Antimicrobial Preservatives Part Two: Choosing a Preservative

Introduction

The second article in this series deals with the many constraints that face the pharmaceutical scientist tasked with developing preservation systems for multi-use oral, topical and parenteral medicinal products. The key role that pH plays in antimicrobial efficacy, as well as general stability considerations (both chemical and physical), will be covered.

Evaluating Performance

Compendial tests^1-3 for antimicrobial efficacy set high performance standards. Furthermore, these requirements are not harmonized and European Pharmacopeia (Ph. Eur.) standards are significantly more challenging than US (USP) or Japanese pharmacopoeias (JP) – see Table 1.

Table 1

It is a regulatory requirement to assess the antimicrobial efficacy of the drug product (in its final container) at the end of the product’s proposed shelf-life. Antimicrobial efficacy needs to be broad spectrum, encompassing bacteria (Gram-positive and Gram-negative), yeasts, fungi and molds; but not viruses. An effective preservative must reduce a microbial population significantly and prevent subsequent re-growth and these effects must be both microcidal and microstatic in nature (see Table 1).

Combining preservatives may help meet performance standards. Benzalkonium chloride (BKC) is ineffective against some strains of Pseudomonas aeruginosa, Mycobacterium and Trichophyton⁴ but combinations with EDTA, benzyl alcohol, 2-phenylethanol or 3-phenylpropanol enhances anti-Pseudomonad activity.⁵ Synergy is also obtained in combination with cetrimide, 3-cresol, chlorhexidine and organomercurials.^6,7 The amino benzoic acid esters (parabens) are more active against Gram-positive than Gram-negative bacteria, and more active against yeasts and molds than bacteria. The antimicrobial activity of these esters increases with increased alkyl chain length (butyl > propyl > ethyl > methyl) but aqueous solubility commensurately decreases and consequently the parabens are often used in combination, e.g. methyl and propyl paraben to ensure adequate solubility in the vehicle. Parabens esters also show some synergy with EDTA,⁸ 2-phenylethanol⁹ and imidurea.¹⁰

The complexity of multi-phase dermal products, formulated as creams, lotions or ointments mean that adequate antimicrobiial efficacy may not be readily attainable due to partitioning of preservative to the lipophilic (oil) phase of the system. The best that might be achieved is a microstatic effect. In practical terms such performance may be acceptable. If the bioburden is low most preservative systems can adequately kill or attenuate growth of most organisms. Current GMP (good manufacturing practice) standards, encompassing operating and sampling procedures, controls on input materials, use of clean room and automated technologies in manufacturing and packaging, when viewed holistically ensure that high standards of microbial cleanliness can be routinely achieved in finished products. Additionally, the state-of-the-art in packaging technology is now such that contamination, prior to use is unlikely. Hence the risk of contamination is probably greatest during patient use of these multidose liquid products. Microstasis may be an acceptable performance standard for non-parenteral products at this stage, if the in-use period is short (< 1-month).

Influence of Product pH

pH can affect the rate of growth of microbes, the interaction of the preservative with cell wall components and the MIC (minimum inhibitory concentration) of many preservatives.^11,12 In general, microbial growth is optimal between pH 6-8. Outside this range growth rate declines. Product pH may reflect the intrinsic pH of the active pharmaceutical ingredient (API), or the product may require pH modification to enhance solubility, stability, palatability or optimal antimicrobial effectiveness (MIC_max). Table 2 lists pH ranges for optimum activity for common preservatives.

Table 2

Excipients in a product may also influence product pH. Hence, pH adjustment to regions less favorable to microbial viability i.e. away from pH 6-8, may not be feasible or must take account of effects on other product attributes as well as the activity of the preservative system. pH effects also reflect the chemical structure of the preservative molecule. For instance, if activity is associated with the non-ionized moiety (i.e. acids, alcohols and phenols) the effect is usually optimal at acidic pH but ultimately reflects the pKa of the individual preservative agent. However, and almost inevitably, there are exceptions. For example, phenol is most active in acidic solutions, despite its high pKa (10.0). Substituted alcohols are also less reliant on pH. Bronopol (2-bromo- 2-nitro-1, 3-propanediol) is not markedly influenced by pH in the range 5.0-8.0, perhaps reflecting that its main activity is via release of formaldehyde, whose microcidal activity is not significantly influenced by pH.²²

