Emulsions as drug carriers for ophthalmic use

Colloids and Surfaces A: Physicochemical and Engineering Aspects 91(1994) 181-190

A

SURFACES

Emulsions as drug carriers for ophthalmic use Shaul Muchtar, Simon Benita* Pharmacy Department, School of Pharmacy, Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem, Israel Received 4 January

1994; accepted

29 March 1994

Abstract The ocular administration

bioavailability of lipophilic

of drugs administered in standard aqueous vehicles is very poor. However, the ocular drugs also exhibits complex problems due to their low aqueous solubility. The wide and

clinically well-accepted usage of emulsions for parenteral nutrition (see PK. Hansrani, S.S. Davis and M.J. Groves, J. Parenter. Sci. Technol., 37 (1983) 145) has raised the possibility of using the oil phase of oil-in-water emulsions as a carrier of poorly water-soluble drugs. However, submicron emulsions are sensitive dispersed systems which need to be physically stabilized, especially following the incorporation of drug molecules’into the oil dispersed globules, the sizes of which range from 100 to 300 nm. Two different drugs were incorporated into the original submicron emulsion which met ocular product requirements. The medicated emulsion formulation was stabilized by a combination of two emulsifying agents (phospholipids and an amphoteric emulsifier) following manufacturing process optimization. Emulsion formulations with extended physical shelf-life were therefore developed and fully characterized. The partition profile of the drugs in the various phases of the emulsions was also determined. Keywords: Emulsion; HU-211; Ocular, Physicochemical

characterization;

1. Introduction

Most ophthalmic drugs are delivered to the eye via an aqueous vehicle. However, such a vehicle exhibits poor ocular bioavailability due to rapid drainage, lacrimation and tear turnover [ 11. Furthermore, many potentially active ophthalmic compounds have a very low water solubility, seriously limiting their ocular application. Such drugs require administration by alternative routes to exert their ophthalmic activity. The various approaches currently investigated to improve the ophthalmic delivery of lipophilic drugs include micelles [ 21, implants, semisolid dosage forms, gels, liposomes [ 31, and nanoparticles [4]. The wide and clinically well-accepted usage of emul*Corresponding

author.

0927-7757/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0927-7757(94)02882-S

Pilocarpine; Stability

sions for parenteral nutrition [IS] has raised the possibility of using the oil phase of oil-in-water emulsions as a carrier of poorly water-soluble drugs. Recently, an intense and long-lasting intraocular pressure (IOP) depressant effect was observed after one single ocular application of a tetrahydrocannabinol (THC) emulsion (0.4% (w/w)) to ocular-hypertensive rabbits [ 61. Oil-in-water submicron emulsions can therefore be considered to be appropriate ocular drug carriers which can improve drug absorption through the cornea and prolong the residence time of the drug in the eye while minimizing potential local and systemic sideeffects. However, submicron emulsions are sensitive dispersed systems which need to be physically stabilized, especially following the incorporation of drug molecules in the oil dispersed globules, the sizes of which range from 100 to 300 nm.

S. Muchtar, S. Benita/Colloids Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190

182

cH,OH

Fig. 1. Chemical structure of HU-211 (the (+)-(3S,4S) enantiomer of 7-hydroxy-A6-tetrahydrocannabinol-l.l-dimethylheptyl).

Furthermore, the use of emulsions in ophthalmic preparations has been limited to a large extent by the fact that the surfactants generally used in emulsions are irritating to the external eye tissues. The objective of the following investigation was to incorporate lipophilic compounds, specifically HU-211 and pilocarpine, in an original emulsion which was stabilized by phospholipids and small amounts of amphoteric surfactants that have virtually no irritating effect on the skin and eye [7]. HU-211 is an isomer of A9-THC which was recently synthesized by Mechoulam and Feigenbaum [S] (Fig. 1). This compound is capable of significantly reducing the intraocular pressure (IOP) without causing any psychotropic side-effects [9]. It is a very lipophilic crystalline compound (99.8% purity) and practically insoluble in water. Pilocarpine, a widely prescribed compound in the management of glaucoma, is mainly used as a hydrochloride salt dissolved in an aqueous solution. The use of the aqueous preparation is limited by the need for frequent instillation (every 6 h) due to very poor absorption and rapid drainage. Attempts to improve pilocarpine therapy by use of an Ocusert@ device [lo] or an aqueous gel were only moderately successful. Pilocarpine base is known to be water soluble and its incorporation into the emulsion system was studied in order to improve its ocular bioavailability.

