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A single intravenous dose of novel flurbiprofenloaded proniosome formulations provides prolonged systemic exposure and anti-inflammatory effect Preeti Verma, Sunil Kumar Prajapati, Rajbharan Yadav, Danielle Senyschyn, Peter R Shea, and Natalie L Trevaskis Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00504 • Publication Date (Web): 15 Sep 2016 Downloaded from http://pubs.acs.org on September 16, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

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A single intravenous dose of novel flurbiprofen-loaded proniosome formulations provides

3

prolonged systemic exposure and anti-inflammatory effect

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Preeti Vermaa,b, Sunil K Prajapatib, Rajbharan Yadava, Danielle Senyschyn, Peter R Sheac and

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Natalie L Trevaskisa,*

8

a

9

Monash University, Parkville, VIC, Australia 3052

Drug Delivery Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,

10

b

11

c

Institute of Pharmacy, Bundelkhand University, Jhansi-284001, Uttar Pradesh, India

Anaesthetic Group Ballarat, 6 Drummond Street North Ballarat, VIC, Australia 3350

12 13 14

*

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Natalie L Trevaskis, PhD

16

Drug Delivery Disposition and Dynamics

17

Monash Institute of Pharmaceutical Sciences, Monash University

18

381 Royal Parade,

19

Parkville, VIC, Australia 3052

20

Tel: +61-3 990 39708, Fax: +61-3 990 39583

21

Email: [email protected]

Corresponding author

22 23

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Table of contents graphic:

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Molecular Pharmaceutics

Abstract:

30

Vesicular and colloidal delivery systems can be designed to control drug release

31

spatially and temporally to improve drug efficacy and side effect profiles. Niosomes (vesicles

32

prepared from nonionic surfactants in aqueous media) are gaining interest as an alternative

33

vesicular delivery system as they offer advantages such as biocompatibility, chemical stability,

34

low cost, high purity and versatility. However, the physical stability of niosomes, like other

35

vesicular systems, is limited by vesicle fusion, aggregation and leakage. Proniosomes

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(dehydrated powder or gel formulations that spontaneously form niosomes on hydration

37

with aqueous media) can overcome these physical stability problems and are more

38

convenient for sterilization, storage, transport, distribution and dosing. Proniosomes have

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mostly been explored for their potential to enhance transdermal and oral absorption. In this

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study we assess, for the first time, the potential for hydrated proniosomes to sustain systemic

41

exposure and therapeutic effect after intravenous delivery. Proniosomes carrying the anti-

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inflammatory drug, flurbiprofen, were prepared by spraying different non-ionic surfactants

43

(span 20, span 40 and span 60 in varying ratios with span 80) and cholesterol onto a sorbitol

44

carrier. The proniosome powders were characterized for surface morphology and flow

45

properties. Niosome formation was assessed at three different hydration temperatures (25,

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37 and 45°C) and the niosomes were assessed for vesicle size, entrapment efficiency and

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sterility. OLP proniosomes prepared with a high ratio of span 80 to span 20 were found to

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spontaneously form vesicles of small size and high drug loading on hydration with aqueous

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media. The OLP derived niosomes successfully sustained in vitro drug release, in vivo

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pharmacokinetics and the anti-inflammatory effect of flurbiprofen in an acute (rat paw

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edema) model of inflammation when compared to a control solution formulation. The study

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demonstrates that hydrated proniosomes can prolong systemic drug exposure over 3 days

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and provide a sustained therapeutic effect. The developed proniosomes represent a novel

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approach to treat acute pain and inflammation with the potential to be administered as a

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single intravenous dose by a clinician at the time of injury or surgery that provides adequate

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relief for several days and reduces fluctuations in therapy. Similar systems loaded with

57

different drugs have potential for broader application in anaesthesia, anti-infective, anti-

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emetic and cancer therapy.

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Keywords: niosomes; proniosomes; parenteral; pharmacokinetics; pharmacodynamics;

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sustained release; anti-inflammatory

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Abbreviations: pharmacokinetics (PK), pharmacodynamics (PD) intravenous (IV), phosphate

62

buffer saline (PBS), scanning electron microscopy (SEM), transmission electron microscopy

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(TEM), high performance liquid chromatography (HPLC), gastrointestinal (GI), red blood cells

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(RBCs), poly ethylene (PE), Akaike information criterion (AIC) and Schwartz information

65

criterion (SIC), oleate laurate proniosome (OLP), oleate palmitate proniosome (OPP), oleate

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stearate proniosome (OSP).

