July 2010 Abridged version, MAIL PO Box Seattle, WA USA. STREET 2201 Westlake Avenue, Suite 200 Seattle, WA USA

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1 A HealthTech Report Stability data on chlorhexidine formulations: PATH summary July 2010 Abridged version, 2012 MAIL PO Box Seattle, WA USA STREET 2201 Westlake Avenue, Suite 200 Seattle, WA USA Tel: Fax:

2 Acknowledgment This report is made possible by the generous support of the American people through the United States Agency for International Development (USAID) under the HealthTech program, cooperative agreement #GPH-A The contents are the responsibility of PATH and do not necessarily reflect the views of USAID or the United States Government. ii

3 TABLE OF CONTENTS Background. 1 Formulation development... 2 Stability results... 3 Conclusions and recommendations 5 References.. 6 iii

4 BACKGROUND Working with a consultant, PATH developed four chlorhexidine (CHX) formulations for use during umbilical cord care. The composition and specification of each of the formulations is described in Tables 1A and 1B. PATH evaluated the formulations for stability and then used a Request for Proposals process to select Frontage Laboratories LLC (Frontage) to conduct further testing. PATH transferred the four formulations to Frontage, where they were compounded based on the preparation process provided by PATH. This report is a summary of the raw data provided in a report from Frontage. During the compounding process, Frontage observed local precipitation in one of the formulations. This formulation, called PATH 003, contained zinc and copper gluconate. Subsequent literature and data review did not reveal any information about usage or stability offered by these two additives in CHX formulations. Therefore, we decided to remove PATH 003 from stability testing. The three remaining formulations PATH 001, 002, and 004 were placed in stability tests. Table 1A. Composition of PATH CHX formulations. Starting Materials PATH 001 PATH 002 PATH 003 PATH % chlorhexidine 7.103% weightby-weight (w/w) 7.103% (w/w) 7.103% (w/w) 7.103% (w/w) gluconate, British (eq. 4% CHX) (eq. 4% CHX) (eq. 4% CHX) Pharmacopeia (BP) (eq. 4% CHX) 50% Benzalkonium chloride, National 0% 0.1% 0.1% 0.1% Formulary (NF) Guar gum NF 0% 0% 0% 1% Zinc gluconate 0% 0% 0.05% 0% Copper gluconate 0% 0% 0.05% 0% Purified water, United States Pharmacopeia (USP) Sodium hydroxide solution, NF for ph adjustment Container closure type Physical state/appearance quantity sufficient (q.s.) to 100 gm q.s. to 100 gm q.s. to 100 gm q.s. to 100 gm ph to 6±0.1 4 ml high-density polyethylene bottle and polypropylene screw closure Clear, colorless solution Gel 1

5 Table 1B. Specifications of PATH CHX formulations. Formulation (Liquid) Attributes Specification Formulation ph 5.5 to 7.0 Aspect Clear, colorless solution CHX 3.6% to 4.4% (or 90% to 110% in CHX gluconate potency assay) Formulation (Gel) Attributes Specification ph 5.5 to 7.0 Viscosity 3,000 cp to 5,000 cp Aspect Clear gel CHX 3.6% to 4.4% (or 90% to 110% in CHX gluconate potency assay) Note: These formulations were prepared at a non-current Good Manufacturing Practices-certified facility only for research and development purposes and not intended to be used for clinical studies. FORMULATION DEVELOPMENT CHX is 1,6-di (4-chlorophenyl-diguanido) hexane, a cationic bis, biguanide of the following formula (Figure 1): The molecular formula of this compound is C 22 H 30 C l2 N 10.2C 6 H 12 O 7 with a formula weight of Figure 1. Chemical structure of CHX gluconate CHX digluconate solution has been used as a topical antiseptic and disinfectant effective against a wide range of bacteria, some fungi, and some viruses. Aqueous solutions of CHX are most stable within the ph range of 5 to 7. At high ph >7, CHX base is precipitated, and in more acid conditions, there is gradual deterioration of activity because the compound is less stable. The hydrolysis product of CHX is p-chloroaniline which is insignificant at room temperature but increases at high temperatures and especially at alkaline ph. 1 The incompatibility of chlorhexidine gluconate with other anionic materials like borates, phosphates, acetates, nitrates, and chlorides restricts the use of buffer salts for maintaining the ph of the solution. Diluted solutions of CHX (<1.0% weight by volume) may be sterilized by autoclaving (115 C for 30 minutes), however autoclaving (sterilization) of solutions greater than 1.0% can result in the formation of insoluble residues and is therefore unsuitable. All diluted solutions to be stored should be either heat treated (sterilized or pasteurized) or chemically preserved to eliminate the possibility of microbial contamination. 1 Regarding storage of CHX solutions, choice of container is important, with best results being achieved with neutral glass or polypropylene. If soda glass is used with CHX solutions, the resultant ph may be above that which is considered optimal for stability (ph 5 to ph 7) because of leaching of alkaline materials from the bottle. Three formulations based on the above specifications for CHX were proposed by PATH and transferred to Frontage. PATH 001 is a liquid formulation containing 7.1% CHX gluconate (eq. 4% CHX) without any stabilizers or preservatives. This is the same formulation as the one used 2