Phenolic preservatives tend to be active over a wider pH range than alcohols or acids, but are less desirable for a number of reasons. Chlorocresol¹⁹ is most effective in acidic solutions but can retain activity at pH regions up to its pKa (9.2). Similarly, 3-cresol²⁵ is effective at pH values below its pKa (9.6). Solution pH does not have a marked effect on the anti-microbial efficacy of 4-chloroxylenol.³¹

Esterification of acids can extend the pH span of antimicrobial activity. The parabens (benzoic acid esters) are active over the range pH 4-8. Efficacy decreases at higher pH due to the formation of phenolate ion (pKa ca. 8.4) and ester hydrolysis at approximately pH 9. Efficacy increases with the longer alkyl chain, but conversely aqueous solubility decreases as hydrophobicity increases.¹³

In contrast to acid preservatives, the quaternary ammonium compounds (QACs) such as benzalkonium chloride (BKC) and benzethonium chloride demonstrate anti-microbial efficacy over a wide pH range (pH 4-10), activity being associated with the ionized (cationic) moiety and optimal at high pH values.^4,11,14 Efficacy is also linked with alkyl chain length (C18 > C16 > C14 > C12). Cetrimide¹⁷ has a slightly narrower effective pH range (7-9), probably caused by the presence of a methyl rather than a benzyl moiety (less effective at stabilizing the charge). High pH causes the microbial cell wall to be negatively charged, thereby favoring the binding of cationic species.

There are no reported pH constraints on the permeation enhancing capability of EDTA, probably a consequence of its multiple pKa values. However, its limited intrinsic anti-microbial efficacy means that it is rarely used on its own, but in combination with other preservatives. ^32,33

pH-related effects can sometimes be more complex than those summarized in Table 1. The antifungal activity of benzoic acid is less susceptible to pH than are its antibacterial effects.¹⁵ The substituted benzoic acid derivative thiomersal, which has a pKa of 3.1 is bacteriostatic and fungistatic at neutral and even mildly alkaline pH’s. However, the antimicrobial activity of the organic mercury component also needs to be taken into account.30 A similar effect is evident with propionic acid²³ and sorbic acids,²⁴ which have appreciable antifungal but little or no antibacterial activity at pH 6.0, but this effect may be concentration dependent.

Biguanide anti-microbials are generally active over the pH range 3-9. However, chlorhexidine is effective over a narrower pH range (5-7) while chlorhexidine base may precipitate from aqueous solutions at pH values greater than 8.¹⁸ Imidurea is effective over the pH range (3-9), although optimum efficacy is seen at acidic pH.²¹ Organomercurial preservatives such as phenylmercuric salts, have broad spectrum bactericidal and fungicidal activities, being more potent with increasing pH. Efficacy against Pseudomonad’s has also been demonstrated at pH 6 or below.^27-29 These preservatives have been utilized in several ophthalmic products having acidic pH values. Activity is enhanced at acidic pH in the presence of sodium metabisulphite, which can enhance activity at low pH, but has the opposite effect at alkaline pH.^34,35 In topical products phenylmercurate salts have been reported as being active at pH 5-8.³⁶

In the ideal world the pH of optimal microbial efficacy is aligned with pH of optimal product stability, solubility or palatability, etc. However, this is rarely the case; pragmatic decisions and compromise are often required such as the selection of a formulation pH which is suboptimal for antimicrobial efficacy but is better aligned with product performance. In those cases, higher concentrations of preservatives may be required (Table 3). Thus, sorbic acid has a pKa of 4.76 and the recommended level of preservative is 0.2% at pH 4.5.²⁴ At this pH, 64.5% of the preservative is unionized (the effective form), the effective concentration of unionized sorbic acid being 0.13%. Table 3 shows the effect of formulation pH on the required concentration of preservative. If the optimal product pH was 6.0, from stability, solubility, palatability perspectives etc the concentration of the preservative would need to be increased 11.9-fold for adequate preservation compared with formulating the product at an optimal pH for preservation , i.e. pH 4.5.