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Molecular Pharmaceutics

Introduction:

69

Vesicular and colloidal delivery systems that control drug delivery and release to

70

improve therapeutic effect, while minimizing side effects and toxicities, have been a major

71

focus of pharmaceutical science for several decades1-4. Liposomes, i.e. vesicles composed of

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one or more phospholipid bilayers, were the first of these delivery systems to make the

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transition from concept to clinic. Liposomes possess many of the desired attributes for

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vesicular delivery systems including biocompatibility, versatility in being able to encapsulate

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both hydrophilic and hydrophobic drugs, and the ability to control drug release temporally

76

and spatially. Liposomes are, however, limited in chemical and physical stability in aqueous

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dispersion, being prone to hydrolysis, oxidation, aggregation, fusion and leakage5. To

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overcome some of the limitations of liposomes there has been recent interest in the

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application of alternate systems such as niosomes.

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Niosomes are uni- or multi-lamellar vesicles prepared from nonionic surfactants in

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aqueous media2,

6, 7

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physical properties to liposomes. They are also biocompatible and able to entrap hydrophilic

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and hydrophobic drugs. The surfactants from which niosomes are prepared are additionally

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more chemically stable, low in cost, higher in purity, readily derivatized and provide greater

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versatility when compared to the phospholipids from which liposomes are prepared8. Like

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liposomes, however, the physical stability of niosomes in aqueous dispersions is limited by

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processes such as vesicular fusion, aggregation and leakage9. Proniosomes represent an

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approach to overcome the physical stability problems of aqueous liposome and niosome

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dispersions, as well as the chemical stability limitations of liposomes10-14. Proniosomes are

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prepared as either liquid crystals of non-ionic surfactants with a gel-like consistency or as dry,

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free-flowing powder formulations in which the surfactants are coated onto water-soluble

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carrier particles (e.g. sorbitol, mannitol, maltodextrin, sucrose stearate etc.). Both types of

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proniosome formulation can be readily converted to noisome suspensions prior to use

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through reconstitution and brief agitation in warm aqueous media13. Proniosomes are most

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often prepared using the slurry method11,

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carrier particles13. Different methods of preparation and types of surfactants alter the

. Niosomes thus share similar structure, preparation techniques and

12

or by spraying surfactants onto water-soluble

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reconstituted niosome shape, size, number of bilayers, entrapment efficiency and stability

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which can ultimately influence drug release and biodistribution in vivo15.

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Niosomes were initially described in the cosmetic industry in the 1970s and have thus

100

been intensly investigated for topical applications, to increase drug contact time and skin

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penetration16. A couple of reports have demonstrated that niosomes, like liposomes, can

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prolong the systemic exposure and alter the organ biodistribution of encapsulated drugs,

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such as anti-cancer and anti-infective agents, following intravenous delivery17, 18. In general,

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there have been relatively few publications that have described the preparation and

105

evaluation of proniosomes and the majority of these reports have focused on in vitro

106

evaluation. Proniosome gels have been developed for topical application and dry powder

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proniosome formulations that can be compressed into table formulations have been

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developed for oral delivery10-14. As far as we are aware the potential for hydrated

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proniosomes to prolong systemic exposure after intravenous delivery has not previously been

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reported. This is despite the enhanced chemical and physical stability of proniosomes when

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compared to aqueous dispersions of niosomes and liposomes as outlined above. Pronisomes

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are also more convenient to sterilize, store, transport, distribute and separated into unit

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doses for intravenous delivery. There are generalized advantages to intravenous dosing such

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as a rapid onset of effect and ability to dose to patients unable to take oral dosage forms such

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as those with difficulty swallowing, delayed gastric emptying or intestinal motility (e.g.

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pediatric, elderly, critically unwell, vomiting, unconscious and sedated/unconscious patients).

