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Case studies compare efficacy testing of preservatives for topical formulations with probiotic actives.
In recent years, many topical probiotic personal care products have been launched into the market (1). In an August 2016 review for Dermatology Times (2), dermatologist Patricia Farris concluded, “The studies reviewed suggest that topical prebiotics, probiotics, and bacterial cell lysates do provide demonstrable skin benefits …At this time, it appears that more studies are warranted to determine if these products are really worth the hype.” These scientific reviews are quick to point out that well-crafted, vehicle-controlled clinical trial results are not generally available for topical semisolids containing live microorganisms. One reason that topical probiotic therapies have not advanced beyond the personal care “post-marketing surveillance regulatory environment” into the controlled clinical trial “new drug approval regulatory environment” is the difficulty in reconciling FDA microbiological requirements for a product containing live microorganisms. More specifically, how can a topical suspension containing more than 50,000 colony forming units (CFU) of probiotic active pharmaceutical ingredient (API) pass United States Pharmacopeia (USP) microbial enumeration testing (USP <61>) (3), tests for specified organisms (USP <62>) (4), and antimicrobial preservative effectiveness (USP <51>) (5)?
Two case studies are being presented here to explore the strategy of adequately preserving the formulation, but using a preservative of sufficiently narrow spectrum to maintain viability/potency of the probiotic active. The first case study uses a probiotic strain of Propionibacterium acnes (P. acnes). P. acnes is a lipophilic, gram-positive anaerobic bacillus that resides in the pilosebaceous unit of human skin. Hundreds of different strains of P. acnes exist. The lipases, proteases, and hyaluronidases secreted by certain strains of P. acnes injure the lining of the pilosebaceous unit and activate production of proinflammatory cytokines that in turn lead to acne vulgaris. Other strains of P. acnes produce no inflammatory response and thus do not induce the symptoms of acne vulgaris. One of these non-inflammatory strains of P. acnes has been chosen to be used as an “active ingredient” in a topical formulation product. In the second case study, the microorganism is a bacteriophage (phage), which is a virus that infects and replicates within a bacterium, ultimately causing bacterial death. The phage used in this study was isolated from the follicular casts obtained from volunteers with facial comedones. Bacteriophages were identified and isolated from the comedones and were then propagated using an amplification process and plated against different P. acnes strains to determine breadth of efficacy to assure the selected phage was suitable to infect and eradicate pathogenic (inflammatory) P. acnes strains.
Formulating a living microorganism is fundamentally different from formulating a small-molecule active topical product. Because the P. acnes probiotic or phage products will be dosed as suspensions, active solubility, solvent compatibility, and penetration across the stratum corneum do not factor in to the development of a topical probiotic. In contrast, the aqueous probiotic formulation does require that pH and osmolality be adjusted to values that assure a favourable environment for the microorganisms to remain viable. In these two case studies, eight different formulations containing preservatives were tested for live microorganism viability over six weeks after compounding.
The low immunogenic strain of P. acnes (probiotic bacterial active) and phage that attacks pathogenic P. acnes (microbiome editor) were provided by Phi Therapeutics. Two gelled aqueous products were made with the intent of formulating cosmetically elegant products: hydroxyethyl cellulose (HEC) at 1.5% w/w and polyacrylic acid polymer or carbomer (Carbopol 980, Lubrizol) at 0.75% w/w, both titrated with propylene glycol until isosmotic (~285 mOsm/k). Both gels were prepared with the following preservative systems: preservative free, methylparaben (0.1%) and propylparaben (0.02%), phenoxyethanol (1.0%), and potassium sorbate (0.2%). In addition, two solution products were made: an 80:20 water:propylene glycol blend (w/w) and an 80:20 water:ethanol blend (w/w). Both solution blends were considered self-preserving. The pH of all the products were taken, but no pH modifiers were added. Active viability and stability testing were conducted every two weeks after addition of active microorganism by diluting the formulated products to a known concentration of active microorganisms and plating them out. P. acnes concentration was calculated using diluted growth promotion plating and back calculating; the number of phage was calculated by plating the dilutions onto bacterial lawns and looking for the presence of kill zones.
When added individually to the probiotic formulations, the parabens, phenoxyethanol, and potassium sorbate completely deactivated the product gelled with HEC or Carbopol by more than 99.95% within the first four weeks of compounding (see Figure 1).
For the probiotic P. acnes strain, as shown in Figure 2, only the 20% propylene glycol solution was reasonably tolerated. The 20% propylene glycol solution initially lost 90% activity at two weeks, but remained at a consistent level of activity from two to six weeks. The 20% ethanol and the Carbopol gelled water (non-preserved) products lost their potency by more than 99.95% within the first four weeks of compounding with the HEC gelled water (non-preserved) product performing no better than 20% propylene glycol product.
The parabens, phenoxyethanol, and potassium sorbate in isotonic HEC formulations did not deactivate the phage six weeks after compounding. Formulating the phage with ethanol or propylene glycol up to 20% solvent did not have any effect on phage activity (see Figure 3).
