Key Considerations for Stabilizing Oxidation-Prone Lipid-Based Drug Delivery Systems

By: Devon Langbein, Application Laboratory Analytical Scientist and Masumi Dave, Pharmaceutical Application Lab Manager - Gattefossé

Introduction

Lipid-based excipients are present in a wide range of drug delivery systems for different routes of administration, including oral, topical, transdermal, and parenteral. By their nature, lipid-based excipients confer unique advantages on the delivery of poorly soluble drug actives as solubilizers and bioavailability enhancers. Also by their nature, some lipid excipients may be subject to oxidative reactions due to high levels of fatty acid unsaturation and/or the presence of polyethylene glycol (PEG) moieties. If left unchecked, oxidative events may affect excipient functionality and drug product stability. Recognizing the sensitivities and oxidative susceptibilities of different lipid classes can help avoid or mitigate unfavorable oxidative events and more importantly aid the development of effective drug products. This article presents a general overview of key oxidative processes in sensitive lipids, steps to minimize unfavorable oxidative reactions, and stability-indicating parameters that can be used to assess the extent of degradation in lipids.

Lipid Chemistry

Lipids are chemically diverse organic compounds that are defi ned by a physical trait, namely their solubility in or miscibility with nonpolar solvents. Naturally occurring lipids include fatty acids, glycerides, phospholipids, waxes, and sterols. Excipients with a wide range of physical and chemical properties (e.g., melting point and hydrophilic-lipophilic balance number [HLB]) can be derived from these natural lipid sources. Many of these lipid-based excipients are constituted by fatty acid esters, comprising a hydrophilic headgroup, such as glycerol, propylene glycol, sorbitan, polyglycerol, polyethylene glycol (PEG), or other ethoxylated derivatives, and lipophilic fatty acids esterified to the headgroup. Consequently, they are amphiphilic entities with surface-active properties to varying degrees depending on the nature of the fatty acids and the hydrophilic headgroup. It should be noted that many lipid-based excipients do not exist as “pure”, single-component compounds but rather as multi-component systems, comprising a distribution of fatty acid chain lengths and/or varying degrees of esterification (e.g., mono- versus di- versus triglyceride).

Oxidative sensitivity of lipid excipients is typically associated with two chemical groups: unsaturated fatty acid moieties and PEG chains. To illustrate the structural differences in the chemical groups, major components of Maisine® and Labrasol® are shown below as examples. Maisine® consists of mono- and diesters of linoleic acid attached to glycerol. Glyceryl monolinoleate (Figure 1) in Maisine®, for example, has two unsaturated bonds and may be prone to oxidation. Regardless, the end user must be cognizant of this inherent sensitivity when incorporating this excipient in an eventual formulation. Labrasol®, on the other hand consists of polyoxylglycerides, obtained from reaction between medium-chain (C8 and C10) triglycerides and PEG-8. Figure 2 depicts a component in Labrasol®, in this case PEG-8 monocaprylate, where the PEG moiety is the likely source of oxidative reactions if left unattended.

Oxidative Processes

The oxidative degradation pathways of lipids are complex, but trends in susceptibility to oxidation are found with lipid-based excipients containing unsaturated fatty acids. Autoxidation is a free radical chain process involving a reaction between molecular oxygen and a lipid free radical from the unsaturated C=C bond, described by different stages: initiation, propagation, and termination.1

The initiation step involves the generation of a free radical in the fatty acid chain in the presence of a radical initiator. Initiators may emerge through the decomposition of hydroperoxide (R–O–OH) impurities in the lipid, which is accelerated in the presence of heat, light, or metal ions such as copper or iron.2 The radical initiator (such as R–O–O· or R–O·) reacts with a labile hydrogen, such as the allylic hydrogen of an unsaturated fatty acid chain, to form a lipid radical. The first reaction in Figure 3 shows the initiation step using the unsaturated C=C bond of oleic acid as an example (radical initiator not shown).