This mixture was composed of 95% phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in a 10: 1 ratio, and small quantities of lysophosphatidylcholine and sphingomyelin (less than 2%). The amphoteric surfactants Miranol MHT (lauroamphodiacetate and sodium tridecethsulfate) and Miranol C2M (cocoamphodiacetate) (Fig. 2) were kindly supplied by Rhone-Poulenc (Cranbury, NJ, USA). These surfactants are derived from carboxylated fatty imidazolines. The two surfactants are similar in chemical structure but have slightly different ionic properties. This is due to the presence of a sulfonate group in the Miranol MHT which makes it more electronegative than Miranol C2M. Both surfactants are electronically neutral zwitterions over a wide isoelectric range. The two Miranols are widely used as surfactants in skin cleansers, baby shampoos and make-up removers. Miranol MHT and Miranol C,M are usually supplied as 34.5% and 50% aqueous solutions respectively. The oil used in the preparation of the emulsions consisted of medium-chain triglycerides (MCT), which were kindly supplied by Societt des Oleagineux (St. Laurent, Blangy, France). The oil is obtained by the hydrolysis of coconut oil and its fractionation into free fatty acid (between 6 and 12 carbon atoms) esterified with glycerol [ll]. This oil was chosen because it is less viscous and 100 times more soluble in water than long-chain triglycerides (LCT). Furthermore, it has the ability

Miranol C,M

2. Experimental 2.1. Materials Surface-active agents

The mixture of phospholipids, Lipoid E-80, was supplied by Lipoid KG (Ludwigshafen, Germany).

Miranol MHT

Fig. 2. Chemical structure of Miranol MHT and Miranol C,M.

S. Muchtar, S. Benitajcolloids Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190

to dissolve large amounts

of liposoluble

drugs

Clll. The tonicity of the emulsion was adjusted with glycerol. The pH was adjusted to the desired value with an aqueous solution of 0.1 N HCl, while c+tocopherol was used as an antioxidant. The glycerol and a-tocopherol were of pharmaceutical grade and were purchased from Sigma (St. Louis, MO, U.S.A.). Two active ingredients were used as drug models in the present work: pilocarpine, and HU-211. HU-211 was generously made available to us by Professor R. Mechoulam, Natural Products Department, The Hebrew University, Jerusalem, Israel.

2.2. Methods

183

Emulsion evaluation Particle

size analysis The mean droplet size and size distribution were determined by use of a photon correlation spectroscopy apparatus (Coulter Counter Supernanosizer MDTM Luton, UK). Each sample was diluted with a filtered isotonic solution (2.25% (w/w) glycerin in water) and was measured at 25°C. Each emulsion was analyzed in duplicate and for each sample, ten determinations were performed. Zeta

potential The zeta potential was measured with use of a Malvern ZetasizerTM (Malvern, UK). Visual observations The degrees of creaming and phase separation were assessed visually at given time intervals. Any other visible changes were recorded.

Emulsion preparation

The purified phospholipid mixture, a-tocopherol and the drug were dissolved while being gently heated in the MCT oil. Glycerol and the amphoteric surfactant were dissolved in the aqueous phase and the pH was adjusted to the desired value. The two phases were heated separately to 70°C and mixed together while heating until the temperature reached 85°C. At this temperature, emulsification was carried out using a high shear Polytron@ mixer (Kinematica, Lucerne, Switzerland). The emulsion was then cooled rapidly and homogenized in a two-stage homogenizing valve assembly (Rannie@, APV Rannie, Inc., Denmark). After rapid cooling, the pH was adjusted to the desired value with HCl solution (0.1 N) and filtered through a 0.45 urn membrane filter (CTE 37 Schleicher and Schuell, Dassel, Germany). After filtration, the emulsion was packed in plastic bottles and sterilized by autoclaving at 121 ‘C for 15 min. Pilocarpine base being sensitive to heat, the corresponding emulsion was only heated to 40°C during the emulsification stage and was sterilized by membrane filtration (0.22 urn) under aseptic conditions. The process was conducted in a nitrogen atmosphere. The influence of the phospholipids and amphoteric emulsifier concentration on the emulsion properties was also studied.

