This material may be protected by Copyright law (Title 17 U.S. Code)
`
`From the Department of Pharmaceutics, Faculty of Pharmacy. Assiut University, Assiut (Egypt)
`
`Preparation and Evaluation of Hydrocortisone
`Multiple Emulsions in Rabbit’s Eye
`
`By M. A. Kassem, M. A. Attia, S, M. Safwat, and M. El-Mahdy
`
`Multiple enmlsione were prepared by the reenmlsifieation of
`primary emulsions using two—step procedure in which surfact—
`ants ( Tween 20 and Span 60) were used. The corresponding
`simple emulsions of 11/0 and o/w were also prepared and used
`for comparison. The in vivo performance was assessed via the
`influence of hydrocortisone 0n the intraocu/ar pressure (101’).
`The parameters of activity used to quantifv the pharnuu‘okine-
`the area under the intramular pressure/
`time curve (AUC); nutximum response ( MR ),' time of max—
`imum response ( TMR) of the mean change in 101’: half value
`duration of the percent mean change in IOP;
`ratio of the
`“ll VD in hours and relative bioavailahilitv percent/or different
`pairs of comparison in difl‘erent emulsion systems were deter-
`mined. To provide a quantitative measure for the individual
`variations, the variance and the coefficient of variation percent
`
`The bioavailability ofh]rdrocortisonefrom emulsions based on
`liquid paraffin, corn oil or eastor oil was found to increase in
`the following order: solution, o/w, w/o/w, o/w/o emulsions. The
`intensity ofdrug action was found to increase in the order of
`solution, w/o/w, o/w, w/o, and o/w/o emulsions. The parameters
`of'activity are dependent on the nature of the oil phase of the
`emulsion. General/v, o/w/o emulsion systems represent an op—
`timal delivery system ma.\7imi:ing drug biom'ailahi/ily and min-
`
`Melujfachcmulsionen warden int Zweisehritt— Verfi/hren dare/t
`wiederholte Emulgierung mit ohetfliichenalrtiven Substanzen
`{ Tween 20 and Span 6(1) hergestellt. Die entspreehenden einfa-
`chen Emu/sionen { W/O and 0/ W) warden ehenfalls hergestellt
`and :u Vergleichsziveclren herangezogen. Die Eigensehaften der
`Zuhereitungen warden in vivo iiher die Wirlrung von Hydro/(or—
`tison auf den Augeniimelulruck bewertet. Zur quantitativen Be-
`urtei/img der Pharntalrolrineti/t warden jo/gende Parameter be—
`stimmt.‘ Fliiehe unter der Augenitmendruclc/Zeit—Kurve, maxi-
`male Wirlcung, Zeitpunlct der maximalen Wirkung, Halbwerts—
`:eit der Mittelwerte der Anderung des Augetzinnendrucks, Quo-
`tient der Wirlcdauer and relative Bioverfi'igbarlreit [In paarwei—
`sen Vergleich verschiedener Entulsioussrvsteme. Als ll/ltlfl indivi—
`dueller Abweichungen warden Varian: and Variations/coeffi—
`:ient bereelznet.
`
`Nae/1 den Ergehnissen nimmt die Bioverfi'igharkeit von llvdro-
`ltortison aus Emulsionen aufder Basis von fliissigem Paraffin,
`ll/Iuiskeimo'l and Ri:inuso"l info/gender Reihenfblge :u.‘ Liisung,
`0/ W—, W/O/ W—, 0/ W/O-Emulsionen. Die Stdrke der Wir/atng
`nimmt in der Folge W/O/W—, 0/ W—, W/O- und 0/-W/O-Emul—
`sionen :11. Die Wit'ltungsparameter sind von der Art der Ol-
`phase abhiingig. 1m allgenzeinen stellen 0/ W/O-Emulsionen op-
`tima/e Freise[zungssvsteme dat; die die Biover/iigharlreit maxi—
`mieren and individuelle Abweichungen minimieren.
`
`llerstellung von Hydrokortison- ll/lehrfaehenmlsionen and deren
`
`Key WONlS.‘ Emulsions. ocular drug delivery - Hvdrocorti—
`sone ~ Multiple emulsions
`
`Ocular bioavailability of many topically applied drugs has been
`increased by using techniques to modify thc response to drugs.