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In many countries patients display a preference for injections over oral dosage forms as it is

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perceived that injections are more effective19-22. In prolonging systemic drug exposure, a

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parenteral proniosome formulation could further enhance treatment by avoiding the need

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for repeat injections or continuous intravenous infusions that can result in fluctuations in

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plasma concentrations, efficacy and toxicity or which are impractical and uncomfortable for

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patients. In this way, a parenteral proniosome formulation that prolongs systemic drug

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exposure has many potential therapeutic applications, including in anaesthetic, analgesic,

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anti-inflammatory,

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treatment20-22.

anti-emetic,

chemotherapeutic,

anti-psychotic

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anti-infective

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The present study aimed to develop a dry, free flowing proniosome formulation of the

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anti-inflammatory drug flurbiprofen that upon reconstitution and intravenous injection would

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prolong systemic exposure and provide sustained anti-inflammatory activity. This formulation

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has potential clinical application as a single dose treatment administered at the time of

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surgery or acute injuries such as fractures, sprains or strains to provide a prolonged anti-

131

inflammatory effect. This will reduce the need for repeat administration of anti-inflammatory

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and analgesic drugs that typically result in fluctuations in plasma drug concentrations and

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limit control of pain and inflammation.

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composition of the proniosome formulations was optimized to form niosomes with uniform

135

and small size, high drug entrapment efficiency and the ability to sustain drug release in vitro.

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Subsequently the optimized proniosome formulations were tested in vivo in pharmacokinetic

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studies and pharmacodynamic studies in a model of acute inflammation (a rat paw edema

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model). The studies confirm the potential of proniosomes as an inexpensive, stable and

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convenient alternative to other vesicular and colloidal delivery systems that can prolong

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systemic exposure and therapeutic effect thus representing a promising single dose

141

treatment for pain and inflammation.

In the first part of this study, the surfactant

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Methods:

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Reagents and Supplies: Flurbiprofen was obtained as a gift sample from FDC Ltd. (Mumbai,

145

India). Cholesterol (CDH, Delhi, India), spans (CDH, Delhi, India), chloroform (Merck, Mumbai,

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India), D-sorbitol (Sigma-Adrich, Bangalore, India), 0.45 µm sterile syringe filter nylon 13 mm

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(SFNY13 X, Axiva, Delhi, India) and dialysis cellophane tubing (MMCO14KDC, Spectrum India,

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Bangalore, India) were purchased from the listed suppliers. In the pharmacokinetic (PK)

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section the suppliers for some of these materials differed with D-sorbitol (Sigma-Aldrich,

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NSW, Australia), spans (Sigma-Aldrich, NSW, Australia), flurbiprofen (Cayman chemical

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company, Michigan, USA), chloroform (Sigma-Aldrich, NSW, Australia), sterile phosphate

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buffered saline (PBS; pH 7.4, Gibco; Dulbecco’s PBS; Thermo Fisher scientific, MA, USA), 0.45

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µm sterile syringe filters nylon 13 mm (Grace Division discovery sciences, VIC, Australia), 3 mL

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sterile syringes (Thermo Fisher scientific, MA, USA), isoflurane (IsoFlo, Abbott, VIC, Australia)

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and polyethylene tubing (PE 8050, Microtube extrusion, NSW, Australia) obtained from the 7 ACS Paragon Plus Environment

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listed suppliers. Acetonitrile (ACN; hyper-grade for liquid chromatography) was purchased

157

from Merck (Bayswater, VIC, Australia). Ultrapure water was obtained from a Milli-Q TM

158

system (EMD Millipore Corporation, MA, USA). All other chemicals were of analytical grade or

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above and sterile PBS (pH 7.4) was used throughout the study.

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Preparation of the proniosome formulations:

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Preparation of the proniosome powder: The surfactants and ratios tested for proniosome

162

formation is given in table 1. The proniosomes were prepared according to the method

163

described by Hu et al13 with some modification. Briefly, the required mass of surfactants,

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cholesterol and drug (flurbiprofen) were dissolved in 1.0 mL of chloroform and sprayed in

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small aliquots (250 µl per aliquot, manually using a pipette) onto the sorbitol powder

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contained in a 100 mL round bottomed flask attached to a rotary evaporator. After

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introduction of each aliquot the flask was rotated in a 60-65 °C water bath under vacuum for

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20-30 min to prepare a dry free flowing powder. The formulation was further dried in

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desiccators under vacuum at room temperature overnight.

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Hydration of the proniosome powder to form niosomes: Proniosome formulations prepared as

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above were tested for vesicle formation at three different hydration temperatures: 25°C,

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37°C and 45°C. Sterile saline (1 mL) at 25°C, 37°C and 45°C was added to the dry proniosome

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powder (20 mg) in a glass vial and the vial was agitated manually for 8-12 seconds. Vesicles

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were then visualized using optical microscopy. Vesicles were formed at all temperatures on

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hydration of the span 20 and span 80 proniosomes while the formulations containing span 40

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and span 60 required a higher temperature (>45°C) for vesicle formation. The span 40 and

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span 60 formulations were not explored further as the high hydration temperature (>45°C)

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required for vesicle formation would not be practical in the clinical setting. The span 20 and

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span 80 surfactant combinations (OLP proniosomes) were thus optimized further for

180

parenteral administration.