Sterility testing The sterility of the emulsion was assessed using the Bactec L6 apparatus (Johnson Laboratories, Towson, MD). This method quantitatively measures radioactive carbon dioxide in Bactec culture vials inoculated with test samples and it is capable of detecting aerobic or anaerobic microorganisms. Stability studies

The stability studies were conducted at various storage temperatures, namely 4, 25, 30 and 37°C. The chemical and physical changes that might occur during storage were monitored by evaluation of the drug concentration and droplet size distribution. The stability of the present emulsion was also examined and compared to a similar emulsion formulation prepared with a non-ionic emulsifier (Pluronic F-68, 1% (w/w) instead of an amphoteric emulsifier) as a function of pH variation and divalent cation concentration. The effect of pH on the mean droplet size and the polydispersity of an emulsion containing 0.35% (w/w) amphoteric surfactant, Miranol MHT, and an emulsion containing 1% non-ionic surfactant, Pluronic F-68 (Tradename, BASF, USA), was measured. The pH was adjusted by means of aqueous 1 N hydrochloric acid solution. Different amounts of CaCl, were added to the

S. Muchtar, S. Benita/Colloids Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190

184

two emulsions described above. After 5 min, visual observations were made and the mean droplet size was measured. Quantitative drug analysis

The analysis of pilocarpine and HU-211 was carried out using a high performance liquid chromatographic (HPLC) method. The experimental conditions are described in Table 1. Emulsion phase partition of drug determination Pilocarpine The pilocarpine concentration in the aqueous phase was determined using the ultrafiltration technique at low pressure. YM-10, 62 mm Amicon ultrafiltration membranes were soaked in deionized water, with several changes of water, for at least 1 h to remove water-soluble contaminants. The membranes were placed in a stirred filtration cell (Model 8200, Amicon, Danvers, MA, USA) operated at room temperature. A portion (40 ml) of the drug solution, or the emulsion to be filtered, was placed into the stirred vessel and 20-40 psi of nitrogen pressure were applied in order to start the filtration. Approximately 2 ml samples of the filtrate were collected until 15-20% of the original liquid had been ultrafiltered. Each sample was then assayed for pilocarpine by HPLC. Prior to filtration, both the drug solution and the emulsion were assayed for pilocarpine content by HPLC. The ultrafiltration technique required validation, as has already been performed by other workers [ 121, before being used for the determination of the phase distribution of the pilocarpine. Membrane adsorption and rejection had to be accounted for in order to accurately measure aqueTable 1 HPLC conditions HPLC

for pilocarpine

and HU-211

parameter

Mobile phase (% (v/v)) Flow rate (ml mini’) Detector wavelength (nm) Column Emulsion sample dilution a Adjusted

with 10% phosphoric

acid.

ous concentrations of pilocarpine. The ultrafiltration membranes were specifically selected for their low non-specific binding. The effects of membrane binding and rejection of pilocarpine were studied by ultrafiltering an aqueous solution of pilocarpine which was maintained at a constant pH of 5.0 at a concentration of 10 pg ml-‘. The recovery curve for pilocarpine from the aqueous solution is shown in Fig. 3. The membrane appeared to be nearly saturated after approximately 5-7% of the total volume had been filtered, as is evident from the leveling-off of the curve. The recovery was 97-98% of the theoretical amount at pH 5, indicating that rejection was negligible. Based on these rejection data, ultrafiltration data for pilocarpine emulsion formulations required only a slight correction, provided that at least 5% of the total volume was filtered to saturate the membrane. For the determination of the drug content in the oily phase, the emulsion was subjected to ultracena

1

0

3

6

Percent

12

9

15

18

Filtered

Fig. 3. Membrane recovery of aqueous a concentration of 100 ug ml-‘.

