Evaluation of the Galenic stability of a Propofol Emulsion after the Expiration date

 

Ali Cherif Chefchaouni^1,2, Ismail Bennani^3,4, Soumaya El Baraka^1,2, Aicha Fahry¹,

Abdelkader Laatiris¹, Naoual Cherkaoui¹, Yasser El Alaoui^1,2, Younes Rahali^1,2

¹Faculty of Medicine and Pharmacy, Mohammed V University of Rabat, Rabat, Morocco.

²Ibn Sina University Hospital Center, Rabat, Morocco.

³Department of Pharmacy, Faculty of Medicine, Pharmacy, and Dental Medicine of Fez,

Sidi Mohamed Ben Abdellah University, Fez Morocco.

⁴Department of Pharmacy, Hassan II University Hospital of Fez, Morocco.

 

ABSTRACT:

Objective: Propofol is an intravenous lipid emulsion indicated for the induction and maintenance of general anesthesia or sedation. Like all emulsions, propofol is thermodynamically unstable. The objective of this study was to evaluate the post expiration stability of a batch of propofol. Methods: The parameters studied to evaluate the stability of the drug were: visual appearance, ph, droplet diameter and zeta potential.For this purpose we had 20 ampoules of Propofol Fresenius with an expiration date of May 2021. The measurements started directly 1 month after the expiration of the batch and lasted 6 months. Results: Visual examination showed no abnormalities suggestive of instability. The pH varied between 6.95 and 7.15 until it decreased to a value of 5.58 at the 6th month after expiration. The diameter of the droplets ranged from 109.52nm to 125.15nm with a maximum of 169.5nm. Conclusion: The results obtained suggest a good galenic stability up to 3 months after expiry and may allow a use beyond the limit of use in case of complete stability study.

 

 

 

INTRODUCTION: 

Propofol (2,6-diisopropylphenol) is a potent intravenous (IV) hypnotic drug commercially launched in 1986 in Europe and 1989 in the United States¹. It has a favorable pharmacokinetic and pharmacodynamic profile, which has made it the most commonly used intravenous anesthetic over the past three decades^2-4. Propofol is used for sedation and anesthesia for almost all types of surgery, but is particularly well suited for anesthesia in ambulatory patients⁵. However, despite the commercial success of propofol emulsions, there remain drawbacks associated with current formulations: emulsion instability⁶, need for antimicrobial agents⁷, hyperlipidemia, and pain on injection⁸.

 

In terms of formulation, propofol is in the form of an oil-in-water (O/W) emulsion due to its low solubility in water⁹, with a characteristic milky white appearance¹⁰. This appearance results from the property of small particles, but those that are large relative to the wavelength of white light, to reflect and refract light evenly when dispersed¹¹. Initially, it was formulated in Cremophor El for human use but the appearance of anaphylaxis of Cremophor EL led to its withdrawal¹².

 

Emulsions designed for IV administration must have extremely small droplet sizes and must be highly stable, as any large droplet size placed in the circulation can enter the pulmonary capillaries and could potentially lead to embolism¹³. They are basically considered to be thermodynamically unstable systems and therefore tend to "break down" during storage through a variety of instability mechanisms. As a result, one of the main challenges encountered during emulsion formulation is to preserve the physical stability of the emulsified system¹⁴. Among the mechanisms that can lead to the degradation of the emulsion are: gravitational separation, aggregation (or flocculation) of droplets, Ostwald maturation and coalescence of droplets¹⁵. The galenic stability of emulsions is closely linked mainly to the zeta potential, which is a negative surface potential surrounding the dispersed globules. As these droplets have a tendency to attract each other, the zeta potential counterbalances this phenomenon by creating repulsive forces between the emulsified globules¹⁶. On the other hand, the relationship between droplet size, which is a key parameter of these emulsions, and stability has been little studied.

 

The objective of this study is to evaluate the galenic stability of a propofol-based lipid emulsion after the expiry date indicated by the manufacturer.

 

MATERIALS AND METHODS:

In total, we had 20 ampoules of Propofol (Propofol Fresenius, batch number: 10LD9330) (10mg/ml) received from the pharmacy of the National Institute of Oncology in Rabat, and having a common use-by date: May 2021. The propofol received is presented in colorless glass ampoule; each 20ml ampoule contains 200mg of Propofol. The active substance is propofol, the other components of this preparation are: refined soybean oil, purified egg lecithin, glycerol, oleic acid, sodium hydroxide and water for injection.

 

Galenic stability measurements were started 1 month after the expiration date and for a period of six months between June and November 2021. These measurements included an examination in the form of visual observation, determination of pH, mean droplet diameter, polydispersity indexand zeta potential. For this purpose, the propofol ampoules were stored under the storage conditions dictated by ICH Q1A (30°C±2°C) for studies under intermediate conditions (6 months).

 

Afterwards, we used dry tubes from the same hospital to take samples from the propofol ampoules.

