 25ml of chloroform to

the mixture and stirring until monophasic again. Then 25ml of water was added and

stirred for about 30

minutes more. This should

produce a biphasic solution

with the lipids at the lower

half. The solution was then

filtered to separate the

polymers from the liquid.

To prevent any further

oxidation, the filtering

process was encapsulated

in a plastic bag filled with

nitrogen. The formulations

containing 5% of the

polymer were most bothersome to filter and were sometimes kept longer than the

formulation containing 2.5% polymer. After about 12 hours of filtering, 10 ml of 0.5%

NaCl solution was poured over the rest of the polymers. However, extremely small

portion of the lipids passed through the filtering paper and therefore they had to be

collected by stirring the filtered polymer-mud (Fig. 7) and either, carefully, pour the

Figure 7 Stirring of the filtered polymer mud after filtering for 12 hours. The lipids can be seen as yellow stains on the polymers.

34

lipids off or use a pipette to collect them. The solution containing the lipids was placed

into a separatory funnel and the lower half, containing the lipids, extracted into a round

bottom flask. The round bottom flask was then installed onto a rotavapor to remove

solvents, leaving only the lipids for the peroxide test.

3.3.12.2 Titration

PV is the most widely used method to measure the extent of primary oxidation in oils.

When measuring the PV, the sample is treated in a solution of acetic acid and a suitable

organic solvent (isooctane in this case) and then subsequently with a solution of

potassium iodide, KI. The iodide ion reacts with the hydroperoxide (ROOH), resulting

in iodine liberation, according to the reactions:

KI + CH3COOH HI + CH3COOK

ROOH + 2HI ROH + H2O + I2

The base produced is taken up by the excess acetic acid. The liberated iodine is then

titrated with a solution of sodium thiosulphate (Na2S2O3) using a starch indicator

(International Fragrance Association, 2011) , according to the reaction:

2NaS2O3+ I2 (purple) Na2S4O6 + 2NaI

(colourless)

Due to small volumes of lipids extracted from the formulations, a mini-peroxide test,

developed by Þormóður (Geirsson, 2008) was used. In the mini-peroxide test 0.3 – 0.5

grams of lipids were dissolved in 10 ml of acetic acid:isooctane (3:2) solution, followed

by 0.2ml of potassium iodide. The reaction was stopped after 60 ±1 second and at least

3 rotational swings. Then 6ml of water was added to stop the reaction and subsequently

1.2 ml of starch. The solution was then titrated with a 0.001 M sodium thiosulphate

solution until no color is observed. It is important that the titration is executed swiftly

and the flask is moved with rotational swings during titration.

35

The equation used to calculate the PV is:

PV (milliequivalents O2/kg sample) =

Where C is molarity of the titrant, expressed as moles/liter

V is volume of titrant used, in ml

W is weight of the lipids, in g.

3.3.12.3 Blending the solutions used in the titration

All of the solutions were prepared as specified in standard operating procedure EBL 5-

40-BL by Lýsi hf. The potassium iodide solution was prepared by dissolving 100 g of

potassium iodide in 70 ml of water. To ensure dissolution, the solution was sonicated

for a few minutes, and to ensure saturation, a few potassium iodide crystals were put

into the solution afterwards. The solution expires two weeks after preparation. The

starch indicator is prepared by measuring 8 g of potato starch dissolved in 4 liters of

boiling deionized water. After a few seconds of boiling, 5 grams of salicylic acid is

added to the solution. The solution expires three months after preparation. The acetic

acid:isooctane solution is prepared by mixing 3 portions of acetic acid with 2 portions

of isooctane. The solution expires five years after preparation. The sodium thiosulphate

solution is prepared by dissolving 1 ampule of 1M sodium thiosulphate in 1 liter of

water, creating a 0.1M solution. The solution is then diluted to give the desired molar

concentration.

The acetic acid:isooctane solution, the starch indicator and the sodium thiosulphate were

all generously donated by Lýsi hf.

3.3.12.4 Statistics

Excel was used to calculate the T-test between the formulations. A two tailed t-test for

unequal variances was used and for a difference to be statistical, the p-value had to be

lower than 0.05 (α=0.05).

36

4. RESULTS

4.1 Formulations 1 - 29

4.1.1 Water content of the CDs

The water content of the CDs used in this project was measured. See Table 2 for the

water content experimental values. The standard deviation did not exceed 0.2% in all

cases.

Table 2 The water content of the CDs used

CD Water content, %

α 11,4

β 14,9

γ 9,8

4.1.2 CD – lipid formulations

To assess which of the parent CDs was the most suitable for creating inclusion complex

with the lipids, formulations with different CD concentrations without polymers were

prepared. The formulations were assessed after being shaken for one week. See Table 3

for the results.

Table 3 Results from the formulation of different cyclodextrins (CD) with cod liver oil (clo) and free fatty acids (ffa) . CD:clo:ffa ratio is 90:9:1 %v/v)

Formulation CD % CD w/v Results

1 γ 10 Monophasic, milky

2 γ 15 Monophasic, milky

3 α 10 Monophasic, milky

4 α 5 Biphasic, lipids on top

5 β 1,5 Biphasic, lipids on top

If the formulation was monophasic an inclusion complex had been formed. If the

formulation was biphasic a complex had not been formed. Formulation 1, 2 and 3 were

superior to the others. Formulation 1 was chosen for further development due to a better

toxicological profile for γ-CD compared to α –CD (Loftsson & Duchêne, 2007) and

lower CD content compared to formulation 2.

37

4.1.3 CD – polymer – lipid formulation

To find which of the polymers had the best stabilizing effect on the CD- lipid solutions,

a number of formulations containing different polymers in various concentrations were

prepared with and without preservatives. If the formulation contained preservatives they

were always identical: 0.02% w/v benzalkonium chloride (BAK) and 0.1% w/v

ethylenediaminetetraaceticd acid (EDTA). This was done to see the effects of

preservatives on a number of factors. See Table 4 for the formulation ingredients. All of

the formulations contained the same amount of NaCl, 5.5% w/v.