`Somc ocular delivery systems extend the duration of drug ac—
`tion by enhancement, of corneal absorption [2, 3, 4]. also by
`using vehicles that retard precorneal drug loss [5. 6]. These
`vehicles includc ointments. latex systems. liposomes, and poly-
`[7]. Controlled drug release can also
`lower peak drug concentration in the systemic circulation [8].
`In spitc of numerous studies of ocular bioavailability. which
`have utilized various vehicles, there are very few studies on the
`nature of the oily phase in multiple emulsions on ocular drug
`absorption and on pharmacological responses in the eye. How—
`ever, it is important, not only to maximize ocular drug absorp-
`tion, but. also to minimize the systemic concentrations of ocu-
`
`Multiple emulsions are emulsions in which globules of the dis—
`persed oily phase encapsulate smaller droplets of the aqueous
`phase and have found application as sustained release delivery
`vehicles and adjuvants for a variety of drugs and biologically
`active materials [9, 10, ll]. Another area ofinterest is the poten-
`tial use ol‘ w/o/w or o/w/o emulsions to facilitate ocular absorp-
`
`tion of drugs. We are presently studyin g the ocular bioavailabil—
`ity of hydrocortisone as a model drug which rises the intraocu-
`lar pressure of the eye as a side effect; released from multiple
`emulsions based on liquid paraffin, corn oil or castor oil using
`surfactants (Twccn 20 and Span 60).
`The use of high surfactant concentrations is not desirable from
`the point of view of toxicity. A physiological property of sur—
`factants is their local analgesic effect [12] when applied topically
`to the cornca of rabbits. Generally. long straight chain com-
`pounds are less irritating than short chain and branched prod-
`ucts. From the toxicological point of View, the oral use of sorbi—
`tan esters of fatty acids and their polyoxyethylene derivatives
`are very low in animals, e.g. Tween 60 [13]. Their use in oral
`emulsions seems eminently safe (Span 60 and Tween 80) [14,
`15].
`
`For potential pharmaceutical purposes, the oils uscd have in-
`cluded refined hydrocarbons such as light liquid paraffin, esters
`of long Chain fatty acids and vegetable oils |16]. Florence et a1.
`[l7] studied the effect of the nature of the oily phase of multiple
`emulsions on the stability of the oil membrane against the leak—
`age ol‘ the entrapped drug and concluded that it depended on
`the nature of the oil used in the preparation of the emulsions.
`
`Pharm. Ind. 56, Nr. 6 (1994)
`Kassem ct a]. — Hydrocortisone
`
`AKN 1020
`
`1
`
`

`

`The use of thickening agent (eg. polyvinylaleohol) in the ex—
`ternal aqueous phase may reduce creaming of multiple emul—
`sions (w/o/w) and improve the stability [18]. Further investi-
`gations on the stability of multiple emulsions would be publi—
`shed in the nearest future.
`
`2. Experimental
`2.1. Materials
`
`Hydrocortisone was kindly provided by Sigma Co. (St. Louis,
`MO, USA); corn oil (Amphora phelex Machan, France); light
`liquid paraffin (USP). castor oil, Tween 20 (polyoxyethylcne
`sorbitan monostearate). Span 60 (sorbifan monopalmitatc) (At-
`las Chemical Ind. Wilmington, DE, USA), propylene glycol
`(Roth, FRG); isotonic xylocaine solution (2 “/1; w/v).
`
`2.1.]. Equipment
`Homogenizer (Type 302; Mechanika Preeyzyjna, Poland); po—
`larizing microscope (Nr. 252700, Reichert, Wien. Austria);
`double beam spectrophotometer (Shimadzu. Kyoto, Japan):
`Schiotz tonometer (Riestcr. FRG); transmitted light microscope
`(laborlux 512795/041654; Leitz, Wetzlar. PRO).
`
`2.2. Procedure
`
`2.2.]. Preparation of emulsions
`Table 1 shows the composition of the different simple and mul—
`tiple emulsion formulations. Simple emulsions were prepared
`using the homogenizer. Aqueous solutions of the hydrophilie
`emulsifying agent (Tween 20) or oily solutions of the lipophilic
`emulsifying agent (Span 60).
