Time-kill Kinetics of a Novel Antimicrobial Silver Wound Gel Against Select Wound Pathogens

New treatments are needed as infection risk associated with diabetic, venous, and pressure ulcers are becoming more prevalent as comorbidities of obesity, aging, and major disease. Postsurgical, burn, and immunocompromised patients are also at an increased risk of wounds and infection. Silver has been utilized in treating various wounds associated with infections and, although highly effective, caution is required for use beyond 2 weeks due to potential silver cytotoxicity. To overcome this obstacle, an antimicrobial wound gel (CelaCare Technologies, Inc, Dallas, TX) was designed to allow low concentrations of a proprietary silver salt combined with acemannan, which has been demonstrated to aid wound healing. Materials and Methods. This study’s objective was to determine the time-kill kinetics of the antimicrobial wound gel vs 4 commercial topical silver products against 6 common wound pathogens and Bacillus subtilis as a spore-forming bacteria. Results. The antimicrobial wound gel achieved a 2.9 log reduction in growth of Pseudomonas aeruginosa within 30 minutes, a 2.3 log reduction in Streptococcus pyogenes within 8 hours, a 2.1 log reduction in methicillin-resistant Staphylococcus aureus within 48 hours, a 2.3 log reduction in S. aureus within 24 hours, a 4.1 log reduction in Escherichia coli within 30 minutes, a 2.9 log reduction in B. subtilis within 60 minutes, and a 3.4 log reduction in Candida albicans within 90 minutes. Overall, the antimicrobial wound gel demonstrated broad antimicrobial coverage against all wound pathogens evaluated, and it was comparable to, or better than, other tested topical silver products containing substantially higher silver concentrations. Conclusion. The broad-spectrum antimicrobial activity of the wound gel indicates it could become a product alternative to current commercial products.

Wounds and wound infections are associated with an increase in hospital length of stay and in mortality.¹ This is especially true in several populations with an increased risk of wounds, poor wound healing, and infection, including patients with diabetes and those with venous and pressure ulcers. Other groups such as obese patients, elderly residents in long-term care facilities (LTCF), as well as postsurgical, burn, and immunocompromised populations, also are at greater risk for infection and delayed healing due to lower immune competency.

The incidence of diabetes (the number one cause of kidney failure, amputation, and blindness), is on the rise.^2,3 In 2014, the Centers for Disease Control and Prevention (CDC) reported that 29.1 million patients in the United States are living with diabetes.² This is alarming for several reasons, including that patients with diabetes are at an increased risk for wounds, with a 4%-10% incidence of developing foot ulcers, and experiencing delayed healing with a higher risk of infection.⁴

Venous stasis ulcers impact 1%-3% of the adult population with repeated cycles of ulceration, healing, and have a 1-year recurrence rate of 18%-28%.⁵ These ulcers are complicated by excessive and prolonged inflammation often related to heavy bioburden of colonizing microorganisms. This represents one of the most important barriers to wound closure and early localized infection.⁵

Obese patients have a higher prevalence of wounds as compared to patients with other comorbidities. This could be due to poor circulation impairing the wound healing cascade. Among 5,240 patients registered in the wound registry between 2005 and 2010, individuals who were obese or overweight (73.1%) comprised the most common comorbid condition, followed by cardiovascular or peripheral vascular disease (51.3%) and diabetes (46.8%).⁶

In the United States, it is projected that the number of people over 65 years of age will surpass 70 million by 2030.⁷ Furthermore, 1.5 million people are currently residents in LTCF and this number is expected to increase with projections of the elderly population in the United States.⁸ Long-term care facility residents are at a high risk of developing health care-associated infections that correlate with increased morbidity and mortality. Pressure ulcers, for example, have been reported to occur in 7%-20% of LTCF residents.^8,9 The prevalence of pressure ulcers increased with the patient’s age, with 71% of ulcers occurring in patients older than 70.9 years. These ulcers are often involved with deep soft-tissue infections that may progress to osteomyelitis and even secondary bacteremic infections reported to have a mortality rate as high as 50%.⁸

Patients who underwent surgical procedures are also at increased risk of pressure ulcers with an overall incidence of 15%. The highest surgical-associated prevalence was observed with hip fracture repair (22%), followed by cardiac surgery (18%), and postsurgical intensive care (11%).¹⁰