pilocarpine

solution

analysis

HU-211

Pilocarpine

Acetonitrile : methanol : water (50 : 30 : 30) 2.0 280 Econosil C-18; 10 ).tm; i.d. 4.6 nm 1: 100 in propan-2-01

0.1% Triethylamine in water, pH 2.5” 2.0 220 Econosil C-18; 10 pm; i.d. 4.6 nm 1 : 100 in methanol

at

S. Muchtar, S. BenitalColloids Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190

trifugation as described below for the HU-211 determination. The pilocarpine content in the oilwater interface was determined by calculating the difference between the total pilocarpine concentration and the concentrations in the oil and water phase. HU-21 I It was impossible to apply the ultrafiltration technique at low pressure on the HU-211 emulsion since it is adsorbed on the Amicon membrane. The emulsion was subjected to ultracentrifugation using a Beckman@ LB 50B ultracentrifuge at 115 OOOgduring 30 min. This resulted in the separation of the oil phase from the emulsion. Aliquots of the oil and aqueous phases were assayed using the HPLC method described above. No clear phase separation was obtained despite the high centrifugal force used, because of the small size of the oil droplets and the fact that the density of the oil is close to that of water. The concentration at the oil-water interface therefore could not be measured by calculating the difference between the total HU-211 concentration and the concentration of the drug in the oil and water phases. Only the combined concentration in the oil and in the oilwater interface could be measured.

3. Results 3.1, Emulsion optimal composition The effects of the Miranol and phospholipid concentrations are shown in Fig. 4 and Fig. 5 respectively. As can be seen, the concentrations of the Miranol at which the smallest droplet size was achieved were 0.75% (w/w) Miranol C2M and 0.50% (w/w) Miranol MHT. The lowest droplet size was achieved at a phospholipid concentration of between 0.75 and 1.5% (w/w). 3.2. Stability assessments The effect of pH on stability for both the emulsion containing the amphoteric surfactant and the emulsion containing the non-ionic surfactant is shown in Fig. 6. As can be seen, the mean droplet size and polydispersity of the emulsion containing

185

Miranol MHT remained constant at a pH value of between 2 and 7. However, in the emulsion containing non-ionic surfactant (Pluronic F-68), a decrease in pH caused a gradual increase in the mean droplet size and a significant change in polydispersity. The effect of divalent cations on both the emulsion containing amphoteric surfactant and the emulsion containing non-ionic surfactant is shown in Fig. 7. It should be noted that the stability of the emulsion containing non-ionic surfactant was even affected at low concentrations of Ca2+ ions (since the mean droplet size increased significantly), and the emulsion was fully separated at concentrations higher than 1 x lop3 M. The emulsion containing amphoteric surfactant was not affected at low concentrations and was moderately affected at Ca2+ ion concentrations higher than 1 x 1O-4 M, exhibiting an increase in the mean droplet size due to droplet coalescence without leading to phase separation. The chemical stability of the HU-211 emulsion as a function of storage temperature is shown in Fig. 8. There was no significant change in HU-211 concentration at 4, 25, or 30°C over a storage period of 14 months. At 37°C there was a slight degradation, beginning after 6 months of storage, resulting in a decrease of lo-12% after 14 months. The results of the HU-211 physical stability evaluation are shown in Fig. 9. It can be seen that up to a storage period of 7 months, the variation in storage temperature did not affect the mean droplet size. The mean droplet size of the emulsion at 37°C began to significantly increase after 7 months. At lower temperatures, a slight but continuous increase was observed. These results indicate that the particle size is time- and temperaturedependent. 3.3. Partition studies The distribution

profiles

of pilocarpine

and

HU-211 in the different phases of the emulsion are

shown in Table 2. 4. Discussion The selection of the emulsifier ‘types was based on pharmaceutical considerations associated with

186

S. Muchtar, S. BenitajColloids Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190 400

1

n C2M IziMHT

300

200

100

0 1

1.5

2

3

5

10

MIRANOLSOLUTIONCONC.%w/w Fig. 4. Effect of Miranol

MHT and C,M concentrations

on the emulsion

mean droplet

PHOSPHOLIPIDSCONC.%w/w Fig. 5. Effect of phospholipids

concentration

on emulsion

mean droplet

size.

size

.j /:/y i!k

S. Muchtar, S. Benita/Colloicis Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190 - 0.5

-1

d

lp ...._.._.._ "'-0

..,_._....