Table 4 Concentrations and results from the CD – polymer – lipid formulations (CD:clo:ffa: 90:9:1 (%v/v)) containing 5.5% w/v NaCl. If Y for preservatives, it contained 0.02% v/w BAK and 0.1% EDTA. CD – cyclodextrin,

Clo – cod liver oil, ffa – free fatty acids, CMC-Na – sodium salt of carboxymethylcellulose, HPMC – hydroxypropyl methylcellulose, PVA – polyvinyl alcohol, PVP – polyvinyl pyrrolidone, Y – yes, N - no

Formulation Polymer Polymer ratio (%w/v)

Preservatives Results

6 Lutrol 2,5 Y Monophasic

7 Lutrol 2,5 N Monophasic

8 Lutrol 5 Y Monophasic

9 Lutrol 5 N Monophasic

10 Carbomer 974 P

1 Y Fatty droplets on top

11 Carbomer 974 P

1 N Fatty droplets on top

12 Carbomer 974 P

0,5 Y Fatty droplets on top

13 Carbomer 974 P

0,5 N Fatty droplets on top

14 CMC-Na 1,5 Y Monophasic

15 CMC-Na 1,5 N Monophasic

16 CMC-Na 0,75 Y Monophasic

17 CMC-Na 0,75 N Monophasic

18 HPMC 1,5 Y Monophasic

19 HPMC 1,5 N Monophasic

20 HPMC 0,75 Y Monophasic

21 HPMC 0,75 N Monophasic

22 PVA 1,4 Y Monophasic

23 PVA 1,4 N Monophasic

24 PVP 2 Y Fatty layer on top

25 PVP 2 N Fatty layer on top

26 Lutrol + HPMC 1,5 + 1 Y Monophasic

27 Lutrol + HPMC 1,5 + 1 N Monophasic

28 None - Y Monophasic

29 None - N Monophasic

38

4.1.4 Testing the formulations

4.1.4.1 Flocculation test, redispersion and smell

Flocculation test was carried out over the course of 192 hours or 8 days as described

above. For simplification the data represented here do not show the degree of

flocculation but only when flocculation occurs. After the flocculation had been

determined, redispersion was assessed. The redispersion values represent the intensity

and length of shaking to get a uniform suspension. The values are in the range of 1 – 5.

Smell is given in the range of 1-6. See Table 5 for results. For the complete flocculation

data and breakdown of how the flocculation was built up, refer to appendix A.

Table 5 Flocculation, redispersion and smell of the formulations redispersion is combined from the intensity and length of shaking. Smell is objective of the observer. NA indicates that no flocculation occurred.

Formulation Hours after flocculation occurs I Smell

6 30 2 1

7 22 1 3

8 22 1 3

9 72 1 1

10 NA 1 6

11 72 2 6

12 0.5 1 6

13 5 1 6

14 NA * 1

15 NA * 2

16 NA * 2

17 NA * 4

18 5 5 4

19 1.5 2 4

20 22 3 3

21 5 2 3

22 22 5 4

23 22 4 3

24 þ5 2 3

25 0.5 1 4

26 192 1 2

27 96 1 6

28 22 1 2

29 0.5 5 4

39

Even though no flocculation was observed in formulation number 10, fatty droplets had

lined the inside of the cylinder after 22 hours. The asterisk at formulations 14-17

indicate that they were incredibly viscous and when shaking them for redispersion (even

though there was nothing to 39otovapor39d) the formulation did not move at all. Figure

6 shows the appearance of five formulations after 8 days in the flocculation test.

Figure 8 Appearance of formulations, from left to right, 9, 10, 14, 18 and 29 after being in the flocculation test for 8 days. The red circle indicate phase separations in formulation 9, 18 and 29 and fatty droplets on the inside of

the cylinder in formulation 10.

40

4.1.4.2 Viscosity

The viscosity was measured, as described above, for the formulations that were eligible,

the ones that were monophasic after being in the shaker for one week (see Table 4).

Figure 9 illustrates the results. For the full data, refer to appendix B.

Figure 9 Viscosity (η) of the formulation in centipoises (cP). Due to big differences between formulations the graph is represented in log values. The graph shows the average experimental values from at least 3 replicates

along with the error bars for standard deviations.

Due to enormous differences between formulations, the graph had to be represented in

log scale. The values read from the visco meter were poorly reproducible: the SD was

sometimes high and the resulting value strongly depended on the parameters of

experiment (the spindle speed). This was especially the case for the most viscous

solutions. As can be seen on Figure 9, formulations 14, 15, 16 and 17 were at least 0.5

log values more viscous than the others. For that reason they were excluded for further

testing.

1

10

100

1.000

10.000

100.000

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

η c

P

Formulation

Viscosity

40

41

4.1.4.3 Surface tension

The surface tension of the formulations was measured as described above. See figure 10

for results. For the surface tension values, refer to appendix C.

Formulation 15* was too thick for it to pour down into the sample vessel and therefore

was not measured. As can be seen on the graph most of the measurements were nicely

reproducible with small standard deviations.

Figure 10 Surface tension (mN/M) of the formulations that were eligible for measurements. The graph shows the average experimental values from at least 3 replicates along with the error bars for standard deviations.

0

10

20

30

40

50

60

70

80

6 7 8 9 10 11 12 13 14 15* 16 17 18 19 20 21 22 23 24 25 26 27 28 29

σ m

N/m

Formulation

Surface tension

42

4.1.4.4 Osmolality

The osmolality of the formulations was measured as described above. As suspected, the

osmolality values were out of the osmometer’s operational range, i.e. exceeded 1200

mOsm/kg. After calculations, 0.35 g (0.7% w/v) of NaCl was chosen for the next

formulations.

4.2 Formulations 30- 39

4.2.1 CD – polymer – lipid formulation

The formulations that showed the best results from the tests above were prepared again

with reduced NaCl content (tonicity equal to 0.7% w/v). The formulation ingredients as

well as their new numbers can be seen in Table 6.

Table 6 Composition of re-prepared monophasic formulations (CD:clo:ffa 90:9:1 % v/v) with 0.7% w/v NaCl. Preservatives used were 0.02% v/w BAK and 0.1% EDTA. HPMC – hydroxypropyl methylcellulose, PVA – polyvinyl

alcohol, Y– yes, N – no

Formulation (old formulation number)

Polymer Polymer conc (%w/v) Preservatives

30 (6) Lutrol 2,5 Y

31 (7) Lutrol 2,5 N

32 (8) Lutrol 5 Y

33 (9) Lutrol 5 N

34 (18) HPMC 1,5 Y

35 (19) HPMC 1,5 N

36 (20) HPMC 0,75 Y

37 (21) HPMC 0,75 N

38 (23) PVA 1,4 N

39 (28) None 0 N

43

4.2.2 Testing the formulations

4.2.2.1 Flocculation test

The flocculation results for the new formulations were very similar to the old ones and

therefore the data is not presented here. For the flocculation data see Table 5 or

appendix A.

4.2.2.2 Surface tension

Since the new formulations were similar to the old ones differing only in NaCl content,

it was assumed that the surface tension would stay approximately the same. To verify

this, two random formulations were tested. The results were notably different from the

old results and therefore the surface tension of all new formulations was to be measured,

as well. Figure 11 shows the results. For the surface tension values and comparison

between the old and new values, refer to appendix D.

Eight of the new formulations had statistically lower surface tension values compared to

the old ones. For the T-test report, refer to appendix G.

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

30 31 32 33 34 35 36 37 38 39

σ m

N/m

Formulation

Surface tension

Figure 11 Surface tension (mN/M) of the new formulations. The graph shows the average experimental values from at least 3 replicates along with the error bars for standard deviations

44

4.2.2.3 Viscosity

Due to big differences in viscosity values between the new and old formulations

obtained from two random formulations, all of them were measured as well. See Figure

12 for results.