Patients with burns are more susceptible to infection. It has been noted that as many as 75% of all deaths following severe burn injuries are related to infection.¹¹ Burn wounds have been reported as the most frequent origin for sepsis due to breakdown of the skin barrier that usually prevents invasion of microorganisms.¹¹

Immunocompromised populations including patients with HIV/AIDS are at increased risk of wound complications associated with infection. As an example, in a comparison study of HIV-positive and HIV-negative patients with burns covering 11%-20% of their total body surface area (TBSA), patients who were HIV-positive were reported to have a 25% mortality rate as compared to 12% for patients who were HIV-negative.¹²

The continual rise in multidrug resistant organisms is becoming more problematic in wound and burn care.^13,14 Multidrug resistant infections, even in noninstitutionalized patients, have been observed with increasing frequency.^15,16 According to a 2013 CDC report, 2 million people per year in the United States are infected with multidrug resistant organisms.¹⁷ With the increase in multidrug resistance and at-risk populations, it is essential to develop cost-effective therapies that have antimicrobial properties along with wound -healing benefits.

Silver is known for its broad-spectrum antimicrobial activity against various microorganisms including antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). In addition, activity against certain viruses, yeast, and fungi has been reported. Silver demonstrates activity at multiple bacterial target sites inducing microbial cell death by binding to the cell wall and disrupting cell wall integrity, as well as binding to DNA and interfering with cell replication and transcription.¹⁸ In addition, limited toxicity toward mammalian cells at currently used concentrations has led to silver-based compositions gaining popularity in advanced wound care.^18,19 Currently, there are more than 10 different types of silver dressings (eg, creams, foams, hydrogels, hydrocolloids, polymer films, and meshes) available to health care professionals. The activity of a silver-containing dressing is related to the amount, type, and distribution of silver in the dressing. As a result, each dressing type may demonstrate different clinical efficacy with different utility and advantages.²⁰

The antimicrobial wound gel (CelaCare Technologies, Inc, Dallas, TX) used in this study is a novel hydrogel designed to allow for low concentrations of a proprietary antimicrobial silver salt via its synergistic activity with acemannan, a purified extract isolated from Aloe vera. Acemannan has been utilized in multiple marketed wound care products for more than 20 years and has demonstrated immunomodulatory properties with positive effects on the healing cascade.²¹ Topical products containing acemannan are approved for management of surgical and traumatic wounds, diabetic ulcers, pressure ulcers, venous stasis ulcers, first -degree burns, and second-degree burns.^22,23

The antimicrobial wound gel has been designed with concentrations of silver substantially lower than reported in commercially available products that range from 55 ppm to 3,000 ppm. The product has been evaluated for cytotoxicity and deemed to be noncytotoxic using a standard protocol at a commercial contract research laboratory in compliance with US Food and Drug Administration Good Laboratory Practices. The antimicrobial wound gel has been evaluated in several verification case studies for multiple wound types. Thus far no skin hypersensitivities have been noted.

The aim of this study was to evaluate the antimicrobial time-kill activity of the antimicrobial wound gel against 6 common wound bacterial pathogens as compared to 4 silver-containing marketed topical products. The pathogens selected represent a broad spectrum of microorganisms commonly identified with health care-associated infection.

S. aureus is estimated to be carried by one-third of all people, and MRSA is responsible for 64% of the total staph infections in intensive care units in US hospitals.^24,25 Thirteen percent of Pseudomonas aeruginosa infections are multidrug resistant, with several classes of antibiotics no longer effective for treatment of pneumonia, sepsis, urinary tract infections, and surgical site infections.¹⁷Streptococcus pyogenes was selected because it is known to cause infections ranging from skin infections to complicated skin and skin-structure infections with increasing resistance against erythromycin.²⁶Escherichia coli has been associated with skin and soft tissue infections in the health care setting, following surgical incision and in patients with neutropenia.^27,28,29Candida albicans was chosen based on data suggesting this dimorphic yeast is the major cause (> 50%) of cutaneous infections in neutropenic patients.²⁸ Lastly, Bacillus subtilis was also selected to evaluate antimicrobial efficacy of silver-containing products against spore-forming bacteria.

