INTRODUCTION:

Fentanyl (N-phenyl-N-(1-(2-phenylethyl)4-piperidinyl) propanamide), commercially available as Fentanyl citrate salt, is a potent synthetic opioid used as an anaesthetic and for the treatment of breakthrough pain^1-4. Fentanyl is approximately 200 times more potent than morphine and has a quick onset (1–2 min) and short duration of action (30– 60 min)⁵. It exhibits its analgesic effects by binding to the μ-opioid receptor, and is metabolized primarily by cytochrome P450 3A4 (CYP3A4) enzyme in the liver⁶. In addition, fentanyl is a small opioid molecule that is given in low doses (100 – 1000µg), and lacks the bitter taste associated with some other opioids^7,8.

The opioid fentanyl is highly lipophillic (octanol: water partition coefficient of ~800:1) and hence is easily absorbed across the mucosal surface, resulting in a faster onset of action. Among the current products on the market, an oral transmucosal effervescentfentanyl buccal tablet (Fentora^®, Cephalon Inc, Frazer, PA, USA) and a sublingual fentanyl tablet (Abstral^®, Orexo AB, Uppsala, Sweden) are available. These are based on a proprietary Ora Vescent^® drug delivery system (Cima Labs Inc, Brooklyn Park, MN, USA) that changes the local pH, the purpose of which is to enhance the absorption of fentanyl through the buccal mucosa. Pharmacokinetic data of Fentora^® revealed that fentanyl was rapidly absorbed within 35–45 minutes and an average onset of analgesia at approximately 15 minutes along with typical side effects of opioid analgesics⁹. Another oral transmucosal fentanyl citrate formulation i.e. lollipop or lozenge (Actiq^®, Abbott Laboratories Inc, Abbott Park, IL, USA) was designed to allow rapid absorption of fentanyl through the mucosa as a result of its lipophilicity^10,11. However, this lollipop formulation stimulates saliva production and swallowing, thus some fentanyl was swallowed and metabolized due to hepatic first-pass metabolism and causing typical opioid dose-related side effects.

Ethypharm (Saint Cloud, France) developed a sublingual tablet consisting of different layers coated onto a neutral core. An alkalinizing layer surrounds the fentanyl citrate layer to increases the solubility of fentanyl and provides optimal oromucosal conditions for rapid dissolution and absorption. Its pharmacokinetics were studied in healthy volunteers and compared with those of the reference product (OTFC). Bio equivalence was demonstrated with OTF Cinaratio of 1:5.The absolute bio availability was estimated to be 70%¹². Another formulation, developed as a buccal-soluble fentanyl film, shows an absolute bioavailability of 71%¹³.

Oral ingestion of opioids is associated with a delay in both onset of action and peak analgesic effect, resulting in inadequate pain control during the first 30 minutes of a breakthrough episode¹⁴. Low oral bioavailability of FC because of its high hydrophobicity (log P=4.05) and also extensive hepatic first pass metabolism hindered its oral use^6,15. Hence oral formulations of fentanyl are not available in the market whereas parenteral, buccal and sublingual formulations are available^16-18. But parenteral formulations may not always be convenient for the patient. Therefore, there is growing interest in developing non-invasive drug delivery system where a rapidly dissolved drug is immediately absorbed into the systemic circulation¹⁹. Oromucosal delivery, via sublingual mucosa as absorption site, is a promising drug delivery route which promotes rapid absorption and high bioavailability, with subsequent almost immediate onset of pharmacological effect. These advantages are the result of abundance of blood supply at the sublingual region allows excellent drug penetration to achieve high plasma drug concentration with rapid onset of an action^20,21.

As mentioned above with sublingual formulations of fentanyl citrate nearly 70 to 71% absolute bioavailability was achieved^{12, 13}. That’s why lipophilic formulations were chosen to overcome the above barriers and among them microemulsion as drug delivery systems have recently gained wide acceptance due to robust formulations perspectives, ease of production and practical enhancement of drug permeability^22-24. These are clear, thermodynamically stable, isotropic liquid mixture of oil, water and surfactant, frequently in combination with a co-surfactant^25,26. This o/w microemulsion formulation enhances the sublingual and buccal bioavailability of fentanyl by facilitating transcellular (across the cell) and paracellular (between the cells) absorption²⁷. Fentanyl being lipophillic drug transported transcellular by a concentration dependent passive diffusion process and also being formulated into o/w type of microemulsion, therefore it is also subjected to transport via the intercellular porous route i.e. paracellular.