`in definite concentrations. were
`used. Multiple w/o/w emulsions were prepared by a two—step
`cmulsilication procedure [1 8]. Hydrocortisone was incorporated
`in the aqueous of the simple emulsions (the inner aqueous
`phase of the multiple w/o/w emulsions) or in the oily phase of
`the simple emulsions (the inner oily phase of the o/w/o multiple
`emulsions).
`
`Table I .' Composition of simple and multiple emulsions. Ex—
`ternal phase volume fraction ratio of primary emulsion is 0.5
`in each case.
`External
`
`phase vol-
`Surfactant
`,
`,
`.
`Emulsion
`Type
`Olly phase type
`type
`ume fraction
`ratio 0
`
`
`o/w
`
`—
`I“)
`Liquid paraffin
`Corn oil
`T
`—
`Castor oil
`T
`
`
`
`
`
`w(o
`
`—
`S'”
`Liquid paraffin
`Corn oil
`S
`—
`Castor oil
`S
`
`
`w/o/w &
`o/w/o
`
`Liquid paraffin
`Corn oil
`Castor oil
`
`TS“
`TS
`TS
`
`0.5
`0.5
`
`1 %, and Span 60,
`*0 Tween 20, 1%. 1” Span 60, 2.5 %. “l 20,
`2.5 “/0.
`
`2.3. Evaluation of emulsions
`Only freshly prepared emulsions of the most stable formulations
`were used in this study to avoid problems of stability. The more
`data concerning the stability of the emulsion systems would be
`published in another paper.
`
`2.3. I . Microscopical examination
`Immediately after preparation of the simple and multiple emul—
`sions, microscopical examination was performed using the po-
`larizing microscope. Microscopic examination was performed
`to inspect the dispersion state of the innermost phase of mul—
`tiple emulsions.
`Photomicrographs of simple emulsions were performed for
`comparison with multiple ones (Figs.
`1 and 3). Figs. 2 and 4—6
`Pharm. 1nd. 56, Nr. 6 (1994)
`Kassem et a1. — Hydroeortisone
`
`“.1
`..
`E‘
`A’
`1‘
`i‘
`
`
`Figs. 1—6: Photomicrographs of emulsions.
`Fig. I (upper left): Diluted simple o/w emulsion based on liquid
`paraffin.
`Fig. 2 {upper right): Multiple w/o/w emulsion based on liquid
`paraffin.
`Fig. 3 {middle left): Diluted simple w/o emulsion bases on li-
`quid paraffin.
`Fig. 4 {middle rig/1t}: Multiple o/w/o emulsion based on liquid
`paraffin.
`Fig. 5 { lower left): Multiple w/o/w emulsion based on corn oil.
`Fig. 6 {lower rig/It): Multiple w/o/w emulsion based on castor
`oil (Q w/o/w 0.5; Q w/o 0.5; Span 60, 2.5 C70, and Tween 20,
`1 00).
`
`show photomicrographs of multiple emulsions of w/o/w and o/
`w/o based on liquid paraffin, corn oil and castor oil, prepared
`with Span 60 (2.5 0/n) and Tween 20 (1%), the phase volume
`ratios of the internal phase as well as the external phase were
`0.5. It is clear from these figures. that the formation of multiple
`drops contain vast numbers of internal droplets. This observa-
`tion is in agreement with Florence and Whitehill [16].
`
`2.3.2. Determination of the partition coefficient
`The partition coefficient of hydrocortisone between liquid par-
`affin, corn oil, castor oil and isotonic phosphate buffer solution
`pH 6.8 was determined as follows: 0.1 of the drug was dissolved
`in 5 drops of propylene glycol and mixed with the isotonic phos—
`phate buffer solution. Equal volumes (50 ml) of each of the
`mentioned oil and isotonic buffer solution were equilibrated in
`a thermostated bath at 37 °C using submersion rotator. After
`equilibration for 24 h the phases were centrifuged and sepa-
`rated. The absorbance at 247 nm was then measured against
`blank prepared in an analogous manner. Drug concentration in
`the oily phase (Co) was determined using the equation
`CO : CL—CW
`
`where C‘ = total drug concentration, CW = drug concentration
`in aqueous phase.