,..’

o”“’

. . . . . .

- 0.2

cJ........

- 0.1

\

‘.

....

..,~

I

! a

\

o..”

I

G 2 k?

- 0.3

,/

l-r

‘;‘ L

- 0.4

.

I

2

.

I

3

.

4

n -..-....-.1

_,__.....

I

.

5

I

.

I

6

10.0

.

7

8

PH Fig. 6. Effect polydispersity.

of pH

on

emulsion

mean

droplet

size

and

4007 3504 kI2

::

83

200 -

Ti

150-

% 9

loo50-

O+f’.

1

1

1

I

-4.0

-3.5

-3.0

-2.5

CaCl2

CONCENTRATION,

Fig. 7. Effect of Ca*+ ion concentration

,

I

-2.0

.

1

-1.5

log M

on emulsion

stability.

the innocuity and clinical acceptability of the excipients. It should be emphasized that information from previous studies regarding the interactions between the amphoteric surfactants and the phospholipids contributed to the selection of the actual emulsifier combination [13]. It was then shown that both amphoteric surfactants can penetrate into the phospholipid monolayers. They are not ejected from the monolayer, even at its high compression, reflecting the strong molecular interactions that occur between the phospholipids and Miranols at the air-water interface. Other workers [14] have already used a similar approach to

187

demonstrate the fact that the existence of molecular interactions between surface-active molecules at the air-water interface would explain the high stability of o/w emulsion formulations using the above-mentioned components and triglycerides. Phospholipids are well-known anionic, biocompatible emulsifiers which are widely used in the formulation of marketed fat emulsions intended for intravenous administration. However, phospholipids are also considered to be poor emulsifying agents since they are unable to stabilize emulsions containing various active drug molecules. They must be combined with other emulsifying agents in order to yield potentially long-lasting, stable, medicated emulsions. Therefore an attempt was made to stabilize the emulsion formulation by adding amphoteric surfactants which are capable of interacting with the phospholipids at the oilwater interface of the emulsified oil droplets on the basis of the monolayer study results [ 131. It can be observed from Fig. 4 that the concentration of Miranol solutions at which the smallest droplet size was achieved was 0.75% Miranol C,M@ and 0.50% Miranol MHT@. These results indicate that both Miranols penetrate into the phospholipid film at the oil-water interface and contribute to the decrease in emulsion mean droplet size and droplet population dispersion (data not shown). Miranol MHT@ exhibited higher surface activity, markedly decreased the mean droplet size and was more effective at lower concentration than Miranol C,M@. Monolayer studies that were previously carried out [ 131 showed that Miranol MHT@ elicited higher efficiency and effectiveness in decreasing the surface tension at the air-water interface than Miranol C,M@. Furthermore, this was also confirmed by the results obtained in the investigation of mixed phospholipids Miranol MHT@ or C,M@ films [13]. This behaviour is probably due to the stabilizing moieties of Miranol MHT@ which are mainly based on sulfate groups that are more electronegative than the acetate groups in Miranol C,M@, resulting in the formation of larger hydrodynamic protective layers around the oil droplets. Moreover, it can be seen from Fig. 5 that the lowest droplet size was achieved at a phospholipid concentration ranging from 0.75 to 1.5% (w/w). At

S. Muchtar, S. BenitalColloidF Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190

188

1.1

*

25C

v

3oc

0

5

10

15

TIME,months Fig. 8. Chemical

stability

evaluation

of HU-211

in an o/w emulsion

at four different

storage

temperatures.

140-

130E H 5;

120-

G

1 0

,

15

10

5

TIME.months Fig. 9. Physical

stability

of HU-211

emulsion

a concentration of 2% (w/w), the droplet size increased, probably due to the repulsion of the amphoteric surfactant from the oil-water interface by the high phospholipid concentration. The polydispersity of oil droplets (data not shown) was minimal at the concentration of 0.75% (w/w), indicating that at this concentration the amount of phospholipid molecules is sufficient to cover the regenerated surface area of the oil droplets without expelling amphoteric surfactant molecules. Finally, the oil content of the emulsion was maintained at 5% (w/w) in order to achieve a viscosity of between

at four different

storage

temperatures.