The propagation stage involves the reaction of the newly formed lipid radical with molecular oxygen (the second reaction shown in Figure 3), leading to the formation of hydroperoxides, which can then react with unsaturated lipids to form further lipid radicals. The reaction of the lipid radical with molecular oxygen is a rapid step compared to the subsequent hydrogen transfer reaction. As a result, the rate of propagation is dictated by the availability of allylic (i.e., labile) hydrogen in the lipid. For this reason, fatty acids with multiple double bonds (i.e., polyunsaturated fatty acids) are much more prone to oxidation than those with only one double bond (i.e.,monounsaturated fatty acids) with other things being equal since they contain more allylic hydrogens and can therefore react more readily in this propagation step. 

Hydroperoxides can alternatively decompose in a series of complicated reaction pathways to form secondary oxidation products. These can vary in nature, from diverse monomeric and oligomeric products to volatile, low molecular weight aldehydes, ketones, alcohols, and alkanes.4 In a lipid-based formulation consisting of potentially sensitive ingredients (e.g., the drug substance itself), other deleterious oxidative side reactions could of course occur.

The termination step involves the reaction of two radicals to form non-radical products. 

PEG moieties are also quite sensitive to oxidative degradation, following a similar initiation-propagation-termination autoxidation scheme as described previously for unsaturated fatty acids.5 Some lipid-based excipients contain PEG moieties, rendering them more hydrophilic and surface-active. The chemistries of these excipients vary; some contain free PEG and/or fatty acid esters of PEG (e.g., Labrasol®, Gelucires®, Labrafils®) while others include ethoxylated hydrophilic headgroups (Kolliphors®, TweensTM). These oxidative processes are accelerated in the presence of heat, light, metal ions, or water.6

Secondary PEG oxidation products include formaldehyde and formic acid7 indicating that the terminal ethylene oxide units in the PEG chain are susceptible to scission. Other degradation products include acetic acid, acetaldehyde, and other PEG-aldehydes, suggesting that scission may also occur within the PEG chain.

Protective Measures Against Oxidation

Certain precautionary steps can be taken in order to prevent, minimize, or control the occurrence of oxidation in lipid-based excipients and formulations and to preserve their integrity. Firstly, it is important to obtain material from a reputable supplier with adequate quality controls in place to fully characterize the excipient as well as certify low levels of impurities (e.g., peroxides, free fatty acids, metals, aldehydes, water). Parameters such as peroxide value and acid value are useful in the assessment of oil quality and will be discussed later in more detail.

Proper storage and handling conditions also need to be met. For lipids, this involves protecting them from exposure to oxygen, light, and moisture. After opening from their original packaging, lipids should be stored in sealed containers with minimal headspace flushed with an inert gas such as nitrogen. Excessively high temperatures should be avoided for long-term storage. When handling and processing lipids, the same protections should be considered. Working under vacuum or nitrogen blanketing is generally recommended in order to minimize exposure to oxygen. Short-term processes or procedures at high temperatures (e.g., autoclaving) may be carried out with proper protection against air and light exposure.

Antioxidants

One of the most effective methods of combating oxidative degradation in lipids is the use of antioxidant additives. Antioxidants inhibit the oxidation process and can be of natural origin (e.g., tocopherols, ascorbic acid, citric acid, rosemary extract) or of synthetic origin (e.g., butylated hydroxytoluene [BHT], butylated hydroxyanisole [BHA], propyl gallate, ascorbyl palmitate). They exhibit different degrees of lipophilicity and hydrophilicity, and the choice of which antioxidant(s) to use and at which levels involves many factors and considerations, including physical state of the dosage form (e.g., “bulk” lipid or emulsion), route of administration, and other formulation components. Examples of commonly used antioxidants classified by solubility and typical use levels are shown in Table 1.

Antioxidants can inhibit oxidation by different mechanisms.1,9,10 Chain-breaking or “true” antioxidants are phenolic compounds that facilitate hydrogen atom transfer to radicals, thereby interfering with the initiation or propagation steps of autoxidation. Through this process, chain-breaking antioxidants lose the hydrogen of the hydroxyl group and form phenoxyl radicals that are relatively stable due to electron delocalization as well as steric hindrance. Examples of chain-breaking antioxidants include BHT, BHA, propyl gallate, and α-tocopherol; their structures are shown in Figure 4.