Figure 12 Viscosity (η) of the new formulations in centipoises (cP). The graph shows the average experimental values from at least 3replicates along with the error bars for standard deviations.

Again, the SD is relatively big and appears to grow as the viscosity increases. The

difference between the old and new formulations as well as the data from the viscosity

measurements can be seen in appendix E.

4.2.2.4 Osmolality

The osmolality values were measured as described above. See figure 13 for results and

appendix F for the osmolality values.

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

30 31 32 33 34 35 36 37 38 39

η c

P

Formulation

Viscosity

45

Figure 13 Osmolality, in milliosmoles/kg solute (mOsm/kg) of the new formulations. The graph shows the average experimental values from at least 3 replicates along with the error bars for standard deviations.

To proceed with the formulation testing, two of eight formulations were decided to be

selected based on the experimental data generated by this step. As stated above, a

formulation with viscosity around 20 has been considered ideal. Based on that,

formulations 34, 35, 36, 37 and 39 can be excluded. The surface tension values are all

quite similar and none can be excluded on the basis of that information. Datum from the

flocculation test points out that formulation 31, 32 and 38 are less stable then the two

others. Therefore, formulations 30 and 33 were chosen for further testing.

4.3 Formulations 30 and 33

4.3.1 Particle size distribution

The particle size distribution was assessed as described above. At maximum

magnification no individual particles could be identified. Scrutinizing the samples

carefully, it was concluded that there was a swarm of extremely small particles, piled

upon each other. Determination of the size of these particles seemed impossible using

available lightscope. Since the particles were much smaller than one interval in the

eyepiece scale, it was concluded that all of the particles were smaller than 1µm, which

perfectly satisfies the European Pharmacopeia requirements.

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

30 31 32 33 34 35 36 37 38 39

mO

sm/k

g

Formulation

Osmolality

46

4.3.2 Peroxide value

The results from the PV test can be seen in Figure 14. For the distinct values, refer to

appendix H.

Figure 14 Peroxide value (meq O2/kg sample) of formulations 30, 33 and “pure cod liver oil plus free fatty acids” at different conditions. Point zero = before formulation, A0 = formulated and shaken for 1 week, A1 = formulated

and kept protected from light and oxygen for 4 weeks, A2 = formulated and kept for 4 weeks protected from light, A3 = formulated and kept for 4 weeks protected from oxygen

As can be seen on Figure 14 all of the samples began at the same time, point zero. At

first formulation 30 protected the lipids best but after that skyrocketed compared to the

others. Formulation 33 seems to be the leader at protecting the lipids from oxidation.

The difference between formulation 30 and “pure cod liver oil plus free fatty acids” was

statistically relevant at condition A0. Formulation 33 was statistically lower at

conditions A0 and A2. Additionally, formulation 30 was statistically lower at condition

A0 compared to formulation 33, and formulation 33 statistically lower than formulation

30 at condition A2. For the T-test reports, please refer to appendix I.

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

Point zero A0 A1 A2 A3

Pe

roxi

de

Val

ue

Condition

Oxidation

30

33

No formulation

47

4.3.3 40-days flocculation

Figure 15 shows a 40-days flocculation test for formulations 30 and 33.

As can be seen on figure 15, a small fraction of the lipids in formulation 30 is not

complexed with the CDs and sits on top as a yellow fatty layer (circled by a red ring). In

opposite, the lipids in formulation 33 seem to be fully complexed with the CDs.

However, a clear border between the sediment and supernatant can be seen which seems

to be vacant of free lipids. Nevertheless, it can be readily redistributed by shaking.

Figure 15 40-days flocculation test. From left, formulations 31 and 33. The red circles indicate phase separation.

48

5. DISCUSSION

5.1 CD-polymer-lipid formulations

Blending of the polymer solutions went smoothly. The method used was obtained from

Fífa Konráðsdóttir, a Ph.D. student and is partly based on a method Bochot et al used to

produce small beads of α-CD and soy oil (Bochot et al., 2007). It appears that the

container the formulation is shaken in for a week to form the complex contributes to the

complex formation. When a smaller container was used for shaking, a formulation with

a fatty layer on top was produced at the end of shaking whereas, in a bigger container,

this layer did not form. Perhaps, container geometry affects noticeably the mixing

intensity and consequently the kinetics of complexation between ingredients.

The CD concentration in formulations 3 and 5 (10% α-CD and 1.5% β-CD respectively)

were chosen as approaching to their solubility (Loftsson & Brewster, 2010). To ensure

full dissolution before use, they were sonicated and heated to increase the solubility.

This was especially important for α- and β-CD. Only the parent CDs were assessed for

their complexing abilities since earlier research conducted at the Faculty of

Pharmaceutical Sciences, University of Iceland indicated the CD derivatives are not

able to form inclusion complexes with the lipids (Geirsson, 2008).

Previous research suggested that α- and γ-CD are superior to β-CD in creating a lipid-

CD complex (Duchêne et al., 2003). The results obtained in the given project supported

this suggestion. The reasion is probably β-CDs low solubility profile, meaning that the

ratio between the CD and lipids is not optimal. Formulation 1 (10% w/v γ-CD), was

chosen for further work since γ-CD has a better toxicological profile compared to α-CD

(Loftsson & Duchêne, 2007) and since lower CD content in comparison to formulation

2 (15% w/v γ-CD) diminishes risk of crust formation on the eyelids of dry eye patients,

leading to irritation (Loftsson. & Jarvinen, 1999).

5.2 Formulations 6-29.

The majority of the polymers used were able to co-produce a monophasic solution with

the CD and the lipids. Polivinyl pyrrolidone (formulations 24 and 25) however failed

49

completely and carbomer 974P (formulations 10-13) was not successful. They were

therefore excluded from further testing with the exception of flocculation, redispersion

and smell test. To facilitate solubilization and limit possible alterations, Lutrol was

refrigerated before mixing with the CD solutions. This is due to Lutrol’s

physicochemical properties: the polymer turns into a gel at relatively low temperatures

(Dumortier, Grossiord, Agnely, & Chaumeil, 2006).

The flocculation data give a representation of how stable the suspensions are and

therefore also the complexes. For formulations 14-17 no flocculation occurred, probably

due to enormous thickness. This viscosity can be traced back to the use of “high

viscosity” CMC-Na instead of CMc-Na of “low” or “medium” viscosity. It is

foreseeable that the values obtained from flocculation, redispersion, surface tension and

osmolality on high viscosity CMC-Na would have been different if the lower viscous

CMC-Na polymers had been used. From the data obtained it seems that a relationship

takes place: as the formulations take longer to flocculate, redispersion becomes easier.