 

1) Preparation of the samples:

First, 36 sample tubes that will be used during the 6 months of study were prepared under a laminar flow hood, with sterile syringes and needles. Then, 20 ml of each emulsion sample was diluted with 1 ml of distilled water in a clean Malvern sample bottle adapted to the Zeta nanosizer v6.12 (Malvern Instrument) to perform the measurements.

 

2) Visual examination:

During each measurement, the prepared sample was observed in order to detect possible phase separations. The reversibility of the creaming was evaluated by 3 successive reversals. The samples were also observed by placing them in front of a black background and then in front of a white background for 5 seconds to look for particles visible to the naked eye. During this visual examination, we can also observe coloration as well as a possible macroscopic precipitation.

 

3) Droplet size and Zeta potential:

The average droplet diameter, the polydispersity index and the zeta potential of the emulsion at each month interval were measured by means of Zeta nanosizer v6.12 (Malvern Instrument) at 25 °C with different mechanisms:

·       Dynamic light scattering for droplet size distributions, and allows detection of droplets ranging in size from 1 nm to 1 mm;

·       Electrophoretic light scattering by measuring the electrophoretic mobility of the emulsion droplets. This mobility is converted into Zeta potential.

 

A dip cell (zen1002, Malvern Instruments) with a pair of parallel Pd electrodes was used to provide an electrical trigger on the charged particles.

 

The American Pharmacopoeia recommends that the average size of lipid globules should be less than 5um and that the percentage of lipid globules with a size greater than 5um should be less than 0.05% ^17,18.

For the zeta potential, it is defined as the potential difference between the surface of the ion layer closely bound to the particle surface and the electroneutral region of the solution ¹⁹. The zeta potential can either be relatively high (25 mV or more, absolute value), or low (below 25 mV, absolute value)²⁰.

 

For each month, 6 dry tubes of propofol were prepared, and for each tube the measurements were made in triplicate to determine the average (Table 1).

 

Table 1: Example of a grid for measuring parameters during month 1

Month	Tube	Mesure 1			Mesure 2			Mesure 3
		Diameter	Polydispersity index	Zeta potential	Diameter	Polydispersity index	Zeta potential	Diameter	Polydispersity index	Zeta potential
1	1
	2
	3
	4
	5
	6

4) pH:

A Corning 445 pH meter was used with a combined glass/SCE electrode to measure pH (pH:9 and pH:4). The meter was checked and calibrated against standard buffers. The electrode was thoroughly rinsed and its calibration checked after each individual sample to avoid errors due to contamination by electrode oil.

 

RESULTS AND DISCUSSION:

1) Visual examination:

On all the prepared tubes containing propofol, neither phase separation nor particles visible to the naked eye were observed during the 6 months of study. An emulsion would have been thermodynamically unstable if the dispersed and continuous phases transform into separate phases, oil and water; by melting or by coalescence of droplets. The European Pharmacopoeia recommends that an injectable lipid emulsion should not show signs of phase separation²¹. No color change or signs of precipitation have been visualized either.No phase separation was also observed after centrifugation (6000rpm for 30min) of the samples 6 months after expiration.

 

2) Mean droplet diameter and polydispersity index:

During the 6months of post-expiration measurements, the mean droplet diameter of the propofol emulsion ranged from 109.52nm to 125.15nm (Figure 1a), with a maximum (169.5nm) recorded in the fifth month after expiration (Table 2). The overall values obtained remain consistent with the United States Pharmacopeia (USP) <729>, which requires that the DMG of an emulsion should not exceed 500nm, and that the large diameter tail, defined as the volume-weighted percentage of fat greater than 5μm or PFAT 5 should be <0.05% of the total dispersed phase. In addition, the USP recognizes that the size of fat globules is critical because, due to mechanical filtration, large globules may be trapped in the capillaries of the lungs²². Two examples of droplet distributiondepending on volume and intensity were presented (Figure 4,5). All samples showed a unimodal droplet size distribution.

 

While the polydispersity index (PDI) varied only slightly (0.14-0.21) during the 6 months of measurements (Figure 1b). PDI is another important dimensionless parameter that describes the width or spread of the particle size distribution. The PDI value can vary from 0 to 1, where droplets with PDIs less than 0.1 imply monodisperse particles, and values greater than 0.1 may imply polydisperse particle size distributions²³.