Other classes of antioxidants include reducing agents and chelating agents. Reducing agents, such as ascorbic acid and ascorbyl palmitate, have a lower reduction potential and are thus more readily oxidized than the species that they are protecting. Chelating agents, such as citric acid, can inhibit metal-promoted oxidation by forming complexes with metal ions, hence limiting their interactions with hydroperoxides. 

Previously, it had been suggested that the more effective antioxidants for bulk oils tended to be more hydrophilic, while the more effective antioxidants for oil-in-water emulsions were more lipophilic. This phenomenon is known as the “polar paradox” and was explained by the postulated sites of oxidation, namely the air-oil interface in bulk oils versus the oil-water interface in emulsions.11 However, exceptions to the polar paradox have been found and alternative explanations offered, suggesting that more parameters than polarity alone (e.g., molecular weight/steric hinderance, the presence of surfactants) factor into an antioxidant’s overall effectiveness in lipids.12 

Antioxidants may exhibit synergy when used in combination. For example, two combined chain-breaking antioxidants or one chain-breaking antioxidant coupled with a chelating agent may offer greater protection against oxidation than either one individually in a given lipid-based system. A combination of α-tocopherol, β-carotene, and ascorbyl palmitate was found to limit oxidation in a sample of purified butterfat stored at 60°C more effectively than any of these antioxidants individually,13 illustrating a synergistic effect. 

Antioxidants are typically added to lipids or lipid-based formulations at concentrations in the 0.01–0.1% (100–1000 ppm) range. It should be noted that antioxidants, especially phenolic antioxidants, may in some cases show prooxidant behavior at high concentrations due to the regeneration of lipid radicals or hydroperoxides.1,14 Additionally, antioxidants (phenolic antioxidants especially) may be sensitive to temperature, degrading and/or vaporizing at elevated temperatures.15 The choice of which antioxidant(s) and usage level to employ may therefore require investigation in the literature and/or comparative studies involving the monitoring of certain stability-indicating parameters. Safety considerations also need to be taken into account when formulating since the type and amount of antioxidant in a formulation require justification. Most common antioxidants, including those listed in Table 1, are referenced in the FDA Inactive Ingredient Database for a variety of dosage forms. For dosage forms requiring multiple process steps, it is critical to add the antioxidant at the earliest stage of processing in order to protect the formulation components throughout all further stages.

Stability-Indicating Parameters

Physical traits, such as color and odor, ought to be monitored during stability studies with lipids; changes in these parameters would suggest the occurrence of oxidation due to the formation of chromophoric or volatile degradation products. Changes in physical state would also be indicative of degradation; this is particularly relevant for PEG-containing semisolid lipid-based excipients such as Gelucire® 44/14.

Peroxide value indicates the amount of peroxide oxygen in milliequivalents per kilogram of substance (unit: mEq O2 / kg); as such, it is a general indicator of oxidative processes in a lipid-based system. Peroxide value is most often determined by iodometric titration with sodium thiosulfate and this method is described in USP General Chapter 401 (Fats and Fixed Oils). An elevated peroxide value indicates the onset of oxidation and upper limits for this parameter are typically specified by lipid-based excipient suppliers or in corresponding compendial monographs. Since it is a measure of primary oxidation, the peroxide value of a lipid-based system could show an increasing trend followed by a plateau or by a decreasing trend (if oxidation is allowed to continue long enough) or could fluctuate over time as radicals in the system are formed and quenched. The sign of an effective antioxidant for a given lipid-based system would be sustained peroxide value readings below a specified threshold.