`The data of drug distribution between the two phases (Table 2)
`were treated according to the equation [19]
`
`Table 2: Partition Coefficient Kp of hydrocortisone in different
`oils.
`
` Type of oil
`
`Liquid paraffin
`Corn Oil
`
`Castor Oil 3.23
`
`2
`
`

`

`2.3.3. Investigation oft/lo in viva performance of
`ophthalmic preparations in healthy eye of rabbits
`To avoid any interference of emulsion stability with the in Vivo
`experiments, freshly prepared emulsions were used. Isotonic
`xylocaine solution (2 % w/v) was dropped into the rabbit’s eyes
`to anaesthetize the cornea. In all cases topical doses each of 50
`pl of ophthalmic solution or tested emulsion were instilled in
`
`Non—medicated formulations were applied to the opposite eye
`which served as control. Each formulation was tested in each
`of 6 rabbits The assigned formulation was applied to the right
`eye, while the control one was applied to the left eye. The intra-
`cular pressure (lOP) of both eyes was measured before and after
`application of both control and test formulations at certain time
`intervals. starting with the experimental eye by using Schiotz
`tonometer. Statistical analysis of the in Vivo data of IOP were
`
`3.1. Bioavoilabilily (AUC) in relation to the
`
`The area under the IOP/time curve was considered from
`two points of view, first. the absolute value of the area
`as a measure of the eye response over all experimental
`points and, second. the variation of the area from one
`test animal to the other, as a measure of the individual
`variations in response to the different delivery systems.
`To provide for a quantitative measure for the individual
`variations both the variance and the coefficient of vari-
`ation percent (cv) were computed, the latter is given by
`
`CV = standard deviation/mean x 100
`
`Table 3 and Fig. 7 reveal a marked difference between
`the solution and the emulsion delivery systems, the latter
`systems give rise to higher values for the AUC; this is
`specially evident for the o/w/o emulsion system. Also,
`here the values of CV reveal the advantage of the emul-
`sion systems in reducing the individual variations. The
`individual variations are less in simple and multiple
`emulsions with an external aqueous phase and least for
`simple and multiple emulsions With an external oily
`
`Simple and multiple emulsion systems do not signifi—
`cantly differ with regard to their effect on the bioavail-
`
`Considering hydrocortisone bioavailability and the sta—
`tistical analysis of the data. the hydrocortisone delivery
`
`Mean change of IOP (%)
`16
`
`I4
`
`12
`
`10
`
`Time (h)
`
`Fig. 7: Percent mean change in 10F of rabbits eye post instilla-
`
`
`tion of hydrocortisone in solution (*) and in different emulsion
`
`
`systems (
`= o/w,
`= w/o, O = w/o/w, O = o/w/o) based on
`liquid paraffin.
`
`systems may be arranged in the following decreasing or—
`der: o/w/o > w/o > w/o/w' > o/w > solution.
`
`The present study shows that the individual variations
`in bioavailability may be reduced by using emulsion de-
`livery systems with an external oily phase in the first
`place or by using multiple emulsion systems in the se-
`cond place.
`
`3. l. I. Biouvailability in relation to the nature oft/re
`oily phase of the emulsion
`To find out whether the nature of the oily phase of the
`emulsion has an effect on the bioavailability from the
`delivery system or not. the same systems were modified
`so that the fraction of liquid paraffin was substituted
`with corn oil or castor oil. Table 3, Figs. 8 and 9 show
`that the bieavailability is dependent on the delivery sys-
`tem. The bioavailability from the emulsions is much
`higher than that from the solution; within the emulsion
`systems it is highest for the o/w/o system. Comparison
`of
`the different emulsion systems
`reveals
`that
`the
`bioavailability from the multiple emulsion is higher than
`that from the corresponding simple emulsions. Also, the
`bioavailability from emulsion systems with an external
`oily phase is higher than that from emulsions with an
`external aqueous phase.
`
`Table 3: Area under curve (AUC) of the mean change in IOP of rabbits eye post installation of hydrocortisone in solution and
`in different emulsion systems.