Microorganisms used for testing. To evaluate the effectiveness of silver-containing antimicrobial products, 6 microorganisms were purchased from American Type Culture Collection (Manassas, VA). These organisms were B. subtilis subsp. Spizizenii (ATCC 6633), C. albicans (ATCC 10231), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), S. aureus (ATCC 6538), and S. pyogenes (ATCC 19615). Methicillin-resistant Staphylococcus aureus (MRSA, WCMC 78044-1), a clinical patient isolate was provided by White County Medical Center (Searcy, AR).

Growth media for microorganisms. Tryptic Soy Broth (TSB, Lot 11361, Exp 11/01/2014) and Tryptic Soy Agar (TSA, Lot 13003, Exp 10/04/2015) were purchased from Hardy Diagnostics (Santa Maria, CA) and prepared under sterile conditions.

Silver-containing antimicrobial wound products. Antimicrobial wound gel (CelaCare Technology Inc, Dallas, TX) was the primary test article. Four commercial silver-containing antimicrobial wound products (SCAWPs) were chosen as positive controls and compared to the antimicrobial wound gel. They were: Curad Silver Solution (SCAWP 1, Medline Industries, Mundelein, IL, 55 ppm), Silver Sulfadiazine 1% Cream (SCAWP 2, Ascend Laboratories, LLC, Montvale, NJ), SilvaSorb Antimicrobial Wound Gel (SCAWP 3, Medline Industries Inc, Mundelein, IL, silver concentration not available per label), and Silver-Sept Silver Antimicrobial Skin & Wound Gel (SCAWP 4, Anacapa Technologies, San Dimas, CA, 200 ppm). Sterile Saline (0.9%) served as the negative control for the experiments.

Equipment used for testing. An ultraviolet-visible spectrophotometer (NanoDrop 2000c, Thermo Scientific, Waltham, MA) was used to measure and adjust optical density (OD) of the working suspension. A 37°C, Model 10 CO2 Incubator (Fisher Scientific, Pittsburgh, PA) was used for culture and to incubate growth of microorganisms. A centrifuge (VanGuard Model V6500, Hamilton Bell Company Inc, Montvale, NJ) was used to inoculate the test articles with microbial working suspensions. A vortex mixer (Vortex Mixer Model MS 3 basic, Fisher Scientific, Waltham, MA) was used for neutralization of the test articles with saline at each time point.

Time-kill kinetic test protocol. The protocol was based on the American Society for Testing and Materials E2315, “Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure.”³⁰ For each microorganism, time-kill assays were performed at least twice with similar results. Mean values of 2 triplicate CFU/mL measurements were plotted.

An isolate of each microorganism was inoculated on TSA and incubated at 37°C for ~24 hours. Then, a 5.0 McFarland suspension of each microorganism was prepared by adding colonies to sterile saline until an OD at 350 nm that ranged between 0.260 and 0.340 was obtained. Then, 200 µL of the 5.0 McFarland suspension was added to 2.0 mL TSB and incubated for 1-2 hours at 37°C to make a microbial working suspension.

The SCAWPs were prepared based on their viscosity and 190 µL of each product was used for testing. Due to the fact that SCAWP 2 is a water-in-oil emulsion, it required a 1:100 dilution in deionized water, resulting in a 30.3 ppm emulsion. Also, 190 µL of diluted SCAWP 2 was tested.

For each time point and microorganism, 6 sterile 15 mL conical tubes were used, containing 190 µL of the antimicrobial wound gel, SCAWP 1, SCAWP 2, SCAWP 3, SCAWP 4, and 0.9% saline (the negative control), respectively.

To expose the microorganism to each test article, 10 µL of the appropriate working suspension was directly added to each of the test articles or saline control contained in each tube at timepoint 0 and then centrifuged at 3000 rpm for 30 seconds. At each time point to be assayed, the test article was neutralized by adding 10 mL of saline to each tube and mixing thoroughly via vortexing and tube inversion.

After neutralization, serial dilutions were performed in a 96-well microtiter plate that contained 180 µL of saline in all rows except the first row. The serial dilution was performed by adding 40 µL neutralized suspension into the first row, then serially transferring 20 µL to the second row, thoroughly mixing and then transferring 20 µL from the second row to the third. Subsequent rows were handled in the same manner until the seventh row, leaving the last row as a sterility control.