As shown in Figure 4, however, the use of a negatively charged polymeric gelling agent (polyacrylic acid polymer) did deactivate the phage by about 95% two weeks after compounding and caused more than 99.95% loss in phage titer by four weeks.
The basis for microbial enumeration testing (USP <61>), tests for specified organisms (USP <62>) and antimicrobial preservative effectiveness (USP <51>) of topically applied non-sterile pharmaceutical products is the statement in US 21 Code of Federal Regulations (CFR) 211.113(a) that “Appropriate written procedures, designed to prevent objectionable microorganisms in drug products not required to be sterile, shall be established and followed” (6). It should be noted that a topical probiotic product should not have greater difficulty in passing USP <61> or USP <62> than topical products containing non-living actives. For topical probiotics, the living microorganism active is not “objectionable” and thus is not required by USP <61> to be below 1000 CFU. Likewise, the absence of pathogens such as Staphylococcus aureus or Pseudomonas aerogenesis as determined by specified organisms of USP <62> is necessary for the safety of any topically applied pharmaceutical. It is passing antimicrobial preservative effectiveness testing (USP <51>) that is the biggest hurdle to the development of a topical product containing a living microorganism as the active.
As seen for the probiotic P. acnes strain used in the first case study, only the addition of 20% propylene glycol was reasonably tolerated by the living microorganism active. The parabens, phenoxyethanol, potassium sorbate, and ethanol, when added individually to the probiotic formulation, deactivated the product by more than 99.95% within four weeks of compounding. The 20% propylene glycol liquid initially lost 90% activity at two weeks, but remained at a very consistent level of activity from two weeks through to six weeks. While a certain segment of the probiotic active appears susceptible to the cell membrane function interference exerted by a glycol, sufficient quantities of viable probiotic remain to manufacture an efficacious topical product. Blending propylene glycol with other glycols to optimize antimicrobial activity has been an area of active research within the field of dentistry (7). The results of the first case study, combined with the literature concerning the bactericidal activity of propylene glycol, indicate that addition of glycols to a fluid suspension of probiotic P. acnes is a reasonable development strategy for complying with 21 CFR 211.113(a).
As expected, the phage was more resilient to the addition of preservatives to the formulation. Addition of ethanol to the formulation did not appear to have any effect on the phage. The parabens, phenoxyethanol, potassium sorbate, and propylene glycol did not appear to deactivate the phage six weeks after compounding. The use of a negatively charged polymeric gelling agent did deactivate the phage by approximately 95% two weeks after compounding and caused more than 99.95% loss in phage titer by four weeks. Obtaining a preserved bacteriophage probiotic topical gel is possible provided that an uncharged gelling agent such as HEC is used as the thickener.
The phage has the capacity to tolerate a wide range of typical pharmaceutical preservatives, and it appears possible to formulate a cosmetically elegant final product if a nonionic gelling agent (such as HEC) is used. However, formulating a final product containing the probiotic P. acnes will be challenging, and certain amounts of care will need to be taken in working with the bacteria and choosing the best method of preserving the final product. Nonetheless, bacterial levels in the 20% propylene glycol aqueous solution have been relatively stable from weeks 2–6, indicating that this is a reasonable development strategy. From the results obtained from these two case studies, it can be concluded that it is possible to formulate a preserved topical product containing living microorganisms.
1. M-CJ Huang and J. Tang, “Probiotics in Personal Care Products,” Microbiol. Discov. http://dx.org/10.7243/2052-6180-3-5 (2015).
2. P.K. Farris, “Are Skincare Products with Probiotics Worth the Hype?” Dermatology Times, Aug. 8, 2016.
3. USP, USP Chapter <61> “Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests” USP 41–NF 36 (US Pharmacopeial Convention, Rockville, MD, 2018), p. 5965.
4. USP, USP Chapter <62> “Microbiological Examination of Nonsterile Products: Tests for Specified Organisms” USP 41–NF 36 (US Pharmacopeial Convention, Rockville, MD, 2018), p. 5971.
5. USP, USP Chapter <51>, “Antimicrobial Effectiveness Testing” USP 41–NF 36 (US Pharmacopeial Convention, Rockville, MD, 2018), p. 5959.
6. CFR Title 21, 211.113(a)
7. T.M. Nalawade, K. Bhat, and S.H.P. Sogi, J. Int. Soc. Prev. Community Dent. 5 (2) 114-119 (2015).
Vol. 42, No. 3
When referring to this article, please cite it as J. Carbol, et al., "Formulating Topical Products Containing Live Microorganisms as the Active Ingredient," Pharmaceutical Technology 42 (3) 2018.
Jason Carbol is manager of Chemistry, Manufacturing, and Controls at Dow Development Laboratories (DDL), a division of Symbio, firstname.lastname@example.org; Pia Isabel Tan was a 2017 Dow Development Institute Intern who is currently a biochemistry major at UCLA; Yug Varma, PhD, is CEO at Phi Therapeutics; and David W. Osborne, PhD, is executive fellow at Dow Development Institute and senior vice-president of Product Development at Arcutis.