2 and 3 cP, which happens to be the optimal viscosity for ocular preparations [ lo,1 51. The results of these preliminary studies led to the identification of an optimal emulsion formulation. The total drug composition of this formulation is depicted in Table 3. The pH of the emulsion was adjusted to the appropriate value according to the active compound stability constraints. Emulsions stabilized with phospholipids are generally sensitive to pH variation [ 15). The emulsion stability depends on the extent of ionization of the anionic phospholipid components in the mixture,

S. Muchtar, S. BenitalColloids Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190 Table 2 Distribution

profile

of HU-211

and

pilocarpine

phase separation. This was also confirmed in the present study for an emulsion composition stabilized by a combination of phospholipids and pluronic F-68. However, it was interesting to note that the detrimental effect induced by Ca2+ ion addition is much less pronounced in the actual emulsions stabilized by a combination of phospholipids-Miranol MHT. Monolayer studies that were previously performed [ 131 indicate that Ca2’ ions can be adsorbed at the oil-water monolayer and can be intercalated between the polar head groups of the phospholipid molecules. This intercalation expands the distance between the emulsifier molecules and disrupts the continuity of the film. The presence of Miranol MHT in the water phase reduces the above effect due to the interaction of the amphoteric molecules with calcium which prevents the adsorption onto the interface. Such an effect could also be visualized in the present study where Miranol MHT probably intercalated with Ca2+ ions, preventing the destabilization of the mixed emulsifier monolayer film at the oil-water interface of the emulsion resulting in a moderate increase in mean droplet size with increasing Ca2+ ion concentration. The results of the chemical (Fig. 8) and physical (Fig. 9) stability assessments indicate that the chemical degradation of the HU-211 emulsion could not be attributed to the relative physical

in emulsion

phases Emulsion phase

Active ingredient

partitioning

HU-211 content,

HU-211 content,

0.12%

49.72” 50.21 -

Oil Water Interface

(% (w/w))

0.24%

51.6” 49.7 _

Pilocarpine content, 1.7% 5.9 19.2 14.9

a In the case of the HU-211 emulsions, it was impossible to separate the oil phase and the interface. Therefore the oil values of 49.72% and 51.6% relate to both.

especially PE. The decrease in the extent of ionization causes a decrease in the zeta potential and thereby reduces the electrostatic repulsion between the oil droplets. Such behavior is also observed in the present study with the emulsion stabilized by a combination of phospholipids and a non-ionic copolymeric surfactant, Pluronic@ F-68 (Fig. 6). However, as expected, the effect of pH on the Miranol-stabilized phospholipidic emulsion was minimal over a wide pH range owing to its amphoteric character. Furthermore, the amphoteric surfactant is self-buffered and shows buffering capacity throughout the pH range 4-10. It is well known from the literature [ 16,171 that the addition of small amounts of divalent cations such as Ca2+ to fat emulsions rapidly leads to Table 3 Emulsion

composition

Ingredient

Emulsion

composition

HU-211

(%) Pilocarpine

Oily phase Oil a-Tocopherol Drug Lipoid E-SO

5 0.02 0.12, 0.24 0.75

5 0.02 1.7” 0.75

Water phase Miranol Glycerin Water to final adjusted

pHb

a Equivalent to 2.0% pilocarpine HCl. b HU-211, pH 6.8; pilocarpine, pH 5.0.