After neutralization and dilution, cell counts were performed by transferring 10 µL from each well to an agar plate and incubating at 37°C for ~24 hours. The following day, colonies were counted and multiplied by the dilution factor to determine the log number of viable microbes. Additionally, 100 µL from each of the neutralized samples was plated on agar plates to give an assay sensitivity threshold of 104 microorganisms per mL.

Silver-containing antimicrobial wound product 1, SCAWP 3, and SCAWP 4 were ineffective at inhibiting P. aeruginosa throughout a 30-minute exposure, and the results were the same as the saline negative control. Silver-containing antimicrobial product 2, 30.3 ppm after 100-fold dilution, produced a 3.5-log reduction at 30 minutes. In addition, the antimicrobial wound gel elicited a 2.9-log reduction at 30 minutes for P. aeruginosa (Figure 1A).

For S. pyogenes, following a 24-hour exposure, SCAWP 2 produced a 1.0-log reduction at 12 hours, SCAWP 1 demonstrated a 2.1-log reduction at 12 hours, and SCAWP 3 had a 2.0-log reduction at 8 hours. The antimicrobial wound gel elicited a 2.3-log reduction at 8 hours. The SCAWP 4 was ineffective at inhibiting S. pyogenes (Figure 1B). 

Tests with MRSA demonstrated a 3.0-log reduction at 48 hours for SCAWP 2; 1.2 at 48 hours for SCAWP 1; 2.5 at 48 hours for SCAWP 4; 1.4 at 48 hours for SCAWP 3; and 2.1 at 48 hours for the antimicrobial wound gel (Figure 2A).

Tests with S. aureus demonstrated log reductions of 1.1 at 24 hours with SCAWP 2; 1.9 at 24 hours with SCAWP 1; 2.6 at 24 hours with SCAWP 4; 1.9 at 24 hours with SCAWP 3; and 2.3 at 24 hours with the antimicrobial wound gel (Figure 2B). 

For E. coli, there were log reductions of 3.3 at 30 minutes with SCAWP 2; 2.9 at 120 minutes with SCAWP 1; 3.5 at 30 minutes with SCAWP 4; 3.0 at 120 minutes with SCAWP 3; and 4.1 at 30 minutes with the antimicrobial wound gel (Figure 3A).

Tests with B. subtilis demonstrated log reductions of 2.8 at 30 min for SCAWP 2, 2.5 at 60 min for SCAWP 1; 1.9 at 90 min for SCAWP 4; 3.1 at 90 min for SCAWP 3; and 2.9 at 60 min for the antimicrobial wound gel (Figure 3B). 

For C. albicans, SCAWP 1 and SCAWP 3 were ineffective through a 120-minute exposure. The data for SCAWP 2 was not available due to insufficient growth at 0 time. Silver-containing antimicrobial wound product 4 and the antimicrobial wound gel elicited 2.9 and 3.4 log reductions, respectively, at 90 minutes (Figure 4). 

In this study, the antimicrobial activity of the antimicrobial wound gel and other silver-containing antimicrobial topical products against selected potential wound pathogens were evaluated using a standard time-kill assay. Time-kill studies have been determined to provide a greater degree of antibacterial agent effectiveness than minimum inhibitory concentration (MIC) assays.²⁶ Dilution with saline was chosen for neutralization because it is an approved method in ASTM E2315, “Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure,”³⁰ and in the authors’ hands, it was comparable to neutralization with thioglycollate broth in its ability to quench the activity of the antimicrobial agents tested (data not shown).