These two are important factors; ideally the formulations should stay monophasic for as

long as possible and redispersion should be relatively easy to ensure a monophasic

solution is readily available. The nature of different layers in the formulation test should

also be considered (appendix A). It is obvious that a monophasic solution is superior to

a two- or a three-layered solution, but a two layered solution containing a water layer on

top of a suspension layer is more desirable to a fatty layer on top of a precipitate. In the

first one, a complex still exists between the lipids and the CD, while in the latter it does

not. It should also be noted that the smell is objective of the observer and varies from

person to person. However it is important to assess the smell as people do not like the

„fishy“smell of the cod liver oil, as well as of the free fatty acids. The formulations that

smell the least are therefore superior to the ones that smell the most. A clinical trial

involving marine lipids suppositories revealed that the cod liver oil smell was

troublesome to two thirds of the participants (Ormarsson et al., 2012). Whether the

same would be the case for eye drops is unknown but has to be considered likely since

they can easily reach the nasal cavity through tear ducts.

The viscosity of the solutions was quite different, ranging from about 5 to 25.000 cP. As

stated above, increased viscosity offers prolonged contact time and therefore

50

bioavailability. However, if the viscosity is too high, patients’ vision is compromised.

Additionally, as the viscosity becomes too high it becomes harder for the CD-drug

complex to partition from the bulk of the media towards, for example, the ocular barrier.

Therefore, a viscosity of about 20 cP has been considered optimal (Kråmer, 2002) and

the formulations that lay around that value are therefore superior to the ones that do not.

The values from the viscosity measurements were not very reproducible as can be

concluded from a rather large SD. The values should be treated deliberately since they

are dependent on experimental parameters, such as the spindle speed. No conclusion can

be made whether the preservatives increased viscosity or not. The formulations

containing the preservatives were more viscous in five cases and less viscous in four

cases.

The surface tension values were quite reproducible as the SD was small. Formulation 15

could not be poured down into the sample vessel due to high viscosity. It is however

strange since there are two formulations that are more viscous than formulation 15. The

reason behind this is unknown, but perhaps a little more patience could have made it

work. No conclusion can be made whether preservative increases or decreases surface

tension. The formulations containing the preservatives had higher surface tension values

in four cases and lower values in four cases.

The fact that the formulations had osmolality values over 1200 mOsm/kg was not a

surprise. A 5.5% w/v NaCl (57.5 g/mole), corresponds to 2.77 g or 0.048 moles of NaCl

in 50 ml of water. To calculate osmolality, the amount of solvent has to be taken into

the equation as well as the total number of dissolved particles with account for their

dissociation. The amount of solvent is 50ml, i.e. 0.05kg, and NaCl is known to fully

dissociate into two ions (Na+ and Cl

-):

= 1.926 mOsm/kg, which

is far above the maximum detection limit for the osmo meter. There is no specific

reason why the formulations had such a large quantity of NaCl other than

thoughtlessness during the formulation. To correct these mistakes, the formulations had

to be remade with less NaCl than before. As stated above, blood and tear osmolality is

287 mOsm/kg (Kråmer, 2002). Osmolality depends on a number of factor, one of them

being the molar concentration of dissolved solute (US Pharmacopeia, 2006). As the

other ingredients in the formulation were a part of the dissolved solute, NaCl was not

51

the only part responsible for the high osmolality but definitely the biggest one.

Therefore, it was decided that a formulation with 0.35 g of NaCl in a 50 ml formulation,

a little hypotonic to tear osmolality, or 243 mOsm/kg, would be chosen for the next

formulations.

5.3 Formulations 30-39.

The formulations chosen to be remade were the ones that had the best results from the

tests above. The formulations containing the CMC-Na polymer (formulations 14-17)

were too viscous and therefore were rejected along with the already excluded

formulations containing PVP (formulations 24 and 25) and carbomer 974P

(formulations 10-13). Formulation 22 was rejected due to high redispersion score as

well as strong smell, formulations 26 and 27 due to high viscosity values and, finally,

formulation 29 due to low stability, high redispersion score and bad smell.

Comparing the surface tension values from the new formulations and the old ones it

turns out the new formulation values are all lower than the old formulation values,

ranging from 0.5 mN/m to 20.1. The surface tension values of new formulations are all,

except formulations 8/32 and 28/39 (old number/new number), statistically lower

(α=0.05) than those of old formulations containing more NaCl. This decrease is due to

Coulombic attraction that draws the ions together and away from the surface, and is in

agreement with previous results (Bhatt, Chee, Newman, & Radke, 2004). A comparison

between the old and new values can be seen in appendix D and the T-test reports in

appendix G.

When comparing the viscosity values of the new formulations with those of the old

ones, no specific trend is revealed as the values fluctuate in relation to each other, see

appendix E.

The measured osmolality values were somewhat hypertonic (average of all formulations

329 mOsm/kg) compared to the calculated value of 243 mOsm/kg. The values are

satisfactory even though they are higher than desired. This is explained by the presence

of other ingredients (like CD, polymer, preservatives) contributing to the total

osmolality. By optimizing the NaCl concentration an ideal osmolality could be derived

but due to lack of time these minor modifications were not performed.

52

5.4 Formulations 30 and 33

The particle size distribution revealed no particles bigger than 1 µm. These results

therefore perfectly comply with the European Pharmacopeia. The available light

microscope operation range was not enough to identify individual particles of such

small size, magnification of at least 10.000 would be sufficient.

Initially, lipid extraction seemed problematic, largely due to failure of lipids to pass

through the filtering paper. However, after a couple of attempts, the lipids were poured

off, and the extraction went smoothly. The amount of lipids extracted, as a ratio of

lipids added to the formulation was surprisingly high. Based on the density of cod liver

oil, ~0.90 g/ml (Loveridge, 2002) the ratio extracted exceeded 100% in some cases (see

appendix H). This means that a considerable amount of impurities, i.e. polymers or

solvents, were present during the execution of the PV. This skews the results from the

peroxide testing, as it would decrease the PV since less-than-calculated amount of lipids

were being titrated. In addition, it seemed as two thirds of the bottles in conditions A2

with formulation 33 titration number 3 (see appendix H) had undissolved free fatty

acids at the end of the titration. This could have led to lower PVs. However, compared

to the other bottles containing formulation 33 in conditions A2, the results were similar.

A number of other factors could also have skewed the results from the titration. The

lack of experience of the investigator, imprecise dilution of the titrant and the conditions

it was kept which could maybe have led to deterioration. Therefore the results should be

taken with some precaution.

The lipids are already oxidized considerably before the encapsulation process, as can be

seen with the PV at point zero. Þormóður assessed the PV of cod liver oil before

encapsulation and got a considerably lower value of roughly 5 (Geirsson, 2008) while

Lýsi hf states that its cod liver oil has a maximum PV of 10 (Lýsi, 2013). The increased

oxidation measured in this study could be, aside from the factors mentioned above, due

to age of cod liver oil used in this project or inappropriate storage conditions.