It is often useful to monitor acid value alongside peroxide value when assessing the performance of an antioxidant. Acid value indicates the amount of potassium hydroxide in milligrams needed to neutralize one gram of a substance (unit: mg KOH/g). It is a direct measure of free fatty acid content in lipids and is obtained via titration with an ethanolic solution of KOH. While described by separate mechanisms, hydrolytic degradation often accompanies oxidative degradation in lipids, especially in hygroscopic (e.g., PEG-containing) lipid-based excipients, and can be catalyzed or accelerated by similar factors (e.g., presence of metal ions, heat).16 Additionally, secondary oxidation products could possibly accelerate hydrolysis in lipids, so preserving oxidative stability may help to limit hydrolytic degradation. Unlike peroxide value, acid value typically only increases with time; again, upper limits are normally specifi ed by suppliers or in compendial monographs. Table 2 and Table 3 show results from a stability study in which peroxide value and acid value were monitored for diff erent lipid-based excipients both with and without the antioxidants BHA and BHT (at 500 ppm each). The samples were stored in 25°C/60% RH conditions until analysis at the indicated time points. These results demonstrate the effectiveness of the combination of BHA and BHT added to the excipients; spikes in peroxide value and acid value were observed for antioxidant-free samples over time, while similar spikes were not recorded for the samples containing the antioxidants.

Chromatographic methods using HPLC or GC can also be useful in assessing the extent of oxidation in lipids and the suitability of antioxidants for a given system. For example, secondary oxidation products (e.g., formaldehyde, acetaldehyde) or antioxidant levels can be monitored directly. Chromatographic methods are used to assay the drug substance and characterize impurities in a formulation, so their use is essential when monitoring the stability of an active pharmaceutical ingredient that is sensitive to oxidation and assessing the effectiveness of an antioxidant.

Conclusion

Lipid-based excipients offer a viable formulation approach for poorly soluble drugs and have been successfully used in a variety of approved dosage forms on the market. An appreciation of the innate sensitivity of certain lipids towards oxidation allows the formulator to anticipate possibly compromising issues regarding excipient and formulation integrity and to protect against them adequately, leading to the successful incorporation of lipid-based excipients in the formulation. The inclusion of an antioxidant is a common and effective measure taken to mitigate the possible degradative effects of oxidation. The selection of a formulation-appropriate antioxidant (or combination of antioxidants) and usage level may require investigation, and certain physical and chemical parameters can be monitored in order to assess antioxidant efficacy.

A list of “dos and don’ts” concerning the protection of lipid-based excipients and formulations is included below.

• Don’t expose lipids to light for prolonged periods during either processing or storage.
• Do use an inert gas such as nitrogen to blanket lipids during processing and to flush the headspace of partially used containers for storage.
• Don’t expose lipids to excessively high temperature or humidity for prolonged periods.
• Don’t use high shear homogenizers to mix lipids without nitrogen blanketing.
• Do use gentle stirring in order to avoid incorporating air/oxygen in lipids.
• Do consider the aqueous solubility versus oil solubility of the antioxidant when choosing an antioxidant.
• Do add the antioxidant(s) prior to processing in order to prevent initiation of oxidation during later process steps.

Author Biography

Devon Langbein is Application Laboratory Analytical Scientist at Gattefossé USA, where he is responsible for developing and carrying out analytical work for R&D projects focused on the use of lipid-based excipients in pharmaceutical dosage forms. He joined Gattefossé in 2017 after gaining over two years of experience at a contract analytical testing company, becoming well versed in a variety of analytical techniques and instrumentation. He completed his bachelor’s degree in chemistry from Cornell University in 2015.

Masumi Dave is Pharmaceutical Application Lab Manager, at Gattefossé USA where she manages a team of analytical and formulation scientists, overseeing R&D projects. The focus of the lab is to characterize, design, and develop novel approaches to the use of lipid-based excipients with the overall goal of providing technical support to pharmaceutical companies. She began her career at Gattefossé USA in 2017 as Application Lab Scientist, following completion of her Ph.D. and Master’s in Pharmaceutics at Long Island University which was preceded by a bachelor’s degree in pharmacy at Rajiv Gandhi University in 2007.

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