The combination product of silver and sulfa antibiotic in micronized form, SCAWP 2, has been used in the management of burn wounds for many years.³¹ Its characteristic form as a water-in-oil emulsion renders it difficult to work with in the laboratory. The kill-time data was not determined for C. albicans due to insufficient growth of the microorganism at timepoint 0. Insufficient growth of C. albicans could be due to the residual inhibitory effect of the high lipid component of SCAWP 2 in the emulsion even after a 1:100 ratio dilution with deionized water at time zero. Microscopic studies have previously shown that the lipid content exerts killing of the microorganism through disruption of the cell membrane.³² In addition, medium-chain fatty acids have been reported to have an inhibitory effect on growth of C. albicans using an MIC assay.³³ This may be partially explained by the fact that C. albicans is an eukaryotic organism. Overall, with the exception of C. albicans, SCAWP 2 demonstrated antimicrobial activity with 1-log to 3-log reductions in all other tested microorganisms. However, it should be noted that SCAWP 2 contains an extremely high concentration of silver (~3000 ppm) combined with the sulfadiazine antibiotic. The large excess of silver is necessary to compensate for losses due to chemical inactivation.³⁴ It was calculated that a patient with a 4 mm full-thickness burn would receive as much as 218,400 ppm silver for a 2-week course of therapy.³⁴ This high concentration of silver from SCAWP 2, and the presence of undesirable counter ions associated with the formulation, may contribute to reports of silver toxicities.³⁴ Moreover, in the clinical setting, SCAWP 2 is short-acting and requires frequent reapplication, which is complicated due to its difficulty in removal.³⁵

Silver-containing antimicrobial wound product 1 is an over-the-counter (OTC) hydrogel product that contains 55 ppm of colloidal silver chloride. This product exhibited antimicrobial activity against gram-positive microorganisms including S. pyogenes, MRSA, S. aureus, and B. subtilis. However, it was ineffective against P. aeruginosa and C. albicans, 2 pathogens that had been classified in the serious threats category by the CDC due to emerging resistance against antimicrobial agents.¹⁷ Yet, the possibility of SCAWP 1 exerting antimicrobial activity against these 2 microorganisms after longer exposure cannot be ruled out, as a previous MIC study indicated the colloidal silver solution to be effective against selected microorganisms including P. aeruginosa.³⁶

Silver-containing antimicrobial wound product 4, an OTC product formulated as an amorphous hydrogel containing 200 ppm of silver was effective against MRSA, S. aureus, E. coli, B. subtilis, and C. albicans. However, it did not exhibit killing within 30 minutes of exposure for P. aeruginosa and was ineffective against S. pyogenes. Like SCAWP 1, SCAWP 4 may require a longer exposure time to be effective against these 2 pathogens.

Silver-containing antimicrobial wound prduct 3 is a prescription and OTC medical device product formulated as a hydrogel with controlled release of ionic silver. For E. coli, both SCAWP 1 and SCAWP 3 required 2 hours for a 2.9-log and 3.0-log reduction, respectively, while other products including the antimicrobial wound gel were active within 30 minutes. Yet, this relatively longer exposure time for SCAWP 1 and SCAWP 3 to kill E. coli is comparative with the exposure time (within 2-3 hours) claimed by the company.

Overall, SCAWP 3 was effective against most organisms except P. aeruginosa and C. albicans. It was not effective against P. aeruginosa within 30 minutes of exposure and did not exhibit antimicrobial activity against C. albicans within 2 hours of exposure. Like other tested silver-containing products, SCAWP 3 may require a longer exposure time to be effective against these 2 pathogens. Moreover, the zone of inhibition study conducted by Echague and colleagues³⁷ reported SCAWP 3 as having relatively weaker antimicrobial effects compared to other products, with the size of the zone inhibition to be ≤ 10 mm for the following organisms: P. aeruginosa, E. coli, and MRSA.

The antimicrobial wound gel, a novel antimicrobial hydrogel formulated utilizing a combination of a proprietary silver salt and acemannan, had antimicrobial activity against all tested microorganisms. In comparison to other commercial products used in this study, the antimicrobial wound gel had higher antimicrobial activity against P. aeruginosa and E. coli, and it had similar or better antimicrobial activity as compared to the other commercial products when tested against MRSA, S. aureus, S. pyogenes, B. subtilis, and C. albicans. The product design for the antimicrobial wound gel allowed for very low silver concentrations due to its combination with acemannan. Acemannan has been reported to positively effect healing and reduce pain and discomfort in a variety of wound types.³⁸ Further, the antimicrobial wound gel is the first antimicrobial topical wound product designed to incorporate a broad spectrum antimicrobial compound with an immunomodulator.