Table 3

Factors that Compromise Preservative Efficacy

Preservatives possess reactive functional groups and may have pH-solubility profiles to be considered when formulating the drug product. The optimal pH for microbial efficacy may not be the same as that required for drug solubility and/or stability, requiring compromise and pragmatic choices. Preservative efficacy can also be affected by interactions with active ingredients, excipients, container/closures or other physicochemical behaviors. Reduction in microbial efficacy can occur during manufacture, throughout the product’s shelf life or during the in-use period. These effects can be ascribed to:

• interactions with other components within the product (drug, excipients, pack or delivery device).
• chemical instability of the preservative.
• physical losses or changes in preservative levels in solution

Possibilities for chemical degradation are manifold, but the risk can be mitigated at the outset by a thorough risk assessment, an understanding of all the product components and by appropriate pre formulation studies to determine interaction propensity. It is important that such knowledge and awareness is generated at the product design stage utilizing QbD (Quality by Design) concepts and approaches. Pharmaceutical products generally have much longer shelf life requirements, i.e. 24-36 months at ambient temperatures than food or beverage products; antimicrobial efficacy must be retained over these periods and during product use. The relatively insensitive nature of preservative efficacy tests^1-3 could mean that modest but inexorable deterioration of antimicrobial effectiveness during shelf-life storage may take time to be manifested or significant. A consequent reformulation and evaluation program can delay product development timelines.

Chemical Stability of Preservatives

Preservative levels during product shelf life need to remain within limits that ensure acceptable antimicrobial efficacy. It should not come as a surprise that most acidic preservatives e.g. benzoic, sorbic and propionic acids are incompatible with strong bases.^15,23,24 Strong oxidizing agents degrade sorbic acid,²⁴ 2-phenylethanol,³⁷ hexetidine,³⁸ EDTA,³² thimerosal,³⁰ propyl gallate³⁹ and butylated hydroxyanisole (BHA).⁴⁰ This latter material is particularly unstable in the presence of peroxides and permanganates and prolonged interaction may even result in spontaneous combustion.⁴⁰ Although, the presence of such reactive materials in a dosage form might be unusual (although benzoyl peroxide is formulated in topical lotions to treat acne), excipients such as povidone, crospovidone, polyethylene glycol and polysorbates may contain residual peroxides.⁴¹ These may be present at low concentrations, but a high excipient-to-preservative ratio could cause significant interaction and degradation.

The antimicrobial efficacy of several preservatives is also compromised by surface-active agents that are common formulation additives, particularly for poorly wetting active ingredients. Benzalkonium chloride,⁴ benzethonium chloride¹⁴ and cetrimide,¹⁷ are cationic in nature and incompatible with anionic surfactants or other excipients such as the negatively charged sodium carboxymethyl cellulose.

Benzyl alcohol,¹⁶ 2-phenoxyethanol,⁴² 4-chloroxylenol²⁰ and 3-cresol²⁵ should not be formulated with non-ionic surfactants. Chlorobutanol⁴³ and 2-phenylethanol³⁷ are also adversely affected by the presence of non-ionic surfactants such as. Polysorbate 80.

Such interactions may not involve conventional chemical transformations, but include more subtle binding phenomena e.g. hydrogen bonding, aggregation and complex formation. Although the overall level of preservative in the product may not change (as measured by chemical analysis) the preservatives may be bound to such excipients and not available in the “free” form; efficacy may be reduced. Determination of preservative efficacy is therefore mandated.^1-3 It is also important that analytical techniques to monitor preservative content in stability testing programs are designed to determine levels in solution in liquid formulations or in the aqueous phase in complex biphasic systems. Preservative needs to be in aqueous solution to evince an antimicrobial effect.

Some preservatives are incompatible with other preservatives. EDTA interacts with thimerosal, propyl gallate and phenylmercuric salts³². Chlorhexidine can interact with benzoic acid¹⁸ whereas cetrimide is incompatible with phenyl mercuric nitrate¹⁷. Such interactions can limit the choice of preservative combinations.