Extensive review of literature reveals lack of information about the bioavailability enhancement of fentanyl using microemulsion as drug delivery systems. Thus the current study was aimed to develop an o/w type of fentanyl microemulsion to enhance its sublingual bioavailability. To achieve this, fentanyl solubility was tested in various vehicles and vehicles with highest solubility for fentanyl were selected as components (oils, surfactants and co-surfactants) of microemulsion. The developed fentanyl loaded o/w microemulsion was investigated for their physiochemical characteristics. Afterwards, the optimized fentanyl microemulsion was evaluated by means of in vitro diffusion study using modified Franz diffusion cell. The stability of developed microemulsion was also investigated.

MATERIALS AND METHODS:

Materials:

Fentanyl citrate USP was purchased from Rusan Pharma Ltd. Gujrat. Labrasol^®, Caproyl PGMC and Caproyl 90 were obtained as a gift sample from Gattefosse Saint Priest (Lyon, France). Monebat^® -20 was obtained as a gift sample from Mohini Organics Pvt. Ltd. Mumbai. Monoolein^®was purchased from Tokyo Chemical Industries Co., Ltd. Tokyo, Japan. Oleic acid pure was purchased from Merck Specialities Pvt., Worli, Mumbai. Propylene Glycol was purchased from Shell Chemicals, Singapore. Glycerin was purchased from KL-Kepong Oleomas, Malaysia. Sesame oil was obtained as a gift sample from Global Merchants. Capmul^® MCM C8, Capmul^® PG 8 and Acconon^® MC8-2was obtained as a gift sample from ABITEC Corporation, Columbus, USA.

Screening of oils, surfactants and co-surfactants for ME:

The solubility of FC in various oils, surfactants and co-surfactants was determined to find out the appropriate oils, surfactants and co-surfactants with good solubilizing capacity forFC in ME. Oils employed were Oleic acid, Monoolein^®, Sesame oil, Capmul^® MCM C8, Capmul^® PG 8, Caproyl PGMC and Caproyl 90. Surfactants and co-surfactants employed were tween 20, labrasol, Acconon^® MC8-2,Propylene glycol and Glycerin. Anexcess amount of FC was added into each 5 ml of oil, surfactants and co-surfactants and the resultant mixtures were sonicated at 37^◦C at an interval of 15 minutes for 60min followed by centrifugation for 30 minat 2500rpm. The supernatant was filtered through a nylon syringe (pore size 0.22µm and diameter 25mm) and FC was determined spectrophotometrically at 257.6 nm by appropriate dilution of filtrate with methanol. Solubility results of FC in various oils, surfactants and co-surfactants are given in Table 1.

Construction of pseudo-ternary phase diagrams²⁸

Pseudo ternary phase diagrams were constructed using water titration method at ambient temperature in order to find out concentration range for ME components. Three phase diagrams were prepared with the 1:1, 1:2 and 2:1 weight ratios of polysorbate 20 to propylene glycol, respectively. For each phase diagram at a specific surfactant (S)/cosurfactant (CoS) mixing ratio (Km), the ratios of oil to the mixture of S/CoS were varied from 1:9 to 9:1. Each mixture of oil and S/CoS was diluted with water, added drop wise, under moderate shaking. After being equilibrated at ambient temperature for 24 h, the mixtures were assessed visually for the clarity of mixture. Phase diagrams were constructed using CHEMIX Ver.3.60 Ternary diagram software. From this ternary phase diagrams, ME compositions were selected that existed into o/w region of ternary phase system.

Preparation of Microemulsion:

The ME for FC was prepared by the water titration method. Based on the ME areas in the phase diagrams, different FC ME formulations were prepared by varying the ratios between S/CoS and at Km= 1:1, 1:2 and 2:1 as given in Table 2. FCadded in the range of 3.2 – 3.97% (w/w) as per solubility into the capmul^® MCM C8, propylene glycol and tween 20. The oil and S_mix mixture was then titrated with drop wise addition of double distilled water with continuous shaking to produce a clear mixture. MEs were optimized with respect to S_mixratio and its concentration effect on in vitrostudies.

Characterization of ME:

According to the regions obtained for o/w ME in the phase diagrams, five ME formulations were selected and evaluated for following parameters:

Measurement of pH:

The pH of the prepared MEs was measured by direct immersion of pHmeter electrode in the formulations at room temperature and all the measurements were carried out in triplicates²⁹.

Measurement of viscosity:

The ME formulations were evaluated for their viscosity at 25±2^◦C using Brookfield viscometer model LV DV-II +PRO [2000 Series] equipped with spindle number S00³⁰.

Measurement of electrical conductivity:

Electrical conductivity of the formulations was measured using a conductivity meter and based on the electrical conductivity, the phase systems of the MEs were determined. The electrode was dipped in the ME sample until equilibrium was reached^31,32.

Drug content:

The quantity of ME containing about 0.8mg of FC, was taken in a 20.0mL volumetric flask. The samples were mixed gently with nearly 10mL of methanol and sonicated (Leela sonic Sonicator, Leele Electronics, Mumbai, Maharashtra) to extract FC completely and then volume was made with methanol. Then absorbance of this solution was taken at 257.6 and analyzed using developed and validated UV spectrophotometric method³³.

In vitro diffusion study:

Franz diffusion cell with an effective diffusion area of 2.009cm² was used for in vitrorelease studies. The dialysis membrane with average flat width 29.31mm, average diameter 17.5mm (Himedia Laboratories Pvt. Ltd.) were mounted carefully in between donor and receptor compartment of diffusion cell. Donor compartment was applied with 0.2g of test microemulsion and the receiver compartment was filled with 24.0ml distilled water pH 6.5. Temperature of receptor medium was maintained at 37±0.5 ̊C with magnetic stirring at 100rpm throughout the experiment. For each experiment, 1ml sample of the receiver medium was withdrawn at predetermined time and then the volume was made up with the equal volume of fresh receiver medium. All samples were filtered through a 0.45µm pore size cellulose membrane filter and analyzed by HPLC method as mentioned in the USP monograph for fentanyl citrate injection³⁴. The mean cumulative values for % drug diffused through the dialysis membrane into the receptor fluid were plotted versus time. The cumulative amount of FC in the receptor fluid per unit area of dialysis membrane, Qt/A (A = 2.009cm²), was plotted against time (t). The steady-state fluxes (J_SS) were calculated from using following formula:

Jss = Q / (A • t)

Where, Q stands for the quantity of compound transported through the membrane in time t

A denotes the area of exposed membrane in cm².

In order to obtain the permeability coefficient Kp (cm/h), the following equation was used;

Kp = Q / [A • t • (Co - Ci)]

Where,

Q stands for the quantity of compound transported through the membrane in time t (min)

Co and Ci are the concentrations of the compound on the outer side (donor side) and the inner side (receptor side) of the membrane respectively

A denotes the area of exposed membrane in cm2

Usually Co denotes as the donor concentration and Ci as 0.

Droplet size and polydispersity index (PDI):

The average droplet size and its distribution (characterized by polydispersity index, PDI) in MEs were measured using dynamic light scattering zetasizer (DLS) (Malvern Zetasizer ZEN3500, UK). All measurements were performed with a scattering angle of 90 ̊ at 25.0 ̊C after diluting the dispersion to an appropriate volume and having dispersion medium viscosity 0.894 mPa.s.

Zeta potential measurements:

The charge on the surface of particles was measured characterized by the nanopartica SZ-100 (Horiba Scientific Ltd., Japan) by measuring the zeta potential of MEs. Small-volume disposable zeta cell and converted to zeta potential by in-built software using the Helmholtz-Smoluchowski equation for measuring the Electrophoretic mobility (μm/s). Zeta potential determinations were carried out in triplicate.

Stability of ME:

MEs were analyzed visually for transparency, phase separation by keeping at 40 ̊C and 75% RH and at room temperature for a period of 3 months. The centrifugation (Laboratory Centrifuge Remi R-8C, India), of formulations at 3,000rpm for 30 min was carried out to assess the physical stability of ME. Clarity, phase separation were investigated to judge the optimal stability of ME formulation.

RESULTS AND DISCUSSION:

Screening of oils, surfactants and co-surfactants for ME:

Solubility of FCin various oils and non-ionic surfactants is shown in Table 1. The solubility of FCwas highest in Capmul^® MCM C8, followed byCapmul^®PG 8,caproyl 90, capmul^®PGMC, Monoolein, Sesame oiland Oleic acid. Besidesthat the drug has a relative high solubility in Capmul^® MCM C8 compared toother oils; it was also selected for the preparation of MEs due to itswell-known bioavailability and permeation enhancing property and biocompatibility^35-37. Choice of the surfactant is critical in formulation of MEs, asit helps in the reduction of the interfacial tension by forming a filmat the oil–water interface resulting in the spontaneous formation ofMEs³⁸. There are literature reports regarding the selection of surfactant on the basis of drug solubility. However, the solubilizationof oil with the surfactant is also an important factor. It is not necessary that, the surfactant having good solubilizing property for drug would also have equally good affinity for the selected oil phase.Non-ionic surfactants were included in the screening of surfactants since they are well-known for their non-irritant nature. They areless affected by changes in pH and ionic strength and are generally regarded as safe and biocompatible. Polysorbate 20 was non-ionic surfactant and had high solubility than other surfactants, so Polysorbate 20 was used to prepare MEs. Co-surfactants are also added to achieve ME systems at low surfactant concentration. Amphiphilic nature, hydrophobic chain and terminal hydroxyl groups of co-surfactants enable them to intermingle with surfactant monolayer at the interface resulting into changes in their packing arrangement which in turn can affect the curvature of the interface and interfacial energy. The incorporation of co-surfactant enhanced the penetration of the oil phase in the hydrophobic zone of the surfactant monomers, which in turn reduced the interfacial tension and increased the flexibility and fluidity of the interface, ultimately leading to increased entropy of the system³⁹. The presence of co-surfactant decreases the bending stress of the interface and imparts the interfacial film sufficient flexibility to take up different curvatures required to form ME over a wide range of composition. PG showed high solubility than other co-surfactants, so it was used for further study. So in this study Capmul^® MCM C8, Polysorbate 20 and PG were selected as the oil phase, surfactants and co-surfactants respectively for the formulations of ME containing FC.

Table 1. Saturation solubility of FC in different oils, surfactants and co-surfactants at 37^◦C (mean ± SD; n = 3).

Sr. No	Components	Solubility (mg/ml)
1	Capmul^® MCM C8	40.45 ± 0.06
2	Capmul^®PG 8	4.09 ± 0.02
3	Caproyl 90	2.917 ± 0.003
4	Capmul^® PGMC	1.9 ± 0.06
5	Monoolein	1.243 ± 0.04
6	Sesame oil	0.204 ± 0.01
7	Oleic acid	0.082 ± 0.003
8	Polysorbate 20	5.662 ± 0.008
9	Acconon^®MC 8-2	5.467 ± 0.05
10	Labrasol^®	4.575 ± 0.08
11	Propylene glycol	44.25 ± 0.052
12	Glycerin	10.327 ± 0.06

Construction of pseudo-ternary phase diagrams:

The pseudoternary phase diagrams were constructed to determine the concentration range of components in the existence range of ME. The pseudoternary phase diagrams were constructed by titration of homogeneous liquid mixtures of oil, surfactant and co-surfactant with water at room temperature as shown in Fig. 1⁴⁰. At Km (S: CoS) values of 1:1, 1:2 and 2:1, mixture of oil, surfactant and co-surfactant blend was varied from 9:1 to 1:9 and vortexed. Each mixture was then slowly titrated with aliquots of distilled water and stirred at room temperature to attain equilibrium. The mixture was visually examined for transparency. All the components were converted to percent weight before constructing the phase diagram⁴¹. The marked area represent all formulations that could self-emulsify in seconds and be infinitely diluted by distilled water indicating that the ME formed are capable of keeping FC solubilized. The shaded areas of phase diagrams shows the ME regions, whereas the non-shaded area display the turbid region. Then within this shaded area that particular ME’s are selected which formed oil in water type of ME’s as mentioned in the Table 2. The formed ME’s are clear, isotropic, transparent and of low viscosity determined by visual inspection.

In vitro diffusion study:

In vitro diffusion studies of optimized FC ME formulations shows successful diffusion through dialysis membrane (Himedia Laboratories Pvt. Ltd.) and the results obtained are presented in Fig.2., and the calculated steady state flux (J_ss) are tabulated in table 4.

FC o/w ME crosses the dialysis membrane mimicking the sublingual mucosa using two different pathways: transcellularly (across the cell) and paracellularly (between the cells). FC being the lipophillic drug transported transcellularly by a concentration dependent passive diffusion process, by facilitated diffusion using a receptor or carrier molecule, or by vesicular transport mechanism. FC being formulated into oil in water type of ME, therefore it is also subjected to transport via the intercellular porous route (paracellular route), across the sublingual route. The presence of oil droplets containing FC along with external aqueous phase appeared in favor of FC permeability. It might be stated that ME could act as drug reservoirs where loaded drug is released from the internal phase to the external phase and finally onto the mucosa.

FC showed better diffusion from ME C2 than ME A2, respectively. For ME C2, the drug exhibited highest steady state flux, whereas it was less for ME A2. The ME C2 exhibited higher steady state flux due to smaller particle size and also having appropriate oil: S_mix proportion which facilitates the diffusion process.