Silver has been utilized for hundreds of years in wound care with its known antimicrobial activity against wound pathogens.³⁹ The antimicrobial activity of silver comes from absorption of silver ions by bacteria or fungi which coagulate intracellular proteins, eventually leading to apoptosis of bacterial cells.³⁹ Silver metal does not exert antimicrobial activity.³⁹ This characteristic therefore requires topical silver commercial products to be designed in various forms to release silver ions. The commonly used forms of silver include: crystalline forms such as nanoparticles, inorganic silver compounds, or silver impregnation into activated charcoal.³⁹

Despite silver’s excellent antimicrobial properties, potential cytotoxicity associated with high concentrations of silver has been a major obstacle limiting the use of silver-containing dressings to no more than 2 weeks with re-evaluation prior to extended treatment.³⁵ Product differences also provide another challenge for providing consistent wound healing among the wide array of commercial silver dressing products. In addition, silver products may no longer provide therapeutic value if a wound is not 30% smaller after 1 month of treatment, clinically resulting in a wound that is less likely to be healed within 3 months.⁴⁰

Silver nanoparticles, for example, could be potentially cytotoxic even at concentrations as low as 15 ppm, and topical silver products currently available on the market utilize either silver nanoparticles (eg, SCAWP 3) and/or contain silver concentrations higher than 15 ppm (eg, SCAWP 1, SCAWP 2, and SCAWP 4).⁴¹ Argyria, a cutaneous discoloration of skin after exposure to high concentrations of silver, is usually seen in patients who are exposed to either silver nitrate or colloidal silver.^39,42 A few rare cases have been reported following the use of silver sulfadiazine or silver-impregnated dressings that released high concentrations of silver into a wound site.³⁹ Besides argyria, other types of silver toxicities have been reported in individual cases associated with use of silver sulfadiazine including renal dysfunction without hepatic and ocular abnormalities, deterioration in mental status, local accumulated silver concentrations in certain patients, and acute leucopenia due to cell maturation arrest.^43-46

There has been debate about whether silver released from dressings promotes or delays wound healing. However, recent findings indicate that silver delays the wound healing process. In 1 study, silver sulfadiazine had an inhibitory effect on keratinocyte and fibroblast growth and induced the retardation of wound closure.⁴⁷ Although some clinical and experimental studies still claim silver released from dressings stimulate or kick-start wound healing by promoting homeostasis, reducing inflammation, and enhancing re-epithelialization and neovascularization, these findings are limited to acute wounds with low levels of infection and minimal systemic or other complications.³⁹ In contrast, some chronic or indolent wounds exposed to silver sulfadiazine or silver-containing dressings may persist for many months with questionable signs of improvement.³⁹ The antimicrobial wound gel with its low silver concentration has substantial antimicrobial benefits, and it is designed to maintain the immunomodulatory properties and healing attributes of acemannan.^21,38

This study was limited to examining the in vitro antimicrobial effects of the antimicrobial wound gel without evaluation of its wound healing properties. To further study the combined benefits of silver and acemannan, clinical studies should be explored to determine the effectiveness of the antimicrobial wound gel in healing a variety of surgical wounds, burns, diabetic ulcers, pressure ulcers, venous stasis ulcers, and reducing infection in full-thickness and partial-thickness wounds.

In conclusion, the antimicrobial wound gel examined in this study demonstrated broad antimicrobial activity against select wound pathogens including MRSA. Head-to-head time-kill results were comparable to, or better than, other tested topical silver products that contained substantially higher concentrations of silver. CelaCare’s novel antimicrobial hydrogel is effective against a variety of microorganisms at a low silver concentration, making it an excellent potential product to reduce infection and to positively impact slow healing wounds.

The authors acknowledge and thank the White County Medical Center, Searcy, AR, and the following iindividuals for their contributions to the time-kill kinetic studies: Mylinda Dill, PharmD, and laboratory technicians Zachary Wilkerson and Gretchen Smith.

Affiliations: Harding University College of Pharmacy, Searcy, AR; University of Pikeville Kentucky College of Osteopathic Medicine, Pikeville, KY; Johns Hopkins Hospital, Baltimore, MD; Community Pharmacist, Tulare, CA; College of Medicine, University of Tennessee Health Science Center, Memphis, TN; and CelaCare Technologies, Dallas, TX 

Correspondence:
YoonJung Lee, PharmD
Harding University College of Pharmacy
Searcy, AR
ylee@harding.edu

Disclosure: Funding for this study was sponsored by CelaCare Technologies, Inc, Dallas, TX. All authors of this manuscript were affiliated with Harding University College of Pharmacy, Searcy, AR at the time the study was conducted.