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Sterilization of proniosome powder for parenteral administration: Once optimal formulations

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were chosen, the proniosomes were prepared for parenteral administration using the same

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spray method as above with a few modifications for sterile preparation. The surfactant and

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drug mixture was filtered through a 0.2 µm membrane syringe filter (Axiva, Delhi, India)

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previously sterilized by autoclaving at 132°C for 8 minutes. The sterile surfactant and drug 8 ACS Paragon Plus Environment

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mixture was then loaded onto the sorbitol carrier contained in a 100 mL round bottom flask

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sterilized by autoclaving at 121°C for 15 minute. Once prepared, the dry free flowing powders

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were filled manually into sterilize glass vials in an aseptic area and then stoppered

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immediately and sealed with a 13 mm aluminum seal.

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Characterization of the proniosome formulations:

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Scanning electron microscopy (SEM) of proniosomes: To prepare the samples for SEM the

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proniosome powder was placed on a double-sided carbon tape, attached on an aluminum

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stub, and stored under vacuum overnight. The next day the proniosome powders were

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coated with Au/Pd at 180 mA for 1 minute using Polaron E5100 (VG MicroTech, Uckfield, UK)

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under an argon atmosphere. The electron micrographs were taken using a field-emission

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SEM, operating at 1 kV (Zeiss DSM 982 Gemini; LEO Electron Microscopy Ltd, Cambridge,

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UK)12.

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Optical microscopy examination of niosome vesicles: For optical microscopy a drop of the

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hydrated proniosomes was placed onto a glass slide under a coverslip, positioned on an

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Olympus IMT2 inverted-stage microscope (Olympus, New Delhi, India) and viewed at 100x

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magnification. Photographs were taken using ECZ Capture software (Topcon, NJ, USA).

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Transmission electron microscopy (TEM) of niosome vesicles: Selected proniosome

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formulations were hydrated with distilled water at 37°C to form niosome vesicles. The

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vesicles were then analyzed by TEM (TECNAI 200kV, TEM; FEI, Netherland). The vesicles were

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placed on a copper grid, with subsequent negative staining (with 2.0% phosphotungustic acid)

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and dried at room temperature followed by TEM analysis at 80 kV as described previously23.

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Niosome vesicle size analysis: To determine the size of the proniosome derived vesicles the

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proniosome powder was hydrated with PBS and vortexed for 2 min. The size of the vesicles

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was then determined by dynamic light scattering based on laser diffraction using a Malvern

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zetasizer (Model nano S Ver. 6.20, UK)24. Out of three measurements at 25°C the average

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vesicle size distribution was determined by intensity distribution.

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Drug entrapment efficiency: To evaluate drug loading capacity, OLP proniosomes with

213

composition described in Table 2, were prepared as described above. A set mass (100 mg) of

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the proniosomes was hydrated in 10 mL milliQ water (EMD Millipore Corporation, MA, USA) 9 ACS Paragon Plus Environment

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at 37°C, vortexed for 2 minutes and centrifuged at 3,000 g and 25°C for 40 minutes25, 26. The

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supernatant was generally cloudy. After the first centrifugation the supernatant was

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collected, diluted 1:10 with milliQ water and centrifuged again at 3,000 g and 25°C for 30

218

minutes to ensure complete removal of the niosomes. The supernatant was then analyzed for

219

free drug content from UV absorbance at 247 nm using a UV-vis spectrophotometer (UV-

220

1700, Shimadzu corporation, Kyoto, Japan)19.

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The % drug entrapment efficiency (DEE) of the niosomes was calculated using the following

222

equation27:

223

%DEE =

224

In vitro drug release study: A 50 mg sample of proniosome powder was added to 5 mL of PBS

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(pH 7.4), vortexed for 5 seconds and filled into a dialysis tube (MMCO14KDC, Spectrum India,

226

Bangalore, India) made with a cellophane membrane that was sealed at each end. The

227

dialysis tube was immersed in 200 mL of PBS (pH 7.4) in a 250 mL glass beaker. The beaker

228

was maintained at a constant temperature of 37 °C and the buffer was stirred with a magnet

229

at a speed of 600 rpm. At set time points between 0 to 24 h, 0.5 mL aliquots (n=3 replicates)

230

were sampled from the glass beaker and replaced with PBS (pH 7.4). Drug concentration in

231

the collected samples was measured from UV absorbance at 247 nm using UV-

232

spectrophotometer as described above and drug release into the buffer subsequently

233

calculated25.