`
`
`Liquid paraffin
`Corn Oil
`Castor oil
`
`AUC
`(mmHg . h)
`
`v
`
`cv
`(0/0)
`
`AUC
`(mmHg - h)
`7—.
`
`v
`
`CV
`('70)
`
`ALJC
`(mmHg - h)
`
`V
`
`CV
`(%)
`
`
`
`
`
`
`
`6.88
`(0.56)
`8.65
`(0.73)
`8.06
`(0.74)
`11.30
`(0.71)
`
`1.85
`
`19.90
`
`3.19
`
`20.60
`
`3.32
`
`22.60
`
`3.01
`
`15.40
`
`8.15
`(0.79)
`10.12
`(0.68)
`9.83
`(0.72)
`12.99
`(0.69)
`
`3.75
`
`23.70
`
`2.78
`
`16.50
`
`3.15
`
`18.10
`
`2.89
`
`13.10
`
`9.60
`(0.72)
`12.98
`(0.71)
`11.50
`(0.71)
`17.00
`(0.65)
`
`3.14
`
`18.50
`
`3.05
`
`13.50
`
`3.00
`
`15.10
`
`2.57
`
`9.42
`
`'7) w/e/w, 0.5; Q w/o. 0.5; Span 60, 2.5 “/0, and Tween 20.1 %. “l 125 w/o, 0.5. and Span 60. 2.5 "/0.
`‘16, and Span 60. 2.5 %.
`
`Pharm. Ind. 56, Nr. 6 (1994)
`Kassem et a1. — Hydroeortisone
`
`3
`
`

`

`Mean change of IOP (%)
`is
`
`14
`
`12
`
`1O
`
`1
`
`2
`
`3
`
`4
`
`6
`5
`Time (h)
`
`7
`
`8
`
`9
`
`10
`
`Fig. 8: Percent mean change in IOP of rabbit's eye post instilla-
`tion of hydrocortisone in solution (*) and in different emulsion
`systems ('1 ) o/W. A : w/o, O 2' w/o/w. O : o/w/o) based on
`corn oil.
`
`Mean change of [OP (%)
`18
`
`16
`
`I4
`
`12
`
`10
`
`t
`
`2
`
`3
`
`4
`
`6
`5
`Time (h)
`
`7
`
`3
`
`9
`
`to
`
`tl
`
`Fig. 9: Percent mean change in IOP of rabbit’s eye post instilla-
`tion of hydrocortisone in solution (*) and in different emulsion
`
`systems (C = o/w, A : w/o. O = w/o/w, O : o/W/o) based on
`caster oil.
`
`Table 4: Ratio of the area under curve (relative bioavailability
`percent) for different palrs of comparlson in systems eontammg
`hydrocortisone.
`Relative bioavailability
`
`(“/0 of rang - 11)
`
`Pairs of comparison
`
`Liquid
`Corn
`Castor
`
`paraffin
`oil
`oil
`
`o/w
`
`with solution
`
`
`
`
`
`with solution
`w/o
`w/o/w with solution
`0/w/o with solution
`
`171 + 203
`
`216
`201
`282
`
`252
`245
`324
`
`239
`
`324
`287
`424
`
`w/o/w with o/w
`W/O/W with w/o
`O/W/o with o/w
`o/w/o with W/o
`w/o
`with o/w
`o/w/o with w/o/w
`
`Pharm. Ind. 56, N1: 6 (1994)
`Kassem et al. — Hydrocortisone
`
`117
`92.2
`164
`131
`126
`140
`
`121
`97.1
`159
`128
`124
`132
`
`120
`0.89
`177
`131
`135
`148
`
`Table 4 presents the relative bioavailability for different
`pairs of comparison. It is obvious that the bioavailability
`of hydrocortisone could be more than doubled by the
`appropriate selection of the emulsion system. Also. it is
`clear that the bioavailability may be increased to the ex-
`tent of about 20—50 0 0 by simple manipulation with the
`nature of the oil and the type of emulsion. The greatest
`increase in bioavailability is observed for hydroeortisone
`in castor oil emulsions. In general castor oil favours drug
`bioavailability more than corn oil and the latter more
`than liquid paraffin. The individual variations are not
`only a function of the type of emulsion but are also de-
`pendent on the nature of the oil. With increasing polar—
`ity of the oily phase of the emulsion. the individual vari—
`ations are suppressed. The bioavailability increased in
`the order solution, o/W, w/o/w. w/o and o/w/o.