The peroxide values in conditions A0 showed that both formulations protected the lipids

from oxidation. Both had statistically lower values assuming two tailed test for unequal

53

variences, formulation 30 with a p-value of 1.64*10-07

compared to lipids kept without

any formulation and formulation 33 with a p-value of 1.8*10-5

. In conditions A2 and A3

something was going on which seemed difficult to interpret. The lipids with no

formulation had lower PV than formulation 30. These results are in contrast with

published data showing that the CDs protect fatty acids from oxidation (Geirsson, 2008;

Kim et al., 2000; Ying et al., 2011). It is possible however that during extraction of the

lipids, considerable oxidation occurred which increased the PV. The plastic bag might,

for example, not been sealed perfectly and/or the heat that was applied with the

rotovapor could have increased the oxidation. As can be seen with the 40-days

flocculation test, a portion of the lipids was not in a complex with the CD anymore. It is

plausible that this could have contributed to increased oxidation of the lipids; compared

to when they had been sealed in a complex with the CD. However, no fatty layer was

observed when extraction of the lipids began so that is apparently not the case.

Additionally it is possible that the lipids without formulation had oxidized so much that

the peroxide values were beginning to decrease, but as discussed in the introduction the

PV follows an inverse parabolic curve. For a complete picture of the oxidation, AV

should be measured and the totox number derived but unfortunately this was out of the

scope of this project. At conditions A2, formulation 33 gave statistically lower PV value

than the lipids without any formulation with a p value equal to 0.014. Additionally, PV

value of formulation 30 was statistically lower in conditions A0 compared to

formulation 33 (p=1.55*10-5

) while PV value of formulation 33 was statistically lower

than that of formulation 30 at conditions A2 (p=8.2*10-6

). Visually though, formulation

33 appeared to be a leader at protecting these lipids.

The 40 day flocculation test showed that formulation 33 was more stable than

formulation 30. The fact that the lipids were, partly, not in a complex with the CD in

formulation 3 after this time could lead to increased oxidation and therefore damaged

product.

54

6. CONCLUSION

It appears that α- and γ-CD at 10% w/v are equally effective in forming a CD-lipid

complex and are superior to β-CD in doing so. γ-CD is however considered to be a

better candidate for eye drop formulation due to a better toxicological profile. From the

results it can be concluded that a combination of γ-CD, and certain polymers are able to

produce a stable, monophasic formulation with cod liver oil and free fatty acids. The

majority of the polymers tested were able to co-produce monophasic solutions with the

lipids and CD.

Two formulations appeared superior to the others after testing the flocculation,

redispersion, smell, surface tension, viscosity and osmolality; one containing 2.5% w/v

poloxamer 407 with 0.02% BAK and 0.1% EDTA w/v, and the other 5% poloxamer

407. Both formulations had optimal viscosity, were relatively stable toward

flocculation, were easily redistributed after standing for an extended period of time,

masked the cod liver oil smell and had an acceptable osmolality value. These two

formulations also complied with the particle size distribution of the European

Pharmacopeia for eye drops. The peroxide values indicate that the CDs might protect

the lipids from oxidation even though the evidence obtained in this project on that was

not conclusive.

Continued research is needed, to see whether or not the CDs protect the lipids from

oxidation, and to what extent, and to see how much the flux of the lipids to, for

example, the cornea of the eye is.

55

7. ACKNOWLEDGEMENT

I would like to thank my supervisor Þorsteinn Loftsson for read through of the thesis

and Dr. Sergey Kurkov for his help during the project and read trough of the thesis.

I would like to thank Dr. Maria Dolores for good advices through the project, Ph.D

students Sunna Jóhannsdóttir and Ingólfur Magnússon for companionship and great tips,

Fífa Konráðsdóttur for helping with preparing the formulations and great tips on the

peroxide value test and Auður Ágústsdóttir for helping finding the right glassware.

I would like to extend my gratitude to Lýsi hf for generously donating vital solutions for

the peroxide test.

I would like to thank both my family and friends for support for the last five years.

Last but not least, I would like to thank my fellow students for the past five years as

they have been both educational and loads of fun.

56

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B

9. Appendices

B

9.1 Appendix A

Flocculation data for formulations 6-29. D – two layered, T – three layered, F – four layered, FL – fatty layer, W – water layer, S – suspension Changes are bolded. When the solutions are

reported as “same”, the ratio between different layers could have changed. Bubbles that are made mainly out of lipids are reported as fatty layer. The ratio reported is in sequence with the layer

reported.

Formulation Time in hours

0.5 1.5 5 22 30 48 72 96 120 144 168 192 Ratio % 6 D. FL, S Same Same Same Same T. FL, W, P Same Same 11:24:71 7 D. S, P Same Same Same Same Same Same Same Same 1:99 8 D. S, P Same Same Same Same Same Same Same Same 1:99 9 D. W, S Same Same Same Same Same 1:98

10 11 D. W, S Same Same Same Same Same 15:85 12 D. W, S Same Same Same Same Same Same Same Same Same T. FL, W, P Same 1:9:90 13 D. W, S Same Same Same Same Same Same Same T. FL, W, P Same 1:3:96 14 15 16 17 18 D. FL, S T, FL, W, P Same Same Same Same Same Same Same Same 6:44:50 19 T. W, FL, S D. FL, S Same Same T. FL, W, P Same Same Same Same Same Same 8:36:56 20 D. FL, S T. FL, W, S Same Same Same Same Same Same Same 3:30:67 21 D. FL, S Same T. FL, W, S Same Same Same Same Same Same Same 7:42:51 22 D. S, P Same T. FL, W, P Dual, S, P Same Same Same Same Same 95:5 23 D. S, P Same Same Same Same Same Same T. FL,S,P. Same 2:88:10 24 T. S, W, S Same Same Same T. FL, W,P Same Same Same Same Same Same Same 18:54:28 25 T. FL, W, S Same Same Same Same D, W, S T, FL, W, P Same Same Same Same Same 1:48:51 26 D.W,S 2:98 27 D.W, S Same Same Same Same 4:96 28 D. W, S Same Same Same Same Same Same Same Same 7:93 29 Dual. FL, S Same Same F. FL, S, W, P Same Same Same Same Same Same Same Same 6:5:48:41

C

9.2 Appendix B

Viscosity of formulations 6-29

Table B 1Viscosity of formulations 6-29. SD – standard deviation. RPM –rounds per minute

Formulation Viscosity (cP)

#1 #2 #3 #4 Average SD Torque Spindle Speed (RPM)

6 8,14 6,21 6,07 5,84 6,57 0,92 14,25 6

7 5,65 5,1 6,17 5,29 5,55 0,41 12,08 6

8 12,69 13,32 14,13 13,77 13,38 0,54 14,73 3

9 22,41 22,95 22,95 22,77 0,25 25,30 1,5

10

11

12

13

14 10.169 11.724 10.148 10.680,33 738,03 34,97 0,3

15 4.157 4.157 4.367 4.227,00 98,99 13,90 0,3

16 23.711 26.790 26.910 25.803,67 1.480,55 85,40 0,3

17 1.178 1.522 1.441 1.380,33 146,84 22,67 1,5

18 279 222 248 249,83 23,14 26,53 0,3

19 338 282 221 280,50 47,78 32,10 0,3

20 475,2 401 466 447,27 33,21 43,67 0,3

21 199,3 172 145 147,2 165,83 21,97 16,73 0,3

22 4,73 3,61 3,64 3,27 3,81 0,55 14,10 12

23 5,01 5,31 5,21 5,18 0,12 10,30 6

24

25

26 362,9 338,8 371,6 357,77 13,87 40,90 0,3

27 351,6 341,9 343,1 345,53 4,32 39,33 0,3

28 334,9 372,6 358,5 355,33 15,55 40,47 0,3

29 4,99 5,49 5,1 5,19 0,21 11,63 6

D

9.3 Appendix C

Surface tension of formulations 6-29.