234

In vitro cytotoxicity assay

235

Cell

236

diphenyltetrazolium bromide) assay. Human histiocytic lymphoma cells (U-937) were

237

purchased from ATCC (VA, USA). Briefly, 5000 cells were plated in 96-well microplates and

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allowed to differentiate into macrophages via addition of 100 μL media containing 100 ng

239

PMA (Phorbol 12-myristate 13-acetate; Sigma-Aldrich, NSW, Australia) per well and incubated

240

for 24h28. Media in triplicate wells was replaced with media (100 μL) containing 10 µg/mL or

241

100 µg/mL flurbiprofen in solution, OLP10 proniosomes or OLP11 proniosomes. Control wells

242

(6 wells) were treated with media only. Cells were incubated with formulations for 24 h, 100

243

µL MTT (5 mg/mL) was then added to each well (except for 3 blank cells) and cells were

Mass of drug entrapped × 100 Total mass of drug added

viability

was

determined

(1)

using

a

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-

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incubated for a further 2 h. The media was replaced with 100 µL dimethyl sulfoxide (DMSO;

245

MP Biomedical, SA, Australia) and following a brief vortex, the color intensity of each well was

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analyzed using a Flourostar microplate reader (Bio-Strategy, Auckland, New Zealand) at 570

247

nm. The percent cell viability was calculated using the following equation:

248

% Cell viability =

249

Where, ABs = Absorbance of formulation treated cells and ABb = Average absorbance of blank

250

cells not treated with MTT and ABc = mean absorbance of control (media only), untreated

251

cells.

252

In vitro hemolytic toxicity assay: The hemolytic toxicity of the niosome dispersions was

253

evaluated using an established method to quantitate hemoglobin release29. Blood from male

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Wistar rats (body weight 225–275 g) was collected from the retro-orbital plexus and placed in

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eppendorf tubes containing 3% w/v sodium citrate (anticoagulant). Red blood cells (RBCs)

256

were separated from the blood by centrifugation at 2000 g for 5 minutes (Mini centrifuge;

257

MCKD07-Zenith, USA) and were suspended in PBS (pH 7.4) to produce a 1.0% v/v RBC

258

suspension. To test for hemolysis, 0.5 mL of a 2.0% m/v dispersion of flurbiprofen in the

259

selected proniosome formulations (OLP10, OLP11 in Table 2) in PBS (pH 7.4) or a 2.0 % m/v

260

solution of flurbiprofen in PBS (pH 7.4) was added to 4.5 mL of normal saline and mixed with

261

1 mL of the RBC suspension. As a positive control 1 mL RBC suspension was also diluted with

262

5 mL distilled water which was considered to produce 100% hemolysis. As a negative control

263

that acted as a blank the RBC suspension was diluted 6 fold with normal saline. All hemolysis

264

test samples were kept in an incubator for 1 hour at 37°C. After 1 hour, the mixtures were

265

centrifuged at 3,000 g for 5 minutes and the supernatants were taken. The supernatants

266

were diluted (1:2) with normal saline and absorbance was measured at 247 nm using a UV

267

spectrophotometer as described above. The absorbance of the supernatant collected after

268

incubation of the RBC suspension with saline was used as a blank and was subtracted from

269

the absorbance of all the samples. The % hemolysis was then determined using the following

270

equation29:

271

Hemolysis(%) =

ABs - ABb × 100 ABc - ABb

(2)

ABF ×100 AB100

(3)

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Where, ABF = Absorbance of formulation treated samples and AB100 = Absorbance of distilled

273

water control

274

Animal studies:

275

Animals: Male Wistar rats (body weight 225–280 g) were used for all studies. The

276

pharmacokinetic studies were approved by the Monash Institute of Pharmaceutical Sciences

277

animal ethics committee and conducted in accordance with the Australian and New Zealand

278

Council for the Care of Animals in Research and Teaching guidelines. The pharmacodynamic

279

and hemolytic toxicity studies were performed under the guidelines compiled by CPCSEA

280

(Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry

281

of Culture, Government of India) with approval no. BU/PHARMA/IAEC/08/008. The animals

282

were kept under standard conditions with free access to food and water at all times during

283

experiments unless otherwise noted.