`
`3.2. Correlation prarameters ofaclivily to drug
`parlilioning between the aqueous and the 0171’ phases
`Figs. 10 and 11 present a semilogarithmic plot of the
`relationship between log partition coefficient for hydro-
`cortisone and the parameters AUC, MR, TMR and
`HVD.
`
`Table 5 presents the values of the correlation coeffficient
`and shows a very poor correlation of partition co-
`etificient to the TMR parameter. Whereas the correlation
`
`4.0‘ Partition cueffitient
`
`3‘9—
`
`2.01
`
`1.0 _
`
`1
`05'
`
`.1
`0.2-
`
`
`,
`0
`l.
`
`4.0— Partition coefficient
`
`
`
`101
`
`2.0-
`
`1.0
`
`0.2-
`
`
`l
`1-
`0
`t
`
`Fig. 10: Semilogartihmic plot of AUC and MR for hydrocorti-
`sone in different emulsion systems in relation to its partition
`coefficient in different oils. 0 : o/w. O = w/o. O : w/o/w. O =
`o/w/o.
`
`4
`
`

`

`0 Easter oil
`
`Table 5: Correlation coefficient (r) for the parameters of activity
`and the log partition coefficient of hydrocortisone in different
`emulsion system based on liquid paraffin, corn oil and castor
`oil.
`
`
`
`
`
`
`Parameters of activity
`MR
`TMR
`(mmHg)
`(11)
`0.963
`0.995
`0.982
`70.995
`0.932
`0.411
`0.974
`0.809
`
`AUC
`(mmHg - 11)
`0.926
`0.904
`0.984
`0.981
`
`HVD
`(h)
`0.947
`0.962
`0.927
`0.994
`
`Type of
`emulsion
`
`O/W
`w/o
`w/o/w
`o/w/o
`
`’ Earn oil
`Liquid paraffin
`
`z.
`
`5
`
`0Castor oil
`
`0Com oil
`
`Liquid paraffin
`
`6 H
`
`5
`
`Fig. 11: Semilogarithmic plot of TMR and HVD for hydrocorti—
`some in different emulsion systems in relation to its partition
`coefficient in different oils. 0 : o/w. O = w/o, O = w/o/w. O :
`
`to the AUC or to the HVD is very high. The correlation
`to MR is fair. Summarizing: in emulsion drug delivery
`systems dedicated for ophthalmic use there is a very high
`correlation between the partition eoelfleient of the drug
`for the two phases and each of the following parameters:
`a) rate of drug absorption (AUC).
`b) extent of drug absorption (MR).
`c) maintenance of drug action (HVD).
`Partition coefficient of the drug presents, thus. an impor—
`tant design parameter in emulsion delivery systems. This
`design parameter can be controlled simply by the proper
`
`4. References
`[1] Gurny. R., l’harm. Acta Helv. 56. 130 (1981) — [2] Bamba.
`M., Puisieux. F, Marty. J. P., Carstensen. J. T.. Int. J. Pharm.
`2. 307 (1979) — [3] March. W. F.. Stewart, R. M., Mandel]. A.
`1., Bruce. L. A.. Arch. Ophthalmol. 100, 1270 (1982) 7 [4] Gold—
`berg, 1.. Ashburn. F. S., Kass. M. A.. Becker. B.. Mer.
`J.
`Ophthalmol. 88, 843 (1979) — [5] Chrai. S. 8.. Robinson. J. R.,
`J. Pharm. Sci. 63, 1218 (1974) 7 [6] Grass, G. M., Robinson. J.
`R.,
`J. Pharm. Sci. 73, 1021 (1984) — [7] Shell. J. W.. Surv.
`Ophthalmol. 29, 117 (1984) 7 [8] Urtti. A., Salmimen, L., Miin-
`alainen. 0.. Int. J. Pharm. 23, 147 (1985) 7 [9] ()motosho, J. A..
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`For the authors: Dr. S. M. Safwat, Faculty of Pharmacy,
`Assiut University, Assiut (Egypt)
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