Table C 1 Surface tension of formulations 6-29. SD – standard deviation

Formulation Surface tension mN/m

#1 #2 #3 Average SD

6 46 46,3 45,5 45,93 0,33

7 51,5 49,1 47,6 49,40 1,61

8 43 43,1 43,1 43,07 0,05

9 43,5 43,1 43,5 43,37 0,19

10

11

12

13

14 70,6 70,3 70,1 70,33 0,21

15

16 69,9 69,3 69,6 69,60 0,24

17 62,8 63,9 64,6 63,77 0,74

18 57,8 58,7 57,1 57,87 0,65

19 57 57,9 56,3 57,07 0,65

20 57,1 56,8 56 56,63 0,46

21 61,3 64,6 64,1 63,33 1,45

22 49,9 51,2 51,2 50,77 0,61

23 51,2 51,5 51,35 0,15

24

25 60,4 59,5 63 60,97 1,48

26 49,7 51,3 52,9 51,30 1,31

27 48,5 45,2 53,5 49,07 3,41

28 61,3 61,3 60,3 60,97 0,47

29 56,9 59,9 59,6 58,80 1,35

E

9.4Appendix D

Surface tension of formulations 30-39 and comparison between them and the old

formulation.

Table D 1 Full data for the surface tension for the new formulations and comparison between the old and new values. SD – standard deviation, RPM – rounds per minute

Formulation Surface tension mN/m

#1 #2 #3 Average SD Old formulation number

Average of the old formulation

Difference between the old value and the new value

30 43,5 43,7 42,4 43,20 0,57 6 45,93 2,73

31 41,5 40,8 41,5 41,27 0,33 7 49,40 8,13

32 43,2 41,9 42,6 42,57 0,53 8 43,07 0,50

33 40,6 39,7 38,7 39,67 0,78 9 43,37 3,70

34 47 46,8 46,2 46,67 0,34 18 57,87 11,20

35 46,5 47,3 46,8 46,87 0,33 19 57,07 10,20

36 50,8 51,7 50,6 51,03 0,48 20 56,63 5,60

37 50,6 50,4 52,1 51,03 0,76 21 63,33 12,30

38 42 44 44,5 43,50 1,08 23 51,35 7,85

39 58,6 60,8 59,3 59,57 0,92 28 60,97 1,40

F

9.5 Appendix E

Viscosity of formulations 30-39 and comparison between them and the old formulation.

Table E 1 Viscosity of formulations 30-39 and comparison between them and the old formulations SD – standard deviation. RPM – rounds per minute, Difference – difference between the old formulation value and the new formulation value

Formulation Viscosity (cP) Viscosity (cP)

#1 #2 #3 #4 Average SD Torque Spindle speed (RPM)

Old formulation number

Average of the old formulation

Difference

30 5,82 5,36 5,57 5,58 0,19 10,93 6 6 6,81 1,22

31 5,4 4,9 4,7 5,00 0,29 10,93 6 7 5,64 0,64

32 19,8 19,3 18,1 17 18,55 0,71 20,12 3 8 13,38 -5,17

33 27,3 27,1 28,9 27,77 0,81 15,27 1,5 9 22,77 -5,00

34 396 399 382 392,33 7,41 40,72 0,3 18 249,83 -142,50

35 352,4 408,5 407,7 394,2 390,70 26,26 40,30 0,3 19 280,50 -110,20

36 169 185 178,6 177,53 6,58 20,76 0,3 20 447,27 269,73

37 122,3 110,2 114 115,50 5,05 11,93 0,3 21 172,03 56,53

38 5,1 6,1 5,5 5,57 0,41 11,65 6 23 5,18 -0,39 39 172 169 177 172,67 3,30 19,07 0,3 28 355,33 182,67

G

9.6 Appendix F

Osmolality of solutions 30-39.

Table F 1 Osmolality values for formulations 30-39. SD- standard deviation

Osmolality mOsm/kg

Formulation #1 #2 #3 #4 Average SD

30 299,25 297,15 298,20 294,00 297,15 2,27

31 304,50 312,33 314,94 315,81 311,90 5,15

32 351,48 345,39 325,38 348,87 342,78 11,87

33 312,33 327,12 331,47 329,73 325,16 8,74

34 317,06 332,00 339,47 341,96 332,62 11,21

35 344,52 347,13 331,47 351,48 343,65 8,61

36 365,40 363,66 369,75 362,79 365,40 3,10

37 335,82 338,43 370,62 341,96 346,71 16,14

38 319,29 325,38 322,77 322,77 322,55 2,50

39 304,50 294,06 307,11 301,89 301,89 5,64

H

9.7 Appendix G

T-test results comparing surface tension values between old and new formulation.

Table G 1 T-test report for formulations 6 and 30

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #6 and #30.