284

Intravenous pharmacokinetic (PK) studies: Male Wistar Rats in the weight range 260-280 g

285

were divided into three groups: (1) control, (2) OLP10 and (3) OLP11 proniosome

286

formulations (composition specified in Table 2). On the first day of the study the rats were

287

anesthetized using isoflurane (IsoFlo, Abbott, VIC, Australia) and kept on a heated pad at 37°C

288

during surgery. The right carotid artery and right jugular vein were then cannulated using PE

289

tubing (PE 8030, Microtube extrusion, NSW, Australia) to enable blood collection and

290

intravenous dosing, respectively, as described previously30. Both the carotid artery and

291

jugular vein cannulas were exteriorized to the back of the neck and placed through a swivel-

292

tether apparatus to enable dosing and sampling. The rats were then allowed to regain

293

consciousness and recover overnight during which time they had free access to food and

294

water in a metabolic cage. Following overnight recovery, the control group rats were

295

administered 2.0 mL/kg of flurbiprofen solution (flurbiprofen dose; 2.5 mg/kg) via IV bolus

296

administration into the jugular vein cannula. The rats in the test groups were administered

297

2.0 mL/kg of OLP10 or OLP11 proniosome formulations (flurbiprofen dose; 2.5 mg/kg) via IV

298

bolus administration into the jugular vein. Whole blood (0.15 mL) was collected from the

299

carotid artery cannula before dosing (blank), at 0.017 (immediately after dosing), 0.083,

300

0.167, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 28, 48 and 72 hours into heparinized (2 IU) eppendorf tubes

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and centrifuged at 3,500g for 5 min to obtain plasma. Plasma samples were stored at -20°C 12 ACS Paragon Plus Environment

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prior to HPLC-MS/MS analysis of drug concentrations as described below. After 24 h sampling

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the rats were euthanized by IV administration via the jugular vein cannula of 1 mL lethabarb®

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equivalent to 325 mg pentobarbitone sodium (Virbac animal health, NSW, Australia).

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Quantification of flurbiprofen in plasma by HPLC-MS/MS

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Plasma sample preparation: To determine flurbiprofen concentration in the collected plasma

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samples, plasma proteins were precipitated via the addition of 300 µL of 100% acetonitrile

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containing 100 ng/mL ketoprofen (internal standard, IS) to a 50 µL aliquot of plasma. Samples

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were vortexed (Vortex mixer, Ratex Instruments, VIC, Australia) for 1.0 min followed by

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centrifugation at 14,000 g for 10 min (Eppendorf Centrifuge 5430 R, NSW, and Australia). A

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300 µL aliquot of the supernatant was subsequently transferred to a vial and evaporated to

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dryness under N2 (gas pressure, 3-4 psi) at 45 ± 50C for 30 ± 10 min (Biotag, TurboTax® LV,

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John Morris Scientific, VIC, Australia). The dried samples were reconstituted with 50 µL of

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10% acetonitrile in milli Q water, vortexed for 1 min and transferred to HPLC vials for analysis.

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HPLC-MS/MS analysis: A Shimadzu LC-MS8030 triple quadrupole type tandem mass

316

spectrometer with an electrospray ionization interface (ESI) and a LC system consisting of a

317

system controller CBM-20A, pump A and B LC-30AD, a SIL-30AC autosampler and a column

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oven CTO-20A at 60oC (all from Shimadzu Scientific Instruments, Kyoto, Japan) was used for

319

analysis. For the ESI the temperature of the desolvation line and heat block were set at 250°C

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and 400°C, nebulizing and drying gas flow rates were maintained at 3.0 L/min and 18 L/min,

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respectively. The flurbiprofen and ketoprofen (IS) samples were run through a Kinetex® C8

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column (1000A, 2.6 µm particle size, 50 mm × 2.1 mm from Phenomenex, CA, USA) with the

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mobile phase set at a flow rate of 0.5 mL/min. The mobile phases were run on a binary

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gradient with mobile phase A (MPA) consisting of miliQ water with 0.1 % formic acid and

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mobile phase B (MPB) consisting of 100% acetonitrile. The gradient used for elution started

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with 10% MPB for 0.8 min, increased to 70% MPB over 0.40 min, held at 70% MPB for the

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next 0.8 min then decreased to 10% MPB in 0.20 min and finally allowed to re-equilibrate at