Table 4. Steady state flux and modeling parameters of optimized FC Microemulsion formulations

Formulation code	In vitro release study
Formulation code	Steady state flux J_SS (µg/cm². h)	Permeability coefficient K_p (cm/hr)
ME A2	278.18 ± 0.39	0.058 ± 0.003
ME C2	361.92± 0.57	0.096 ± 0.005

Figure 2. Percent cumulative drug diffused verses time profiles of FC through optimized A2 and C2 microemulsion formulations

Droplet size and polydispersity index (PDI):

Droplet size of optimized ME C2 ME was found to be 96.9nm. The concentration of surfactant in the S_mix ratio of 2:1 was higher in C2 formulation, therefore reduced particle size was observed. As a generalization, the droplet size is inversely proportional to emulsion stability. Thus smaller particle size i.e. 96.9 nm for C2 microemulsion formulation would be more stable as shown in Fig.3.

The polydispersity index (PDI) of optimized C2 ME was found below 0.45 which confirmed narrow size distribution of oil droplets. Generally, for narrow distribution PDI ranges from 0.01 to 0.5 and for broad size distribution, PDI > 0.7^42,43. As PDI of C2 ME was 0.263 which confirmed narrow size distribution of oil droplets.

Figure 3. Particle size distribution plot of optimized C2 ME microemulsion formulation

Zeta potential (ZP) measurements:

ZP is an indicator of the stability of ME. This ZP is the charge present on the dispersed phase (oil globule) at the shear plane of the electric double layer in the aqueous solution. As per the principle of o/w ME, a uniform layer of tween 20 (surfactant) has formed surrounding the oil globule of Capmul MCM C8. As the literature study reveals that absorbed layer of large molecules shifts the shear plane to a farer distance from the particle surface and this leads to a reduction of the measured ZP⁴⁴. That means in case of highly charged particle surface, a relatively low ZP will be measured and despite the low ZP the system will be stable⁴⁵. Thus the tween 20 (mol wt 1128) had shifted the shear plane to a farer distance from the surface of oil globule and resulted into low ZPvalue as observed for C2 ME formulation.The ZP measurement for optimized C2 ME was found to be -8.5 mV and graph of intensity (a.u.) vs ZP (mV) was exhibited in the Fig.4.

Figure 4. Zeta potential plot of C2 ME microemulsion formulation

Stability study:

In stability studies, the ME exhibited no precipitation of drug, creaming, phase separation, and flocculation on visual observation and was found to be stable after centrifugation (3000 rpm for 15 min) at 40 ̊C and 75% RH and at room temperature.

CONCLUSION:

In conclusion, the permeability of fentanyl citrate from the microemulsion is determined in the present study to be with a steady state flux 361.92 µg/cm².h. This fentanyl citrate microemulsion can be further researched for its applicability in sublingual film formulation. The direct relationship between the surface area of the film and improved permeability of fentanyl citrate via microemulsion drug delivery will have a combined effect for treatment of breakthrough pain.This aspect provides confidence for therapeutic administration of fentanyl citrate microemulsion for the management of breakthrough pain.

CONFLICTS OF INTEREST:

The authors report no conflicts of interest regarding this investigations.

ACKNOWLEDGEMENTS:

The authors are thankful to Centre for Advanced Research and Innovation (CARIn), Zim Laboratories, Kalmeshwar Dist. Nagpur (M.S.), India for providing the instrumentation and facilities and License for working with Narcotic opioid analgesic drugs. The authors are sincerely thankful to Government of India, Ministry of Science and Technology, Department of Science and Technology (DST), New Delhi for their thorough support. The authors are also thankful for our analytical research developmental laboratory team members for providing timely help.

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Received on 24.09.2021 Modified on 26.04.2022

Research J. Pharm. and Tech 2023; 16(3):1319-1326.

DOI: 10.52711/0974-360X.2023.00217

Formulation code	Composition [Capmul^® MCM C8: Smix (1:1) (Tween 20:propylene glycol): water: FC]	Composition [Capmul^® MCM C8: Smix (1:2) (Tween 20:propylene glycol): water: FC]	Composition [Capmul^® MCM C8: Smix (2:1) (Tween 20:propylene glycol): water: FC]
ME A2	5.76:23.05:67.22:3.97	-	-
ME A3	9.68:23.43:63.89:3.2	-	-
ME B2	-	5.77:23.08:67.33:3.82	-
ME C2	-	-	2.88:10.08:83.16:3.93
ME C3	-	-	8.65:20.19:67.29:3.87