#6 #30

Mean 45,93333 43,2

Variance 0,163333 0,49

Observations 3 3

Hypothesized Mean Difference

0

df 3

t Stat 5,857143

P(T<=t) one-tail 0,004961

t Critical one-tail 2,353363

P(T<=t) two-tail 0,009922

t Critical two-tail 3,182446

Table G 2 T-test report for formulations 7 and 31

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #7 and #31

#7 #31

Mean 49,4 41,26667

Variance 3,87 0,163333

Observations 3 3

Hypothesized Mean Difference

0

df 2

t Stat 7,014507

P(T<=t) one-tail 0,009862

t Critical one-tail 2,919986

P(T<=t) two-tail 0,019725

t Critical two-tail 4,302653

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulation #8 and #32

#8 #32

Mean 43,06667 42,56667

Variance 0,003333 0,423333

Observations 3 3

Hypothesized Mean Difference

0

df 2

t Stat 1,325825

P(T<=t) one-tail 0,158029

t Critical one-tail 2,919986

P(T<=t) two-tail 0,316059

t Critical two-tail 4,302653

Table G 3 T-test report for formulations 8 and 32

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #9 and #33

#9 #33

Mean 43,36667 39,66667

Variance 0,0533 0,9033

Observations 3 3

Hypothesized Mean Difference

0

df 2

t Stat 6,552123

P(T<=t) one-tail 0,011255

t Critical one-tail 2,919986

P(T<=t) two-tail 0,02251

t Critical two-tail 4,302653

Table G 4 T-test report for formulations 9 and 33

I

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #18 and #34

#18 #34

Mean 57,86667 46,66667

Variance 0,643333 0,173333

Observations 3 3

Hypothesized Mean Difference

0

df 3

t Stat 21,46625

P(T<=t) one-tail 0,000111

t Critical one-tail 2,353363

P(T<=t) two-tail 0,000221

t Critical two-tail 3,182446

Table G 5 T-test report for formulations 18 and 34

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #19 and #35

#19 #35

Mean 57,06667 46,86667

Variance 0,643333 0,163333

Observations 3 3

Hypothesized Mean Difference

0

df 3

t Stat 19,67043

P(T<=t) one-tail 0,000144

t Critical one-tail 2,353363

P(T<=t) two-tail 0,000287

t Critical two-tail 3,182446

Table G 6 T-test report for formulations 19 and 35

Table G 7 T-test report for formulations 20 and 36

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #20 and #36

#20 #36

Mean 56,63333 51,03333

Variance 0,323333 0,343333

Observations 3 3

Hypothesized Mean Difference

0

df 4

t Stat 11,87939

P(T<=t) one-tail 0,000144

t Critical one-tail 2,131847

P(T<=t) two-tail 0,000288

t Critical two-tail 2,776445

Table G 8 T-test report for formulatins 21 and 37

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #21 and #37

#21 #37

Mean 63,33333 43,2

Variance 3,163333 0,49

Observations 3 3

Hypothesized Mean Difference

0

df 3

t Stat 18,24449

P(T<=t) one-tail 0,00018

t Critical one-tail 2,353363

P(T<=t) two-tail 0,000359

t Critical two-tail 3,182446

J

Table G 10 T-test report for formulations 23 and 38

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #23 and #38

#23 #38

Mean 52,06667 43,5

Variance 1,563333 1,75

Observations 3 3

Hypothesized Mean Difference

0

df 4

t Stat 8,151545

P(T<=t) one-tail 0,000616

t Critical one-tail 2,131847

P(T<=t) two-tail 0,001233

t Critical two-tail 2,776445

t-Test: Two-Sample Assuming Unequal Variances

Difference between formulations #28 and #39

#28 #39

Mean 60,96667 53,63333

Variance 0,333333 18,80333

Observations 3 3

Hypothesized Mean Difference

0

df 2

P(T<=t) one-tail 0,050485

t Critical one-tail 2,919986

P(T<=t) two-tail 0,100969

t Critical two-tail 4,302653

Table G 9 T-test report for formulations 28 and 39.

K

9.8 Appendix H

Data for the peroxide value.

Condition & Formulation

Flask Measurem. g Oil ml titrant PV Average SD g Extracted

% extracted

0 point 1 1 0,435 6,5 14,94

2 1 0,303 5,3 17,49

3 1 0,3288 5,4 16,42 16,29 1,05

A0

30 1 1 0,3637 3,6 9,90

1 2 0,3638 3,8 10,45

1 3 0,3665 4,2 11,46 2,9 63%

2 1 0,3793 5,8 15,29

2 2 0,3482 5,25 15,08

2 3 0,3448 4,9 14,21 2,88 63%

3 1 0,3742 5,1 13,63

3 2 0,3563 5 14,03

3 3 0,3952 4,95 12,53 12,95 1,86 3,04 66%

33 1 1 0,26 5 19,23 0,26 6%

2 1 0,3724 6,9 18,53

2 2 0,3923 8,4 21,41

2 3 0,3903 8,2 21,01

2 4 0,4521 8,85 19,58 4,48 98%

3 1 0,1708 2,9 16,98 19,46 1,49 0,84 18%

NF 1 1 0,31 10,4 33,55

1 2 0,3361 11,6 34,51

1 3 0,3056 9,4 30,76

1 4 0,3279 9,7 29,58

1 5 0,322 10 31,06 31,89 1,84

A1

30 1 1 0,321 14 43,61

1 2 0,3023 14,85 49,12

1 3 0,315 15,65 49,68 3,53 77%

2 1 0,3608 15,2 42,13

2 2 0,3728 16 42,92

2 3 0,317 13,7 43,22 2,39 52%

3 1 0,3517 14,2 40,38

3 2 0,337 13,75 40,80

3 3 0,3011 11,9 39,52 43,49 3,41 2,46 54%

33 1 1 0,3552 16,1 45,33

1 2 0,3212 15,7 48,88

1 3 0,4014 20,8 51,82 3,27 71%

2 1 0,3079 9,4 30,53

L

2 2 0,3081 11 35,70

2 3 0,4042 13,2 32,66 3,81 83%

3 1 0,3195 12,5 39,12

3 2 0,3028 12,1 39,96

3 3 0,3055 12,1 39,61 40,40 19,73 4,61 100%

NF 1 1 0,3685 18 48,85

1 2 0,3346 14,2 42,44

1 3 0,31 13 41,94

1 4 0,3147 14,6 46,39

1 5 0,388 18 46,39

1 6 0,4191 22 52,49 46,42 3,62

A2

30 1 1 0,3938 31 78,72

1 2 0,3961 30 75,74

1 3 0,3177 22,5 70,82 4,29 94%

2 1 0,315 24,5 77,78

2 2 0,3195 25,25 79,03

2 3 0,3224 24 74,44 4,47 97%

3 1 0,3263 30 91,94

3 2 0,3257 29,5 90,57

3 3 0,3358 31,2 92,91 81,33 7,78 3,90 85%

33 1 1 0,34 19,5 57,35

1 2 0,3474 20,5 59,01

1 3 0,34 11,5 33,82 5,03 110%

2 1 0,3312 21 63,41

2 2 0,3021 18 59,58

2 3 0,424 24,5 57,78 5,71 124%

3 1 0,303 17 56,11

3 2 0,329 20 60,79

3 3 0,508 25 49,21 55,23 8,41 5,32 116%

NF

1 1 0,3382 26 76,88

1 2 0,3388 22 64,94

1 3 0,3487 23,6 67,68

1 4 0,3193 20,4 63,89

1 5 0,329 19,4 58,97

1 6 0,3494 19,2 54,95

1 7 0,3273 21,5 65,69

1 8 0,3404 22,3 65,51

1 9 0,3435 24 69,87 65,37 5,86

A3

30 1 1 0,3975 18 45,28

1 2 0,3525 14,35 40,71 2,02 0,44

2 1 0,44 17 38,64

2 2 0,3415 12,65 37,04

2 3 0,2205 9,15 41,50 1,87 0,41

M

3 1 0,3598 10,5 29,18

3 2 0,3925 11,5 29,30

3 3 0,3182 7,4 23,26 35,61 7,06 2,28 0,50

33 1 1 0,316 10,6 33,54

1 2 0,1471 6,15 41,81 3,41 0,74

2 1 0,3019 3,1 10,27

2 2 0,3214 6,4 19,91

2 3 0,3303 4,8 14,53 3,68 0,80

3 1 0,3765 11 29,22

3 2 0,4375 13 29,71

3 3 0,338 10,2 30,18 26,15 9,77 4,90 1,07

NF 1 1 0,3165 12,8 40,44

1 2 0,3196 8,7 27,22

1 3 0,3343 9,4 28,12

1 4 0,3239 10,5 32,42

1 5 0,3028 9,5 31,37

1 6 0,3352 10,2 30,43

1 7 0,3274 10,7 32,68

1 8 0,3243 9,7 29,91

1 9 0,3836 12,5 32,59 31,69 3,59

Table H 1 Data for the peroxide values for formulations 41 and 44 and no formulations in conditions A0, A1, A2 and A3.