Most available preservatives seem ostensibly to possess stable chemical structures. This may explain why reports of intrinsic chemical instability (i.e. that do not involve interaction with other product components) are not widespread. Paraben^13,44,45 preservatives are susceptible to base-catalyzed ester hydrolysis at high pH, degrading by classic pseudo-first order kinetics, shorter chain analog such as methylparaben being least stable.¹³ Stability in solution is not markedly affected by pH up to about pH 6.5, but degradation rates increase at pH 7.5 and above.⁴⁶ As parabens show antimicrobial efficacy over the pH range 4-8 caution may be advisable where product pH is likely to be higher than neutral. Unfortunately, there are few if any alternative preservatives that are efficacious at neutral to slightly alkaline pH values. In the light of the predictable and well characterized degradation kinetics of these agents scientifically relevant accelerated (high temperature) stability studies at the formulation development stage may predict long term stability in the final product. The formulation scientist may then have the option to include overages to compensate for any losses. The guiding principle with respect to inclusion levels is that these be minimal but commensurate with adequate preservative efficacy at the end of shelf-life.⁴⁷ If the formulation pH is sub-optimal from an antimicrobial effectiveness perspective higher concentrations than typically accepted may be appropriate (Table 3).

Higher product pH values (>pH 8) are often inimical to microbial viability but effects on preservative stability also need consideration. Despite its many advantages as a preservative sorbic acid is relatively unstable in semi-solid and liquid preparations. The principal degradation pathway is via auto-oxidation resulting in acetaldehyde and β-carboxyacreloin end-products as well as numerous other volatile aldehydes, e.g. malonaldehyde, acrolein, crotonaldehyde and related furans (2-methylfuran, 2-acetyl-5-methylfuran, 2,5-dimethylfuran).⁴⁸ Sorbic acid may be stabilized by phenolic anti-oxidants, for example 0.02% w/w propyl gallate39 but presence of an anti-oxidant in the formulation needs to be justified.

Macromolecules can be adversely affected by preservatives. Benzyl alcohol causes aggregation of recombinant human interferon (rhIFN), recombinant human granulocyte stimulating factor rhGCSF) and, human interleukin-1 receptor antagonist (rhIL1ra).⁴⁹ Several biopharmaceutical products mandate that the diluent for constitution must not contain preservative(s) because of potential adverse interactions with the anti aggregant Polysorbate 80, present in most recent biopharmaceuticals, particularly monoclonal antibodycontaining products.

Preservatives for multidose insulin preparations must be chosen carefully. Insulin zinc suspensions cannot contain phenol as it destroys the crystallinity of the insulin; mixtures of parabens are used instead. In contrast, neutral protamine insulin requires the presence of phenol or meta-phenol to form and preserve the crystal form that provides the long-acting effect.⁵⁰

Physical Stability of Preservatives

Preservative content in medicinal products can be depleted during manufacture, storage or use. The parabens.^13,45,46 benzoic acid,¹⁵ benzyl alcohol,¹⁶ 2-phenoxyethanol,⁴² 3-cresol,²⁵ chlorocresol¹⁹ and chlorbutanol⁴³ are all volatile to greater or lesser extents. This renders them susceptible to processing losses by sublimation or evaporation during product manufacture or throughout product life. 3-Cresol²⁵ and phenol²⁶ are not suitable as preservatives for preparations that need to be lyophilized due to their volatility. In addition, if any of the container/closure components are permeable to gases, e.g. plastic bottles or elastomeric closures, then this can result in the depletion of these volatile preservatives during long term product storage.

Polyvalent ions may cause precipitation of certain preservatives from solution, due to H-bonding,for example:

• sorbic acid²⁴ and chlorhexidine¹⁸ can be “salted out” by Ca²⁺ ions.
• chlorobutanol⁴⁴ and chlorhexidine¹⁸ interact with Mg²⁺ ions.
• bronopol²² and phenylmercuric nitrate²⁹ can be precipitated by Al³⁺ ions.
• Fe³⁺ ions can salt out BHA⁴⁰ and butylated hydroxytoluene (BHT).⁵¹
• EDTA³² is precipitated by most polyvalent cations.

The overall level of the preservative in the product may remain unchanged but free concentration in solution is diminished due to precipitation or adsorption, reducing antimicrobial efficacy. Analytical techniques to monitor preservative content need to reflect such possibilities viz determine the level of preservative in solution, in the aqueous phase in a complex liquid such as a cream or lotion.