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10% MPB for 1 min until the next injection. HPLC-MS/MS detection was performed with the

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ESI in negative ion mode by monitoring the signals of the parent ion to daughter ions

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transitions from 243 to 198.8 ([M-H]-1 - COOH-1) for flurbiprofen and 253 to 208.7 ([M-H]-1-

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COOH-1) for ketoprofen. The retention times for flurbiprofen and ketoprofen were 1.77 and 13 ACS Paragon Plus Environment

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332

1.72 min, respectively. The analytical and preparation methods used for quantification of

333

flurbiprofen in plasma samples were validated by determining the recovery of flurbiprofen

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from spiked plasma samples (n=4-5) and the accuracy and precision of measurement at low,

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medium and high concentrations of flurbiprofen on three different days. For all samples

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within the concentration range 20-1000 ng/mL the accuracy and precision were found to be

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within ±10% of expected with few exceptions at the lowest limit of quantitation (LLOQ) (at

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±15-20%).

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Non-compartmental pharmacokinetic (PK) analysis: The elimination rate constant (slope),

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half-life (i.e. 0.693/k) and mean residence time (MRT) were calculated from 2 to 8h for

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control and 8 to 72h for proniosomes, to allow a reliable comparison of the terminal half-life

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for the three formulations since the 24 h concentration was below the LLOQ for the control

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formulation. The Vss (volume of distribution at steady state) was calculated via multiplying

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clearance and MRT. The area under the plasma-concentration time curves from time zero to

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8 h, 24 h, 72 h and infinity (AUC0-8h, AUC0-24h, AUC0-72h and AUC0-∞, respectively) were

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calculated using the trapezoid rule to the last measured time point (Clast) and extrapolated to

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infinity by dividing Clast by the elimination rate constant, k.

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PK Modeling: PK modeling was performed to describe the time course of plasma drug

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concentrations and to compare the disposition kinetics of all three formulations using

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WinNonlin® Pro (Version 5.3, Pharsight Corp., Mountain View, CA). Initially two and three

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disposition compartment linear models were evaluated to fit the plasma PK profiles.

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However, the three compartment model was not able to describe the plasma PK profiles. All

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three formulation (control, OLP10 and OLP11) PK profiles were simultaneously fitted well to

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the two compartment linear model. After IV bolus dosing into the central compartment

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(plasma), drug was assumed to distribute to and from the central to peripheral compartment

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with distribution rate constants of k12 and k21, and eliminate from the central compartment

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with a first order elimination rate constant, kel. Model performance was evaluated by Akaike

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information criterion (AIC) and Schwartz information criterion (SIC)31.

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The differential equations and initial condition for the two compartment model were as given

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below.

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d (Plasma) = - kel ⋅ Plasma - k12 ⋅ Plasma + k21 ⋅ Peripheral dt

Initial condition = Dose/Vc

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(4)

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Molecular Pharmaceutics

d (Periphera l) = k12 ⋅ Plasma − k21 ⋅ Peripheral dt

Initial condition = 0

(5)

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Pharmacodynamic (PD) study in rats: The anti-inflammatory activity of the flurbiprofen drug

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delivery systems was evaluated using a carrageenan-induced rat paw edema model. This

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model was chosen to evaluate the potential of the proniosomes to inhibit acute inflammation

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since inflammation generally occurs relatively rapidly and subsides within 72 to 96 hours in

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this model32, 33. Twelve male Wistar rats (body weight 225 to 275 g), housed in standard cages

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with free access to water and a standard laboratory diet, were divided into four groups.

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Group 1 served as a vehicle control (normal saline only) whereas Group 2-4 were

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administered flurbiprofen solution (control formulation) or proniosome formulations OLP10

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or OLP11. The flurbiprofen solution, OLP10 and OLP11 were solubilized in phosphate buffer

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saline (pH 7.4) at a concentration of 1.25 mg/mL flurbiprofen. Rats were administered 2

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mL/kg of the formulations (resulting in the dose of 2.5 mg/kg flurbiprofen) or normal saline

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(vehicle control) via bolus intravenous injection into the tail vein 10 minutes prior to

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induction of paw edema.