N

9.9 Appendix I

Results from excel for the T-test PVs between formulation 30, 33 and no formulation in

conditions A0, A1, A2 and A3.

t-Test: Two-Sample Assuming Unequal Variances

Difference betwen A0 #30 and #33

#30 #33

Mean 12,95231 19,45586

Variance 3,902593 2,660232

Observations 9 6

Hypothesized Mean Difference

0

df 12

t Stat -6,94467

P(T<=t) one-tail 7,75E-06

t Critical one-tail 1,782288

P(T<=t) two-tail 1,55E-05

t Critical two-tail 2,178813

Table I 1 T-test report for formulations 30 and 33 cond. A0

t-Test: Two-Sample Assuming Unequal Variances

Difference between A1 #30 and #3

#30 #33

Mean 43,48696 40,40053

Variance 13,12875 51,05628

Observations 9 9

Hypothesized Mean Difference

0

df 12

t Stat 1,15574

P(T<=t) one-tail 0,13514

t Critical one-tail 1,782288

P(T<=t) two-tail 0,270281

t Critical two-tail 2,178813

Table I 2 T-test report for formulations 30 and 33 cond. A1

t-Test: Two-Sample Assuming Unequal Variances

Difference between A2 #30 and #33

#30 #33

Mean 81,32843 55,22961

Variance 68,25513 79,65407

Observations 9 9

Hypothesized Mean Difference

0

df 16

t Stat 6,437905

P(T<=t) one-tail 4,1E-06

t Critical one-tail 1,745884

P(T<=t) two-tail 8,2E-06

t Critical two-tail 2,119905

Table I 3 T-test report for formulations 30 and 33 cond.

t-Test: Two-Sample Assuming Unequal Variances

Difference between A3 #30 and #33

#30 #33

Mean 35,61321 26,14679

Variance 57,02715 109,184

Observations 8 8

Hypothesized Mean Difference

0

df 13

t Stat 2,07683

P(T<=t) one-tail 0,029103

t Critical one-tail 1,770933

P(T<=t) two-tail 0,058206

t Critical two-tail 2,160369

Table I 4 T-test report for formulations 30 and 33 cond.

O

A2 A3

t-Test: Two-Sample Assuming Unequal Variances

Difference between A0 #30 and "No formulation"

#30 No formuluation

Mean 12,95231 31,89184

Variance 3,902593 4,233421

Observations 9 5

Hypothesized Mean Difference

0

df 8

t Stat -16,7383

P(T<=t) one-tail 8,21E-08

t Critical one-tail 1,859548

P(T<=t) two-tail 1,64E-07

t Critical two-tail 2,306004

Table I 5 T-test report for formulations 30 and Nf cond. A0

t-Test: Two-Sample Assuming Unequal Variances

Difference between A1 #30 and "No formulation"

#30 No formulation

Mean 43,48696 46,41658

Variance 13,12875 15,74764

Observations 9 6

Hypothesized Mean Difference

0

df 10

t Stat -1,44978

P(T<=t) one-tail 0,088873

t Critical one-tail 1,812461

P(T<=t) two-tail 0,177747

t Critical two-tail 2,228139

Table I 6 T-test report for formulations 30 and Nf cond A1

t-Test: Two-Sample Assuming Unequal Variances

Difference between A2 #30 and "No formulation"

#30 No formulation

Mean 81,32843 65,37438

Variance 68,25513 38,75694

Observations 9 9

Hypothesized Mean Difference

0

df 15

t Stat 4,626748

P(T<=t) one-tail 0,000165

t Critical one-tail 1,75305

P(T<=t) two-tail 0,000329

t Critical two-tail 2,13145

Table I 7 T-test report for formulations 30 and nf, cond A2

t-Test: Two-Sample Assuming Unequal Variances

Difference between A3 #30 and "No formulation"

#30 No formulation

Mean 35,61321 31,68683

Variance 57,02715 14,56212

Observations 8 9

Hypothesized Mean Difference

0

df 10

t Stat 1,327631

P(T<=t) one-tail 0,106904

t Critical one-tail 1,812461

P(T<=t) two-tail 0,213809

t Critical two-tail 2,228139

Table I 8 T-test report for formulations 30 and nf, cond A3

P

t-Test: Two-Sample Assuming Unequal Variances

Difference between A0 #33 and "No formulation"

#33 No formulation

Mean 18,71359 31,89184

Variance 10,37855 4,233421

Observations 6 5

Hypothesized Mean Difference

0

df 9

t Stat -8,21008

P(T<=t) one-tail 8,99E-06

t Critical one-tail 1,833113

P(T<=t) two-tail 1,8E-05

t Critical two-tail 2,262157

Table I 9 T-test report for formulations 33 and Nf, cond A0

t-Test: Two-Sample Assuming Unequal Variances

Difference between A1 #33 and "No formulation"

#33 No formulation

Mean 40,40053 46,41658

Variance 51,05628 15,74764

Observations 9 6

Hypothesized Mean Difference

0

df 13

t Stat -2,08851

P(T<=t) one-tail 0,028494

t Critical one-tail 1,770933

P(T<=t) two-tail 0,056988

t Critical two-tail 2,160369

Table I 10 T-test report for formulations 33 and Nf, cond A1

Table I 11T-test report for formulations 33 and Nf, cond A2

t-Test: Two-Sample Assuming Unequal Variances

Difference between A2 #33 and "No formulation"

#33 No formulation

Mean 55,22961 65,37438

Variance 79,65407 38,75694

Observations 9 9

Hypothesized Mean Difference

0

df 14

t Stat -2,79684

P(T<=t) one-tail 0,007134

t Critical one-tail 1,76131

P(T<=t) two-tail 0,014269

t Critical two-tail 2,144787

Table I 12 T-test report for formulations 33 and Nf, cond A3

t-Test: Two-Sample Assuming Unequal Variances

Difference between A3 #33 and "No formulation"

#33 No formulation

Mean 26,14679 31,68683

Variance 109,184 14,56212

Observations 8 9

Hypothesized Mean Difference

0

df 9

t Stat -1,41792

P(T<=t) one-tail 0,094948

t Critical one-tail 1,833113

P(T<=t) two-tail 0,189896

t Critical two-tail 2,262157