 

Figure 1a: Evolution of the average droplet size

 

Figure 1b: Evolution of the polydispersity index

Table 2: Descriptive statistics for mean droplet diameter (nm), polydispersity index, zeta potential (mV), and pH

Month	Mean droplet diameter (nm)				Polydispersity index
	Mean	Standard Deviation	Minimum	Maximum	Mean	Standard Deviation	Minimum	Maximum
Month 1	109.52	25.92	90.90	156.03	0.14	0.07	0.07	0.21
Month 2	118.24	15.58	92.82	130.00	0.21	0.07	0.08	0.26
Month 3	116.97	22.18	90.94	148.30	0.19	0.08	0.08	0.25
Month 4	119.94	19.96	102.67	156.07	0.20	0.06	0.11	0.25
Month 5	125.15	30.46	91.26	169.50	0.20	0.07	0.08	0.29
Month 6	112.09	14.45	90.69	131.40	0.19	0.06	0.09	0.23

 

Month	Zeta potential (mV)				pH
	Mean	Standard Deviation	Minimum	Maximum	Mean	Standard Deviation	Minimum	Maximum
Month 1	-48.63	1.41	-49.80	-46.20	7.13	0.15	7.00	7.40
Month 2	-48.08	0.45	-48.90	-47.60	7.15	0.10	7.00	7.30
Month 3	-45.15	1.42	-47.80	-44.10	6.93	0.12	6.70	7.00
Month 4	-38.67	0.30	-38.90	-38.20	6.75	0.10	6.60	6.90
Month 5	-37.70	0.50	-38.50	-37.10	6.37	0.21	6.10	6.70
Month 6	-35.95	0.85	-37.60	-35.20	5.58	0.20	5.30	5.90

 

3) pH and Zeta potential:

During the first, second, and third months after expiration, the average pH measured was between 6.95 and 7.15 until decreasing to a value of 5.58 at the 6^th month after expiration (Figure 2). In the case of our emulsion and for optimal stability, the pH would be between 7 and 8.5³. In another study studying the stability under normal conditions of use of two propofol formulations which differ by the additive content, the pH varied from 7 to 8.5 in the formulation containing edetate disodium, and from 4.5 to 6.4 in the emulsion containing sodium metabisulfite²⁴. Furthermore, it is well known that an o/w emulsion prefers a basic pH to develop²⁵. The effect of pH reduction on the stability of phospholipid-stabilized emulsions has already been described^24,26.

 

Concerning the zeta potential, all the values obtained were negative. They ranged from -48.63mV to -35.95 mV over the six months of post-expiration measurements (Figure 3). Emulsion droplets with zeta potentials of -40 to -50mV are considered stabilized charged ^24,27. On the other hand, a value below 30mV in absolute value favors aggregation, instability, flocculation or coagulation of the emulsion²⁸. The results obtained after the three months of expiration can be considered similar to those previously reported for parenteral emulsions stabilized by phospholipids ²⁹with a value of about -50 mV at pH 8 and a progressive decrease with the decrease of the pH. During the 6 months of expiration, it was found in the results obtained also a decrease in zeta potential and pH.

 

 

Figure 2: Evolution of the pH

 

Figure 3: Evolution of Zeta potential

 

 

Figure 4: Droplet size distribution according to volume (month 1 after expiration)

 

Figure 5: Droplet size distribution according to intensity (month 6 after expiration)

 

4) General discussion:

The expiration date guarantees the stability and efficacy of a drug until that date in its original container. In reality, it represents only an assurance that the activity indicated on the label will last at least until that time³⁰. Nevertheless, FDA regulations do not require determining how long drugs remain active after the expiration date, allowing manufacturers to arbitrarily set expiration dates without determining the actual long-term stability of drugs³¹. Few studies have investigated the stability of an injectable lipid emulsion over time.

 

The stability of injectable lipid emulsions is a challenge in parenteral administration. The droplet size of this type of emulsion can have a direct impact on the toxicity and stability of this type of formulation. Increasing droplet size is the first indication of formulation stability problems. In addition, droplets larger than 5μm can be trapped in the lungs and cause pulmonary embolism³². It represents together with droplet distribution the most important characteristics of an injectable lipid       emulsion³³. Unlike the United States Pharmacopeia (USP), the European Pharmacopoeia does not propose droplet size limits for injectable lipid emulsions.

 

For zeta potential, high potential (negative or positive) emulsions are electrically stabilized while low zeta potential emulsions tend to coagulate or flocculate, which may result in poor physical stability. In general, when the zeta potential of an emulsion is high, the repulsive forces exceed the attractive forces, resulting in a relatively stable system³⁴.

 

The results obtained concerning the propofol emulsion could eventually admit galenic stability that could go up to 3 months after the expiration date.We then suggest a stability study in its entirety includinga sterility test as well as a bacterial endotoxin detection test should be performed, the dosage of the active substance should also be carried out to detect a possible degradation ofthe active principle over time: to consider a post-expiry use of the lipid emulsion in emergencies, particularly in the event of a shortage of the propofol emulsion. Nevertheless, special monitoring must be established if the use of the emulsion after its expiry date is envisaged, as these forms are thermodynamically unstable by nature.

 

CONCLUSION:

The study of the stability of the emulsion of propofol using different parameters: pH, the diameter of the droplets, and zeta potential made it possible to conclude a galenic stability of 3 months after the date of expiry. Completing a global stability study will probably allow post-expiry use with positive economic benefits for hospital structures.

 

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Accepted on 09.08.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(11):4993-4998.