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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
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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
4 5 6
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
*
15
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
36
(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
39
mostly been explored for their potential to enhance transdermal and oral absorption. In this
40
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-
42
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,
46
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
49
media. The OLP derived niosomes successfully sustained in vitro drug release, in vivo
50
pharmacokinetics and the anti-inflammatory effect of flurbiprofen in an acute (rat paw
51
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
56
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-
58
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
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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
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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:
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Vesicular and colloidal delivery systems that control drug delivery and release to
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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
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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
81
aqueous media2,
6, 7
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physical properties to liposomes. They are also biocompatible and able to entrap hydrophilic
83
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
89
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
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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
113
doses for intravenous delivery. There are generalized advantages to intravenous dosing such
114
as a rapid onset of effect and ability to dose to patients unable to take oral dosage forms such
115
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-
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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
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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
138
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
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treatment for pain and inflammation.
In the first part of this study, the surfactant
142 143
Methods:
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Reagents and Supplies: Flurbiprofen was obtained as a gift sample from FDC Ltd. (Mumbai,
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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
159
above and sterile PBS (pH 7.4) was used throughout the study.
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Preparation of the proniosome formulations:
161
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,
164
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
166
contained in a 100 mL round bottomed flask attached to a rotary evaporator. After
167
introduction of each aliquot the flask was rotated in a 60-65 °C water bath under vacuum for
168
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.
170
Hydration of the proniosome powder to form niosomes: Proniosome formulations prepared as
171
above were tested for vesicle formation at three different hydration temperatures: 25°C,
172
37°C and 45°C. Sterile saline (1 mL) at 25°C, 37°C and 45°C was added to the dry proniosome
173
powder (20 mg) in a glass vial and the vial was agitated manually for 8-12 seconds. Vesicles
174
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
176
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)
178
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.
181
Sterilization of proniosome powder for parenteral administration: Once optimal formulations
182
were chosen, the proniosomes were prepared for parenteral administration using the same
183
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)
185
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
193
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
196
SEM, operating at 1 kV (Zeiss DSM 982 Gemini; LEO Electron Microscopy Ltd, Cambridge,
197
UK)12.
198
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
200
Olympus IMT2 inverted-stage microscope (Olympus, New Delhi, India) and viewed at 100x
201
magnification. Photographs were taken using ECZ Capture software (Topcon, NJ, USA).
202
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
204
vesicles were then analyzed by TEM (TECNAI 200kV, TEM; FEI, Netherland). The vesicles were
205
placed on a copper grid, with subsequent negative staining (with 2.0% phosphotungustic acid)
206
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
209
was then determined by dynamic light scattering based on laser diffraction using a Malvern
210
zetasizer (Model nano S Ver. 6.20, UK)24. Out of three measurements at 25°C the average
211
vesicle size distribution was determined by intensity distribution.
212
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
214
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
216
supernatant was generally cloudy. After the first centrifugation the supernatant was
217
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.
221
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
225
(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
238
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
246
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
254
Wistar rats (body weight 225–275 g) was collected from the retro-orbital plexus and placed in
255
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
301
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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Molecular Pharmaceutics
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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
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spectrometer with an electrospray ionization interface (ESI) and a LC system consisting of a
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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
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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
Molecular Pharmaceutics
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1.72 min, respectively. The analytical and preparation methods used for quantification of
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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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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 =
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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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The non-compartmental pharmacokinetic parameters for all three formulations were
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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
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high temperature required for vesicle formation would not be convenient or compatible with
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preparation for parenteral administration. In contrast, vesicles formed at all three
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temperatures for the OLP formulations (span 20 and span 80 combinations) with only a few
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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
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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
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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
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efficiency for the OLP formulations was then measured (Table 2). The entrapment efficiency
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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
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efficiency. The surface morphology of the OLP10 and OLP11 proniosome powders was 16 ACS Paragon Plus Environment
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Molecular Pharmaceutics
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subsequently studied using SEM (Figure 2). The surface study clearly distinguished the
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formulation from the sorbitol carrier, since it appeared smoother and to have fewer “fine
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structures” like stubbles and sharp corners, whereas the sorbitol powder showed crystals
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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
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(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
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dynamic light scattering data in Table 2.
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The suitability of the OLP10 and OLP11 formulations for in vivo administration via
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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
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dispersions and were inspected visually against a black and white background to ensure
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clarity and the lack of any particulate matter. No particulate matter was observed. Sterility
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testing was performed using three vials of each formulation by a direct inoculation method
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following Indian Pharmacopoeia guidelines. The culture tubes were observed after 24 hours
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and up to 7 days post inoculation for any growth or turbidity. No growth or turbidity was
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observed in any of the tubes up to 7 days after preparation, suggesting the sterility of the
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formulations.
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The comparative cytotoxicity of flurbiprofen solution, OLP10 and OLP11 proniosome
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formulations against U-937 cells (monocytes/macrophages) was determined via MTT assay
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(Figure 4A). There was no significant difference in cell viability on incubation with flurbiprofen
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solution or flurbiprofen in either proniosome formulation (OLP10 and OLP11). The cell
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viability was >76% on incubation with proniosome formulations for 24 hours, even at highest
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tested concentration of 100 µg/mL flurbiprofen. This data confirms the biocompatibility of
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the proniosome formulations and lack of cytotoxicity. The hemolytic toxicity of the chosen
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OLP10 and OLP11 formulations was also evaluated in vitro and confirmed suitability for
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intravenous injection (Figure 4B). The proniosome formulation OLP11 caused significantly
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lower (p