Adsorption by excipients, especially those with large surface areas or onto container/closure systems can also bind and remove preservative(s) from solution. Table 4 lists examples.

Table 4. Preservatives Susceptible to Adsorption

Physical and chemical interactions can make the antimicrobial preservation of antacid-containing formulations challenging. The pH of such products is typically neutral to slightly alkaline, i.e. pH 7-8, where intrinsic preservative activity can be low. Additionally, the presence of polyvalent cations (e.g. Al ³⁺, Ca ²⁺, Mg ²⁺) in antacid products can precipitate the preservative. Adsorpton on to the insoluble antacid is also possible. Such effects are additive and contribute to loss of preservative efficacy making antacid suspensions notoriously difficult to preserve.. This is reflected in lower acceptance criteria for Antacids (category 4 products) in USP <51> 1 viz ‘No increase (in bacteria, yeasts and molds) from the initial calculated count at 14 and 28 days’ (footnote to Table 1).

Multiphase semi-solid products such as creams, ointments, foams and lotions, as well as some parenteral and nasal/ophthalmic products, can have aqueous and oily phases stabilized by surface active agents. Viscosity enhancers can also be included as suspending agents. Such agents can remove preservatives from the solution phase as described earlier.

Preservatives can partition between oil and aqueous phases and at the interface containing the surface-active agent (depending on partition coefficient) reducing aqueous concentration and compromising the antimicrobial effect. Such behaviors can reduce efficacy of the parabens preservatives, particularly the longer chain analogues such as butyl paraben.⁴⁶ Chlorhexidine activity can be reduced because of micelle formation.¹⁸ Some preservatives can form ion-pairs (or in situ salt formation) with corresponding drug substances , e.g. timolol with sorbic acid. This has been proposed as a mechanism for enhancing the ocular bioavailability of timolol but the consequences for impact on the efficacy of the preservative system has not been reported.⁶⁸

Some preservatives can form cocrystals with drug substances. Such occurrences can be reported positively in the literature but unanticipated cocrystal formation is typically deleterious, removing free preservative from the aqueous phase in liquid oral or cream/ lotion products. Many of the preferred approaches used to form cocrystals are akin to those employed during preparation of semisolid and suspension formulations, for instance high energy mixing and homogenization. Many preservatives, including alcohols, phenols and carboxylic acids have the appropriate synthons to act as effective coformers. Examples include Nalidixic acid forming co crystals with phenolic coformers⁶⁹ and fluoxetine forming cocrystals with benzoic acid.⁷⁰ Even preservatives that do not possess obvious synthons may act as coformers. Methylparaben has been reported as forming cocrystals with the anti-malarial quinidine⁷¹ and with β-lactam antibiotics.⁷²

The chlorinated preservatives, chlorobutanol,⁴³ chloroxylenol²⁰ and chlorhexidine¹⁸ can partition onto polymeric suspending agents by competitive displacement of water of solvation. Similarly, the antimicrobial efficacy of 2-phenoxyethano⁴² is reduced in the presence of cellulosic suspending agents such as methylcellulose, sodium carboxymethyl cellulose and hydroxypropyl methylcellulose.⁶¹

The possibilities for reduced anti-microbial efficacy in multi-phase systems, has engendered efforts to devise in silico predictive approaches to determine the impact of formulation parameters on preservative activity. The influence of partition coefficients, binding constants (surfactants and polymers), and oil-in-water ratios have all been investigated but with limited success.¹² To date the pragmatic approach, involving optimizing the preservation system and inclusion levels by conventional assessment techniques remains the desired approach.

Conclusions

Preservatives, either singly or in combination remain necessary to prevent microbial contamination of multi-use aqueous liquid or semisolid medicinal products by opportunistic pathogens. Non-inclusion can result in serious patient health consequences. There are a limited number of approved preservatives that can be included in such multiuse oral or topical medicinal products and the number is constrained even further in parenteral products. The optimal conditions for preservative efficacy (pH, physical and chemical stability) may not be the same as for the drug product.Compromisesmay be necessary to ensure an optimal product shelf-life.

Acknowledgements

Dr. Paul Newby and Dr. Don Singer, GSK for their review and comments on this manuscript.

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