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Prior to induction of paw edema the rats were weighed and marked on their right hind

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paw behind the tibia-tarsal junction34, 35. Paw edema was then induced 10 minutes following

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the intravenous injection of the control or flurbiprofen formulations via injection of 0.1 ml of

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1% w/v carrageenan (in 0.9% NaCL) into the right hind paw at the sub planter region. The paw

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volume was measured using a liquid plethysmometer36 before and at 0.167, 0.50, 1.0, 2.0,

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4.0, 6.0, 8.0 and 24h after carrageenan injection. Paw volumes did not differ significantly

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across the groups prior to induction of paw edema. The % inhibition of edema induced by

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carrageenan was calculated using the formula below:

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% Inhibition of edema =

385

Where,

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Vcontrol= mean paw volume of rats in vehicle control group

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Vtreated= paw volume of rat in test group

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Statistical analysis

VControl - VTreated × 100 VControl

(6)

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389

The non-compartmental pharmacokinetic parameters for all three formulations were

390

compared using a one-way ANOVA with post-hoc analysis. Significance was at a level of p
45°C). These formulations were therefore not explored further as the

401

high temperature required for vesicle formation would not be convenient or compatible with

402

preparation for parenteral administration. In contrast, vesicles formed at all three

403

temperatures for the OLP formulations (span 20 and span 80 combinations) with only a few

404

clumps visible by optical microscopy at 45°C. Some representative optical photomicrographs

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for formulations OLP10 and OLP11 are shown in Figure 1 A-D.

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The OLP proniosome formulations were then prepared using sterile manufacturing

407

techniques to enable parenteral administration as described in the methods. The size

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distribution of the niosome vesicles formed upon dilution of the proniosome formulations

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with PBS pH 7.4 was measured using dynamic light scattering intensity distribution profiles.

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All OLP formulations yielded niosomes that were relatively uniform in size and in the size

411

range 150-290 nm (Table 2). In general the size decreased with increasing span 80 content

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such that the OLP10 and OLP11 formulation derived niosomes exhibited the smallest size of

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all the formulations (188 nm and 153 nm, respectively). The percentage drug entrapment

414

efficiency for the OLP formulations was then measured (Table 2). The entrapment efficiency

415

was generally high for most formulations with the exception of OLP1 for which it was 59.8%.

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Based on these findings formulations OLP10 and OLP11 were chosen for further

417

analysis as they prepared niosomes of the smallest size and with high drug entrapment

418

efficiency. The surface morphology of the OLP10 and OLP11 proniosome powders was 16 ACS Paragon Plus Environment

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subsequently studied using SEM (Figure 2). The surface study clearly distinguished the

420

formulation from the sorbitol carrier, since it appeared smoother and to have fewer “fine

421

structures” like stubbles and sharp corners, whereas the sorbitol powder showed crystals

422

with sharp edges and fine structures (data not shown). The size and shape of the hydrated

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niosomes formed for the OLP10 and OLP11 formulations was then confirmed by TEM analysis

424

(Figure 3). The niosomes were spherical, homogeneous and of uniform size (Figure 3). The

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size range (145-320 nm) of the vesicles visualized via TEM was relatively consistent with the

426

dynamic light scattering data in Table 2.

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The suitability of the OLP10 and OLP11 formulations for in vivo administration via

428

intravenous injection was subsequently confirmed by sterility, clarity and cytotoxicity testing.

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The sealed vials of proniosome powders were hydrated with sterile PBS to form niosome

430

dispersions and were inspected visually against a black and white background to ensure

431

clarity and the lack of any particulate matter. No particulate matter was observed. Sterility

432

testing was performed using three vials of each formulation by a direct inoculation method

433

following Indian Pharmacopoeia guidelines. The culture tubes were observed after 24 hours

434

and up to 7 days post inoculation for any growth or turbidity. No growth or turbidity was

435

observed in any of the tubes up to 7 days after preparation, suggesting the sterility of the

436

formulations.

437

The comparative cytotoxicity of flurbiprofen solution, OLP10 and OLP11 proniosome

438

formulations against U-937 cells (monocytes/macrophages) was determined via MTT assay

439

(Figure 4A). There was no significant difference in cell viability on incubation with flurbiprofen

440

solution or flurbiprofen in either proniosome formulation (OLP10 and OLP11). The cell

441

viability was >76% on incubation with proniosome formulations for 24 hours, even at highest

442

tested concentration of 100 µg/mL flurbiprofen. This data confirms the biocompatibility of

443

the proniosome formulations and lack of cytotoxicity. The hemolytic toxicity of the chosen

444

OLP10 and OLP11 formulations was also evaluated in vitro and confirmed suitability for

445

intravenous injection (Figure 4B). The proniosome formulation OLP11 caused significantly

446

lower (p