6 in the randomized controlled trials conducted by Johns Hopkins University and other partners in Nepal and Bangladesh. PATH 002 is a liquid formulation containing 7.1% CHX gluconate (eq. 4% CHX) with benzalkonium chloride (BAK) as a preservative. CHX is a cationic molecule so it is generally compatible with other cationic materials such as quarternary ammonium salts (cetrimide and BAK) although compatibility will depend on the nature and relative concentration of the second cationic species. 1 BAK is a mixture of alkylbenzyldimethylammonium chlorides which has been reported to be safe as a preservative at concentrations up to 0.1%. 2 BAK is added in the assumption that this chemical might add stability to the CHX solution in order to achieve the minimum of a 2-year shelf life required by the drug authority in Bangladesh and other countries. PATH 004 is a gel formulation containing 7.1% CHX gluconate (eq. 4% CHX) with BAK as a preservative and guar gum as a thickening (viscosity enhancer) agent. Guar gum 3 is hydrocolloid derived from a leguminous plant. The structure of guar gum (Figure 2) consists of a linear backbone of linked units of mannose with galactose. The ratio of mannose to galactose is 2:1. Guar gum is an economical thickener and stabilizer. The very high viscosity attained at low concentrations makes guar gum an excellent thickener in the food industry. The other advantage of using guar gum is that it is non-ionic so it is stable over a wide ph range. Figure 2. Chemical structure of guar gum. STABILITY RESULTS PATH 001, PATH 002, and PATH 004 CHX gluconate topical formulations were compounded at Frontage s lab and put on 6-month stability at three conditions of 5 C, 25 C/60% relative humidity (RH), and 40 C/75% RH. The samples were tested for appearance, ph, CHX potency, and p-chloroaniline at each time point as per protocol created by PATH. The assay method for CHX gluconate is based on USP 29-NF24 and for p-chloraniline is based on USP31-NF26. All three formulations maintained the CHX potency, p-chloroaniline (impurity level), and the ph within the acceptable range during the entire stability study. PATH 001 and PATH 002 showed similar potency profiles (Table 2) as well as identical trends in ph (Table 3), suggesting that BAK was not imparting additional stability to the CHX formulation in PATH 002. PATH 004 showed a different ph and potency profile. PATH 004 showed a decreasing trend in potency (Table 2) during the entire stability study with a sudden drop occurring at the 6 month time point. The inconsistency in potency number(s) indicates the difficulty in assaying viscous samples because the gel formulation is viscous due to the high molecular weight of guar gum (200,000 to 250,000) and causes difficulty in transferring the contents for analytical assay. This may have possibly resulted in low potency numbers. The other possibility for a higher drop in 3

7 potency in PATH 004 relative to PATH 002 is that the guar gum contains acidic impurities 4 as a carryover from the extraction/purification process which is potentially causing the degradation of CHX. This can be seen from the impurity (p-chloroaniline) level (Table 4) of PATH 004 that is higher than in the PATH 002 formulation. However, it should be noted that this impurity level is still well within the USP specifications for all the formulations. Finally, large-scale production of gel formulation containing guar gum requires specialized equipment (high-pressure homogenizer) which could result in additional equipment and labor costs in the manufacturing process. High-pressure homogenization is essential to the quality and stabilization of lotion/gel/viscous formulations since it is a very effective way to create the exact required product texture while at the same time a very stable product compared to the traditional devices such as agitators, stirrers, rotor-stator devices, or colloid mills. The result is a homogeneous effective product with superior stability and shelf life. Table 2: Six-month CHX potency assay on CHX formulations (specifications: 90.0% to 110.0%). Product/Condition Initial 1M 3M 6M PATH 001, 5C 95.2 * PATH 001, 25/ * PATH 001, 40/ PATH 002, 5C 95.4 * 91.9, PATH 002, 25/ * PATH 002, 40/ PATH 004, 5C 95.3 * , 91.5 PATH 004, 25/ * PATH 004, 40/ , 89.5 * Not performed as per project agreement. Table 3. Six-month ph data on CHX formulations (specifications: 5.0 to 7.0). Product/Condition Initial 1M 3M 6M PATH 001, 5C 6.0 * PATH 001, 25/ * PATH 001, 40/ PATH 002, 5C 6.0 * PATH 002, 25/ * PATH 002, 40/ PATH 004, 5C 5.6 * PATH 004, 25/ * PATH 004, 40/ * Not performed as per project agreement. 4