0.50 MHT or 0.75 C,M 2.25 100

189

0.50 MHT or 0.75 C,M 2.25 100

190

S. Muchtar, S. BenitalColloids Surfaces A: Physicochem. Eng. Aspects 91 1994) 181-190

instability of the submicron emulsion, which was moderate at low storage temperatures. In contrast, the physical instability at high storage temperatures may be associated with the chemical degradation of HU-211 since it began earlier (after 6 months) than the physical instability (which began after 7 months) and probably altered the physicochemical properties of the mixed emulsifier film at the oil-water interface of the oil droplets. It can be deduced from the stability results reported in this study that HU-211 submicron emulsions remain practically stable, both physically and chemically, when stored at between 4 and 30°C over a period of at least 15 months. It should be added that pilocarpine emulsion stability was monitored at 4°C over a 2 year period. No marked chemical or physical change was noted. It was surprising to note that about 50% of HU-211, which is a very liposoluble drug, was localized in the external aqueous phase of the emulsion (Table 2). It is believed that HU-211 had been rather solubilized in the micelles formed by the excess of surfactants used in the actual emulsion formulation. The presence of the micelles was corroborated by low angle X-ray scattering measurements and the exact nature of the micelle is currently being investigated (data not shown). Moreover, this was confirmed by independent studies on HU-211 solubility in aqueous phase containing various concentrations of Miranol. It was found that HU-211 solubility in aqueous 2.25% glycerin solution was 0.038 mg ml-’ while in a Miranoll MHT aqueous micellar solution, the HU-211 solubility was 0.55 mg ml-‘. The HU-211 did not present any affinity for the oil-water interface of the emulsion, suggesting that this molecule did not exhibit any surface activity. The results of the partition and phase distribution profile of the pilocarpine emulsion (Table 2) confirmed that pilocarpine, which is a watersoluble compound, is located mainly in the water phase (79.2%). However, the fraction of drug located at the oil-water interface and in the oily phase is not negligible, and it may have improved the ocular bioavailability and the prolonged antiglaucoma effect over that of pilocarpine hydrochloride solution as shown in independent clinical studies (data not presented).

5. Conclusion It can be concluded from the overall results presented in this study that the use of amphoteric surfactants combined with phospholipids improved the physical properties and stability of the emulsions which can be exploited as promising therapeutic ophthalmic delivery systems. Furthermore, toxicity evaluation and pharmacological efficacy studies have already been performed and will be reported in the near future.

References 111 P.

Fitzgerald, J. Hadgraft and C.G. Wilson, J. Pharm. Pharmacol., 39 (1987) 487. 121 M.F. Saettone, B. Giannaccini, G. Delmonte, V. Campigli, G. Tota and F. LaMarca, Int. J. Pharm., 43 (1988) 67. c31 D. Meisner, J. Pringle and M. Mezei, Int. J. Pharm., 55 (1989) 105. c41 T. Harmia, P. Speiser and J. Kreuter, J. Pharm. Acta Helv., 62 (1987) 322. c51 P.K. Hansrani, S.S. Davis and M.J. Groves, J. Parenter. Sci. Technol., 37 (1983) 145. C61 S. Muchtar, S. Almog, MI. Torracca, M.F. Saettone and S. Benita, Ophthalmic Res., 24 (1992) 142. c71 H.P. Ciuchta and K.T. Dodd, Drug Chem. Toxicol., 1 (1978) 305. cs1 R. Mechoulam, and J.J. Feigenbaum, in G.P. Ellis and G.B. West (Eds.), Progress in Medicinal Chemistry, Vol. 24, Elsevier, Amsterdam, 1987, Chapter 5. c91 R. Mechoulam, J.J. Feigenbaum, N. Lander, M. Segal, T.V.C. Jarbe, A.J. Hiltunen and P. Consroe, Experimentia, 44 (1988) 762. Cl01 V.H.L. Lee and J.R. Robinson, J. Ocular Pharmacol., 2 (1986) 67. Cl11 A. Bach and V.K. Babayan, Am. J. Clin. Nutr., 36 (1982) 293. Cl21 D.L. Teagarden, B.D. Anderson and W.J. Petre, Pharm. Res., 5 (1988) 482. M.Y. Levy, S. Benita and A. Baszkin, t-131 S. Muchtar, Colloids Surfaces B: Biointerfaces, 1 (1993) 149. Cl41 M.Y. Levy, S. Benita and A. Baszkin, Colloids Surfaces, 59 (1991) 225. J. Pharm. Sci., 61 [I51 S.P. Loucas and H.M. Haddad, (1971) 185. Cl61 S. Muchtar, M.Y. Levy, S. Sarig and S. Benita, Sci. Tech. Pharm., l(l991) 130. Cl71 S.S. Davis and M. Galloway, J. Clinic. Hosp. Pharm., 11 (1986) 33.