8 Table 4. Evaluation of six-month impurity level (p-chloroaniline) in CHX formulations (specifications: total impurity no more than 3%). Product/Condition Initial 1M 3M 6M PATH 001, 5C % * % % PATH 001, 25/ % * % % PATH 001, 40/ % % % % PATH 002, 5C % * % % PATH 002, 25/ % * % % PATH 002, 40/ % % % % PATH 004,5C % * % % PATH 004, 25/ % * % % PATH 004, 40/ % % % % * Not performed as per project agreement. The physical appearance data for the three formulations however was quite different. PATH 001 maintained the clear, colorless aspect throughout the 6-month stability study; PATH 002 and PATH 004 turned pale yellow to light brown at higher temperatures. This change in color is likely due to the interaction of CHX with chloride from BAK resulting in the light-brown coloration of the two formulations. This interaction has been well documented in literature 5,6 where combining CHX and BAK catalyze browning reactions in formulations that are in vitro. The discoloration in appearance is acceptable as long as it does not affect the potency and efficacy of the CHX formulations. The stability test demonstrated that CHX assay was in the acceptable range despite the discoloration. We can conclude therefore that the discoloration did not adversely affect CHX s potency. In addition, it was confirmed with USP and other literature data that yellow to light-brown appearance is a common characteristic of CHX gluconate. Current product specifications should be changed to accommodate the appearance characteristics of CHX gluconate if used in combination with BAK. Since the stability data did not indicate any advantage of adding BAK to PATH 002 and PATH 004, we explored performing USP 51 which is a preservative efficacy test with PATH 002 to determine whether BAK was offering added value as a preservative. Discussions with the Société Générale de Surveillance, a bioanalysis testing lab, revealed that since the concentration of CHX in the formulations is very high (4%), CHX will probably kill any bacteria with or without BAK, thereby making the role of BAK indistinguishable. CONCLUSIONS AND RECOMMENDATIONS All three CHX formulations passed the stability test based on ph, CHX potency, and impurity level (p-chloroaniline). Although PATH 002 and 004 showed discoloration, this did not negatively affect the potency of the formulations. We believe it is important to address ways to reduce the discoloration observed in CHX formulations containing BAK since the change in appearance (color) may have an impact on consumer confidence and hence product acceptability. One option would be to reduce the 5

9 amount of BAK (0.02% to 0.05%) and evaluate the stability. The other possibility would be to remove BAK from future formulations since the stability test data indicated that addition of BAK did not offer any improvement over CHX formulations that do not contain BAK. The latter action also could reduce the cost of the product by reducing the number of inactive ingredients and the compounding step(s) in the manufacturing process. For this reason, we recommend that BAK should be removed from all future product formulations of 4% CHX. Finally, although all the three formulations were within the acceptable range in potency, ph, impurity, and appearance, PATH 004 (gel) presented a decreasing tendency in potency. This indicated a difficulty in assaying viscous samples. Therefore, if a gel formulation is selected, it will be necessary to identify/create a reliable assay method to evaluate the gel formulation for quality assurance/quality control. Also, in the gel production process, the manufacturer should be equipped with specialized production equipment to create good homogeneity in the gel formulation, and skilled personnel should be readily available to handle the equipment. These factors have implications on the production cost. The target price for CHX gel may not be able to accommodate this type of production cost. REFERENCES 1 Joslyn LJ. Gaseous chemical sterilization. In: Block SS, ed. Disinfection, Sterilization and Preservation, 4 th Edition. Media, PA: Lippencott Williams & Wilkins; 1991: Final report on the safety assessment of benzalkonium chloride. The Official Journal of the American College of Toxicology. 1989;8(4): Srichamroen A. Influence of temperature and salt on viscosity property of guar gum. Naresuan University Journal. 2007;15(2): Kawamura Y. Guar gum, chemical and technical assessment. Prepared for: The 69 th Joint Food and Agriculture Organization of the United Nations/World Health Orgainzation Expert Committee on Food Additives Available at: Accessed July 6, Nordbø H. Ability of chlorhexidine and benzalkonium chloride to catalyze Browning reactions in vitro. Journal of Dental Research. 1979;58: Bain NJ. Chlorhexidine in dentistry a review. New Zealand Dental Journal. April 1980;76: