colwidth="63pt" />

Formulation
T. rubrum
T. mentagrophytes
T. tonsurans
T. verrucosum
E. floccosum

SDA Control
No growth

Org. Control
No halo
No halo
No halo
No halo
No halo

PCO
(0.5 ml)
(0.5 ml)
(0.5 ml)
nm+
nm+

1%/Lidocaine
12.92 mm
14.41 mm
14.41 mm

0.1% -
(1.0 ml)
(1.0 ml)
(1.0 ml)
nm+
nm+

(Liquid)
8.69 mm
10.76 mm
14.36 mm

In the above table, nm+ indicates not measurable due to total inhibition.

TABLE 3

Inhibition Halos per Formulation (Bacteria and Fungi)

Formulation
E. coli
S. aureus
P. aeruginosa
B. subtilis
A. niger
C. albicans

TSA Control
No growth

Org. Control
No halo
No halo
No halo
No halo
No halo
No halo

Perillartine 1%
(1.0 ml)
(1.0 ml)
(1.0 ml)
(1.0 ml)
(1.0ml)
(1.0 ml)

(Liquid)
4.61 mm
4.65 mm
3.63 mm
4.51 mm
3.53 mm
2.54 mm

While lidocaine was used as an anesthetic agent in the above embodiment, it is understood that other topical anesthetic agents are encompassed by the invention to the extent that the formulation retains its antimicrobial activity. Some non-limiting examples of topical anesthetic agents that can be used include pramoxine, phenol, prilocaine, benzocaine, dibucaine, bupivacaine, proparacaine, epinephrine, capsaicin, menthol, methyl salicylate, tetracaine, camphor, cocaine, dyclonine, and combinations thereof. Moreover, according to another embodiment of the invention, the formulation including the anesthetic agent can be also used on skin that has no wounds.

In-Vivo Studies

While studying the effects of an alcohol-free formulation containing perillyl alcohol at different concentrations using a porcine model, pathogenic microorganisms A. baumannii and Methicillin-Resistant Staphylococcus aureus (MRSA USA300) were used to inoculate the wounds. One side of the pig was inoculated with MRSA USA300 and the other with A. baumannii (ATCC 19606), as illustrated in FIG. 5.

A. baumannii is a Gram (−) bacteria, which is known as a significant hospital pathogen that has developed significant resistance to antimicrobial agents, and a leading cause of hospital acquired infections, specifically bloodstream infections (BSI) (Jasna Hrenovic et. al., Applied and Environmental Microbiology p. 2860-2866 May 2014 Volume 80 Number 9). It has also been shown to be multi-drug resistant to carbapenem and ampicillin-sublactam antibiotics (CASR) (Teena Chopra et. al., Antimicrobial Agents and Chemotherapy p. 6270-6275 Dec. 2013 Volume 57 Number 12). MRSA USA 300 is methicillin-resistant S. aureus, a Gram (+) microorganism that is a common cause of hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) (Jakub M Kwiecinski et. al., Curr Opin Microbiol. 2020 February; 53:51-60. doi: 10.1016/j.mib.2020.02.005). This opportunistic pathogen is the main cause of sepsis and endocarditis due to blood-stream infections.

As a result of the in-vivo study above, the expectation was that the Perillyl Alcohol would be deactivated, as would be evidenced by growth of the microorganisms in the exposed skin, and the bacterial count would grow, or at the very least remain the same to show a bacteriostatic effect. This assumption was based on the mechanism of action proposed for Perillyl Alcohol as discussed below.

The antiseptic actions of oxygenated terpenoids, such as Perillyl Alcohol, are proposed at the cell membrane level. Considerable research demonstrates that oxygenated terpenoids disrupt the membrane via physicochemical changes as they enter the lipid layer. The strongest inhibitory effects exhibited were on microbial oxygen uptake and oxidative phosphorylation. Additional evidence points to reduction of the membrane order, affecting lipid bilayer packing, and increasing membrane small ion permeability or loss of chemiosmotic control. Thus, oxygenated terpenoids act by adversely affecting three fundamental properties of lipid bilayers, namely stability, order, and permeability.

However, to our surprise, the complete opposite effect and unexpected result was observed. Based on the proposed mechanism of action of perillyl alcohol and derivatives, the red blood cells would have been expected to be attacked, and through this process inactivate the disinfectant. The reason is that the red blood cell membrane is essentially a two-dimensional (2D) structure, comprised of a cytoskeleton and a lipid bilayer, tethered together. The lipid bilayer includes various types of phospholipids, sphingolipids, cholesterol, and integral membrane proteins, such as band-3 and glycophorin. So based on the proposed mechanism of action of the monoterpene, the bilayer of the red blood cells should have been affected by the perillyl alcohol and should have resulted in inactivation of the disinfectant effect.

Nevertheless, perillyl alcohol showed to be selective towards cells from microorganisms and differentiate over other cells or proteins present that could deactivate its disinfectant properties. Furthermore, blood contains several enzymes that offer potential metabolization of perillyl alcohol. Prior systemic studies using perillyl alcohol have failed to deliver the compound via its metabolization before it can reach the therapeutic effect. It has been shown that perillyl alcohol metabolizes quickly in rats where it was not detected. In separate studies, it was shown that in humans, dogs, and rats, it rapidly metabolized showing that it could have been affected by enzymes.

Treatment Regimen (Porcine Model)

Forty-eight (48) rectangular wounds measuring 10 mm×7 mm×0.5 mm deep were made in the paravertebral and thoracic area with a specialized electrokeratome fitted with a 7 mm blade. Wounds were blotted dry with sterile gauze pads. The wounds were separated from one another by 15 mm of unwounded skin and were individually dressed. As illustrated in FIG. 5, four (4) wounds were randomly assigned to each of the six (6) treatment groups (A-F), and they were then inoculated according to the experimental design shown in FIG. 5. One side of the animal was inoculated with Methicillin Resistant Staphylococcus aureus (USA 300) and the other side with Acinetobacter baumannii (ATCC 19606).

Immediately after performing the incisions, the wounds were inoculated with the appropriate bacterial strain as described in the “Wound Inoculation” section below and treated within 30 minutes of inoculation. Wounds were divided into six (6) groups per bacteria of four wounds each, treated with the appropriate treatments provided by sponsor (see design above FIG. 5). All wounds were covered, individually, with a polyurethane film dressing (Tegaderm; 3M, St. Paul, MN) which were secured along the edges using surgical tape. All dressings were covered and secured by wrapping the animal with self-adherent elastic bandages (Coban; 3M, St. Paul, MN). The dressings were left in place for 24 hours to determine the treatment efficacy in decreasing bacterial proliferation in the wounds. After 24 hours, the dressings were removed, and four wounds were recovered for each treatment group as described below.

Microbiological Techniques
Wound Inoculation

A fresh culture of Methicillin Resistant Staphylococcus aureus (NRS 384/USA 300) and Acinetobacter baumannii (ATCC 19606) were used in this study. The challenge inoculum suspension was prepared by scraping the overnight growth from a culture plate into 4.5 mL of sterile water. This resulted in a suspension concentration of approximately 108 colony forming units/mL (CFU/mL) for each bacterium. Serial dilutions were made until a concentration of 106 colony forming units/mL (CFU/mL) was achieved as determined by optical density measurements. The inoculum was vortexed, and 25 mL of the suspension was inoculated into each wound. In addition, serial dilutions of the suspension were plated onto culture media to quantify the exact concentration of viable organisms used for this experiment.

Recovery Methods
Wound Assessment

Four wounds from each treatment group (A-F) were recovered 24 hours after treatment. A sterile surgical steel cylinder (22 mm inside diameter) was placed around the wound area. One (1) mL of all-purpose neutralizer solution was pipetted into the cylinder and the site was scrubbed with a sterile Teflon spatula for 30 seconds. Serial dilutions were made from all culture samples and the extent of microbiological contamination assessed using the Spiral Plater System (Spiral Biotech, Norwood, MA). This system deposits a 50 μl aliquot of the scrub bacterial suspension over the surface of a rotating agar plate. ORSAB Screening Agar Base with Selective Supplement was used to isolate Methicillin Resistant Staphylococcus aureus USA 300 from the wounds, and Leeds Acinetobacter medium was used to isolate Acinetobacter baumannii (ATCC 19606). All plates were incubated aerobically overnight (16-24 hours) at 37° C., after which the number of viable colonies were counted.

Observations

Observations were taken during the treatment application and during the assessment time at 48 hours. Within 30 minutes of inoculation, 500 μL of the appropriate treatments were deposited into each wound respectively and spread with sterile spatula.

Results

After counting the colonies, the data was tabulated and the Log of colony forming units/mL (Log CFU/mL) for Methicillin Resistant Staphylococcus aureus (MRSA) USA300 and Acinetobacter baumannii ATCC 19606 (AB19606) determined. The arithmetic mean of the Log (CFU/mL) and standard deviation were calculated for each treatment. The effect of the treatments on the challenge pathogen was determined. Table 4 and Table 5 present the raw data.

Inoculation with Staphylococcus aureus (MRSA) USA300

When examining each treatment group, it was determined that the wounds treated with Perillyl Alcohol liquid gel 1% (treatment D) showed the lowest Log CFU/mL (7.39±0.35) MRSA USA 300 compared to other treatment groups (see FIG. 6). This was followed by treatment (C) Perillyl Alcohol liquid gel 0.5% (7.67±0.36), treatment (B) Perillyl Alcohol liquid gel 0.25% (8.00±0.06), treatment (A) Perillyl Alcohol liquid gel 0.1% (8.04±0.17), and treatment (E) Vehicle liquid gel (8.12±0.15) of MRSA USA 300 recovered from wounds. Untreated group wounds resulted in a greater Log CFU/mL (8.91±0.35) of MRSA USA300.

After 24 hours all treatment groups assessment reduced bacterial count compared to untreated group (treatment F), as shown in FIG. 6. Wounds treated with treatment D (Perillyl Alcohol liquid gel 1%) showed the largest reduction of MRSA (1.52±0.18 Log CFU/mL reduction) compared to treatment F (Untreated group). These values corresponded to a 96.98% reduction of MRSA at 24 hours when compared to wounds treated with treatment F (Untreated group). Bacterial reduction in wounds treated with Treatment B (Perillyl Alcohol liquid gel 0.25%) and treatment A (Perillyl Alcohol liquid gel 0.1%) had similar percentage reduction (87.70% and 86.51%, respectively) of MRSA after 24 hours assessment compared to untreated wounds (treatment F).

Inoculation with Acinetobacter baumannii (ATCC 19606)

Wounds treated with treatment D (Perillyl Alcohol liquid gel 1%) showed the lowest Log CFU/mL (3.10±0.56) of AB19606 when compared to other treatment groups (see FIG. 7). This was followed by treatment (C) Perillyl Alcohol liquid gel 0.5% (4.03±0.01), treatment (B) Perillyl Alcohol liquid gel 0.25% (4.71±0.32), treatment (A) Perillyl Alcohol liquid gel 0.1% (5.49±0.30), and treatment (E) Vehicle liquid gel (6.45±0.43) AB19606 recovered from wounds. Untreated group wounds resulted in a greater Log CFU/mL (7.38±0.28) of AB19606.

After 24 hours all treatment groups assessment shows reduced bacterial count when compared to untreated wounds (treatment F), as shown in FIG. 7. Wounds treated with treatment D (Perillyl Alcohol liquid gel 1%) showed the largest reduction of AB19606 (4.28±0.28 and 3.35±0.13 Log CFU/mL reduction) compared to untreated wounds (treatment F) and Vehicle liquid-gel (treatment E), respectively. These values corresponded to a 99.99% and 99.96% reduction of AB19606 after 24 hours treatment application when compared to untreated wounds (treatment F) or treated with the vehicle liquid-gel (treatment E), respectively.

Wounds treated with treatment D (Perillyl Alcohol liquid gel 1%) showed the lowest Log CFU/mL of Methicillin Resistant Staphylococcus aureus (MRSA) USA300 and Acinetobacter baumannii ATCC 19606 when compared to other treatment groups (see FIG. 8). The same trend was observed when the concentrations of Perillyl Alcohol liquid gel increased.

The following trends were noted. Wounds treated with Treatment D (Perillyl Alcohol liquid gel 1%) had the lowest amount of Methicillin Resistant Staphylococcus aureus (MRSA) USA300 and Acinetobacter baumannii (ATCC 19606) recovered 24 hours after treatment application. Wounds treated with Treatment D (Perillyl Alcohol liquid gel 1%) and Treatment C (Perillyl Alcohol liquid gel 0.5%) showed a larger reduction Methicillin Resistant Staphylococcus aureus (MRSA) USA300 and Acinetobacter baumannii (ATCC 19606) as compared to untreated wounds (1.52±0.18 and 4.28±0.28 Log CFU/mL reduction, respectively).

After 24 hours, assessment wounds which were treated with Treatment D (Perillyl Alcohol liquid gel 1%) showed 96.98% and 99.99% reduction of Log CFU/mL of Methicillin Resistant Staphylococcus aureus (MRSA) USA300 and Acinetobacter baumannii (ATCC 19606), respectively when compared to untreated wounds (treatment F). All Perillyl Alcohol liquid gel at different concentrations were more effective against Acinetobacter baumannii than MRSA (FIG. 8).

Overall, the Perillyl Alcohol liquid gel is the most effective in reducing bacterial counts against Acinetobacter baumannii Gram (−) bacteria than Staphylococcus aureus (MRSA) USA300 Gram (+) bacteria. These formulations would also be effective against Pseudomonas aeruginosa Gram (−) bacteria (another common pathogen with higher incidence in infections).

TABLE 4

MRSA USA 300 S. aureus Data

Bacteria
Dilution
Count
CFU/mL
Log CFU/mL

S. aureus
−4
44
8.79E+06
6.94

MRSA USA300

Dilu-

Log

Treatment
Wound
tion
Count
CFU/mL
CFU/mL

A - Perillyl
1
−5
48
9.59E+07
7.98

Alcohol
2
−5
60
1.20E+08
8.08

liquid-
3
−5
86
1.72E+08
8.24

gel 0.1%
4
−5
35
7.00E+07
7.84
STDV

Mean
1.14E+08
8.04
0.17

B - Perillyl
1
−5
49
9.79E+07
7.99

Alcohol
2
−5
58
1.16E+08
8.06

liquid-
3
−5
52
1.04E+08
8.02

gel 0.25%
4
−5
41
8.20E+07
7.91
STDV

Mean
1.00E+08
8.00
0.06

C - Perillyl
1
−4
114
2.28E+07
7.91

Alcohol
2
−5
23
4.60E+07
7.66

liquid-
3
−5
44
8.79E+07
7.94

gel 0.5%
4
−4
73
1.46E+07
7.16
STDV

Mean
4.28E+07
7.67
0.36

D - Perillyl
1
−5
23
4.60E+07
7.66

Alcohol
2
−4
38
7.60E+06
6.88

liquid-
3
−5
20
4.00E+07
7.60

gel 1.0%
4
−4
131
2.62E+07
7.42
STDV

Mean
3.00E+07
7.39
0.35

E - Vehicle
1
−5
81
1.62E+08
8.21

(liquid-
2
−5
40
8.00E+07
7.90

gel)
3
−5
74
1.48E+08
8.17

4
−5
82
1.64E+08
8.21
STDV

Mean
1.39E+08
8.12
0.15

F - Untreated
1
−6
47
9.39E+08
8.97

2
−6
62
1.24E+09
9.09

3
−6
39
7.80E+08
8.89

4
−6
24
4.80E+08
8.68
STDV

Mean
8.60E+08
8.91
0.17

TABLE 5

A. baumannii Data

Bacteria
Dilution
Count
CFU/mL
Log CFU/mL

A. baumannii
−4
41
8.20E+06
6.91

ATCC 19606

Dilu-

Log

Treatment
Wound
tion
Count
CFU/mL
CFU/mL

A - Perillyl
1
−2
185
3.70E+05
5.57

Alcohol
2
−3
27
5.40E+05
5.73

liquid-
3
−3
21
4.20E+05
5.62

gel 0.1%
4
−2
56
1.12E+05
5.05
STDV

Mean
3.61E+05
5.49
0.30

B - Perillyl
1
−2
54
1.08E+05
5.03

Alcohol
2
−2
31
6.20E+04
4.79

liquid-
3
−2
28
5.60E+04
4.75

gel 0.25%
4
−1
94
1.88E+04
4.27
STDV

Mean
6.12E+04
4.71
0.32

C - Perillyl
1
−1
52
1.04E+04
4.02

Alcohol
2
−1
54
1.08E+04
4.03

liquid-
3
−1
52
1.04E+04
4.02

gel 0.5%
4
−1
55
1.10E+04
4.04
STDV

Mean
1.07E+04
4.03
0.01

D - Perillyl
1
−1
39
7.80E+03
3.89

Alcohol
2
0
60
1.20E+03
3.08

liquid-
3
0
32
6.40E+02
2.81

gel 1.0%
4
0
21
4.20E+02
2.62
STDV

Mean
2.52E+03
3.10
0.56

E - Vehicle
1
−3
79
1.58E+06
6.20

(liquid-
2
−4
26
5.20E+06
6.72

gel)
3
−4
40
8.00E+06
6.90

4
−3
49
9.79E+05
5.99
STDV

Mean
3.94E+06
6.45
0.43

F - Untreated
1
−4
87
1.74E+07
7.24

2
−4
92
1.84E+07
7.26

3
−4
81
1.62E+07
7.21

4
−5
31
6.20E+07
7.79
STDV

Mean
2.85E+07
7.38
0.28

Without alcohol, the Perillyl Alcohol liquid-gel reduced both microorganisms in one treatment. The 1% formulation containing surfactants and alcohol-free showed a 96.98% and 99.99% reduction of Log CFU/mL of MRSA and A. baumannii after a single 24-hour treatment, respectively, compared to untreated wounds (FIG. 8). Thus, according to the present invention, Perillyl Alcohol remains active in the presence of bodily fluids, blood, and soil (organic or inorganic), with no alcohol. Of particular importance is that this level of reduction in microorganisms is possible in only one treatment.

Furthermore, this also represents a unique behavior for treatment of wounds. This reduction in Gram (+) and Gram (−) microorganisms translates into a shorter treatment for infections on wounds. Since the reduction is quite significant after a 24-hr period, it means that under clinical conditions less applications of Perillyl Alcohol completely render the wound virtually free from infection leading to better outcomes. It also provides relief from the extended use of saline solutions in trauma centers, which is the only current treatment to the disinfection of open wounds.

Most relevant is the fact that the main disinfectant for wounds is the prescription of oral (systemic) antibiotics which only act against bacteria and will generally take days to be effective. In marked contrast, Perillyl Alcohol, as well as other oxidized monoterpenes, have been shown to be active against pathogenic yeast and fungi, as well as dermatophytes and can act immediately preventing potential amputations.

The formulation remains active against yeasts, such as Candida spp. (e.g., C. albicans, C. glabrata, C. parapsilosis). It is known that a main cause of bloodstream infections is Candida spp., which produces Candidemia. Candida infection can spread from your bloodstream to other parts of your body (such as your eyes, kidney, liver, and brain). This phenomenon is known as Invasive Candidemia.

Microorganisms treated with the formulations are listed on Tables 6-9 below.

TABLE 6

Gram Negative

Enterobacter spp.

Escherichia coli

Enterococcus faecalis

Serratia marcescens

Klebsiella pneumoniae

Haemophilus influenzae

Pseudomonas spp.

Pseudomonas aeruginosa

Pseudomonas cepacia

Proteus mirabilis

Porphyromonas gingivalis

Prevotella intermedia

TABLE 7

Gram positive

Acinetobacter spp.

Acinetobacter baumannii

Staphylococcus spp.

Bacillus subtilis

Bacillus cereus

Methicillin Resistant

Staphylococcus aureus

(MRSA)

Staphylococcus aureus

Staphylococcus epidermidis

Streptococcus pyogenes

Streptococcus pneumoniae

Micrococcus yunnanensis

TABLE 8

Fungi

Aspergillus spp.

Trichophyton rubrum

Aspergillus spp.

A. brasiliensis

A. flavus

A. fumigatus

T. mentagrophytes

T. tonsurans

T. verrucosum

E. floccosum

TABLE 9

Yeast

Candida spp.

Candida albicans

Candida glabrata

Candida parapsilosis

Candida auris

Candida tropicalis

Issatchenkia orientalis

Relating to the fungal species targeted by our research are those most recovered from humans; these include:

- 1. T. mentagrophytes, T. rubrum and T. verrucosum as causative agents of Tinea barbae
- 2. T. tonsurans as causative agent of Tinea capitis
- 3. T. mentagrophytes, T. rubrum and E. floccusum as causative agents of Tinea pedis
- 4. T. mentagrophytes and T. rubrum as causative agents of Tinea corporis
- 5. T. mentagrophytes, T. rubrum and E. floccusum as causative agents of Tinea cruris
- 6. T. mentagrophytes and T. rubrum as causative agents of Tinea manuum
- 7. T. mentagrophytes, T. rubrum and E. floccusum as causative agents of Tinea unguium

For example, a solution of perillartine (1,4 cyclohexadiene-1-carboxaldehyde syn-oxime) was used to treat different dermatophytes with the results shown in Figure V below combining different volumes to mimic dilution:

TABLE 10

Inhibition Halos per Formulation (Dermatophytes)

Formulation
T. rubrum
T. mentagrophytes
T. tonsurans
T. verrucosum
E. floccosum

SDA Control
No growth

Org. Control
No halo
No halo
No halo
No halo
No halo

Perillartine 1%
(0.5 ml)
(0.5 ml)
(0.5 ml)
(0.5 ml)
(0.5 ml)

(Liquid)
14.78 mm
20.84 mm
nm+
nm+
17.50 mm

(1.0 ml)
(1.0 ml)
(1.0 ml)
(1.0 ml)
(1.0 ml)

16.70 mm
17.37 mm
nm+
nm+
15.34 mm

In the above table, nm+ indicates not measurable due to total inhibition.

When treating microorganisms in samples of saliva from healthy volunteers, we found that although perillartine is a semi-synthetic sweetener it also acts as an antibacterial in the mouth, as shown in Table 11.

TABLE 11

Inhibition Halos per Formulation (Bacteria in Mouth)

Formulation
Volunteer #1
Volunteer #2
Volunteer #3

TSA Control
No growth

Perillartine 1%
4.33 mm
5.19 mm
4.61 mm

(Liquid)

Chlorhexidine
5.33 mm
5.70 mm
5.43 mm

gluconate

0.12%

(Liquid)

Listerine
0.91 mm
1.10 mm
0.83 mm

(Liquid)

Finally, the third group of microorganisms included in our research was tested by using the agar diffusion-disk technique. Four different bacteria and two different fungi were pour plated individually and the anti-microbial activity was detected either by inhibiting all growth or by showing a distinguished halo around a diffusion-disk previously impregnated with the formulation. Halo formation was measured with a caliper and biological activity was considered according to size. The formulation containing perillartine was exposed to aliquots of 1.0 ml of each organism, from an initial inoculum's concentration greater than 10⁴cfu/ml. Details of specific applications and formulations are set forth below in Table VII, which summarizes the various results obtained.

TABLE 12

Inhibition Halos per Formulation (Bacteria and Fungi)

Formulation
E. coli
S. aureus
P. aeruginosa
B. subtilis
A. brasiliensis
C. albicans

TSA Control
No growth

Org. Control
No halo
No halo
No halo
No halo
No halo
No halo

Perillartine 1%
(1.0 ml)
(1.0 ml)
(1.0 ml)
(1.0 ml)
(1.0 ml)
(1.0 ml)

(Liquid)
4.61 mm
4.65 mm
3.63 mm
4.51 mm
3.53 mm
2.54 mm

Formulations Tested

The formulations tested contain solvents that are not considered polymers, surfactants, such as N-methyl pyrrolidone (NMP) is used as a solubilizing agent. These series of preferred formulations are liquid under refrigeration (approximately 4° C.) and when warmed above 25° C. become a semisolid solution or liquid-gel

(Table 13).

TABLE 13

Preferred Formulation Alcohol-free Solution of (S)-Perillyl Alcohol

Solvent:
5% NMP:5% Solutol HS15:14% Pluronic F-127:76%

Water

Concentration:
0.1 to 1%

Physical State:
Solution

Another version of the preferred formulations contains Hydroxypropyl-β-Cyclodextrin (HPbCD). HPbCD is considered an enabling excipient, not a solvent, preservative, emollient, sunscreen, emulsifier, gelling agent, thickening agent, humectant, sequestrant, or polymer. (Table 14)

TABLE 14

Preferred Formulation Alcohol-free Solution of (S)-Perillyl Alcohol

Solvent:
20% HPbCD:3% Povidone K90:77% Water

Concentration:
0.1 to 1%

Physical State:
Solution

The above solution listed on Table 14, also has the property of remaining liquid while stored under refrigeration, and when warmed up to room temperature will become a liquid-gel.

When comparing CHG to the effectiveness of POH, other preferred formulations provided proof of comparable effectiveness when evaluating minimum-inhibitory concentration and minimum bactericidal concentration (MIC/MBC) results (Tables 16-18). The inclusion of IPA is mandatory by the Food and Drug Administration's (FDA) requirements as part of the comparison with the chlorhexidine solution. As shown in the study for open wounds in a porcine model (above), IPA is not necessary to achieve the antimicrobial effect. The IPA was formulated to maintain it below flammability levels. CHG on the other hand must be formulated using 70% IPA to provide antimicrobial effectiveness. One of ordinary skill in the art would appreciate that other disinfectant alcohols such as but not limited to ethanol, or n-propanol can also be used alone or in combination with IPA in accordance with the teachings of the present invention.

The antimicrobial effectiveness of POH diminishes in more dilute solutions. Therefore, challenges to its effectiveness were performed at 0.1% concentration and found to be reasonable against all microorganisms. At higher concentrations, over 90% concentrations it was observed to present immediate effects eradicating the microorganisms completely. However, POH at concentrations equal to and higher than 4% will cause skin irritation and thus are not practical for clinical applications. Therefore, the highest concentration proposed is 3% to maintain a practical clinical range.

TABLE 15

Preferred Formulation 0.1 to 3% Solution

of (S)-Perillyl Alcohol and 15% IPA

Solvent:
1% Yellow 6:0.8% Carbopol 940 (Polyacrylic acid)

(Lubrizol):15% Isopropanol:0.08% Isopropyl

myristate in Water, final pH 6 adjusted with NaOH

Physical State:
Opaque orange thixotropic gel

TABLE 16

MIC Alcohol Containing Formulation

(1% Perillyl Alcohol/15% IPA)

Minimum Inhibitory

1% POH/

Concentration
CHG
15% IPA
Vehicle

Candida albicans
1:1024
1:16
<1:2

ATCC #10231

Enterococcus faecalis
1:8192
1:16
<1:2

ATCC #29212

Escherichia coli
1:8192
1:16
<1:2

ATCC #25922

Klebsiella pneumoniae
1:4096
1:16
<1:2

ATCC #10031

Micrococcus yunnanensis
1:8192
1:16
<1:2

(formerly luteus)

ATCC #7468

Proteus mirabilis
1:512
1:16
<1:2

ATCC #7002

Pseudomonas aeruginosa
1:2048
1:16
<1:2

ATCC #27853

Serratia marcescens
1:512
1:16
<1:2

ATCC #14756

Staphylococcus aureus
1:8192
1:16
<1:2

MRSA ATCC #33591

Staphylococcus epidermidis
1:8192
1:16
<1:2

ATCC #12228

TABLE 17

MBC Formulation (1% Perillyl Alcohol/15% IPA)

Minimum Bactericidal

1% POH/

Concentration
CHG
15% IPA
Vehicle

Candida albicans
1:1024
1:16
<1:2

ATCC #10231

Enterococcus faecalis
<1:2048
1:8
<1:2

ATCC #29212

Escherichia coli
1:64
1:8
<1:2

ATCC #25922

Klebsiella pneumoniae
1:4096
1:16
<1:2

ATCC #10031

Micrococcus yunnanensis
<1:2048
1:8
<1:2

(formerly luteus)

ATCC #7468

Proteus mirabilis
1:512
1:8
<1:2

ATCC #7002

Pseudomonas aeruginosa
1:1024
1:8
<1:2

ATCC #27853

Serratia marcescens
1:256
1:8
<1:2

ATCC #14756

Staphylococcus aureus
1:4096
1:8
<1:2

MRSA ATCC #33591

Staphylococcus epidermidis
1:8192
1:8
<1:2

ATCC #12228

TABLE 18

Time-Kill (1% Perillyl Alcohol/15% IPA).

Microorganism
ChloraPrep ®
1% POH/15% IPA

(Exposure: 30 sec,
(Lot Number:
(Lot Number:

1, 3, 5, 10 minutes)
83028)
ASLP 05072015)

Candida albicans ATCC #10231
99.9999%
99.9999%

Enterococcus faecalis

ATCC #29212

Micrococcus yunnanensis

ATCC #7468

Staphylococcus aureus MRSA

ATCC #33591

Staphylococcus epidermidis

ATCC #12228

Proteus mirabilis ATCC #7002
99.9999%
99.9997%*

Pseudomonas aeruginosa
99.9992%
99.9992%

(ATCC 27853)

Escherichia coli ATCC #25922
99.9999%
99.9992%*

Klebsiella pneumoniae

ATCC #10031

Serratia marcescens

ATCC #14756

*Due to neutralization being achieved, in part by dilution, the lowest reliable dilution in the Time Kill Assay is 10⁻².

It is important to point out that CHG 2% is a solution that contains 70% isopropyl alcohol (IPA) which is a superb antiseptic in compositions containing greater than 65% concentration. Some studies have concluded that the overwhelming antiseptic effect from CHG is derived from the isopropyl alcohol. A comparative study between a formulation containing 70% isopropyl alcohol versus a 2% CHG commercial solution showed equivalent effectiveness between both products affording an approval by the Food and Drug Administration (Table 19).

TABLE 19

Results from 70% IPA in vehicle versus 2.0%

ChloraPrep ® (in 70% IPA).

Body
Mean Log₁₀CFU/cm²Reduction from Baseline

Area
Treatment
30 sec
10 min
6 hours

Abdomen
IPA in vehicle
2.66
3.38
2.42

ChloraPrep ®
2.91
3.35
2.23

Vehicle
1.03
1.39
1.26

Normal saline
0.64
0.78
0.94

Groin
IPA in vehicle
3.56
4.28
3.01

ChloraPrep ®
3.36
4.2
2.72

Vehicle
1.24
1.83
1.98

Normal saline
1.2
1.43
1.6

*Randomized, paired comparison study - 320 volunteers.

The formulations according to the present invention can be used for application of the antimicrobials to wounds, and in addition to semisolid delivery systems (e.g., gels, cream, ointment, foam), formulated as immediate release the invention also includes extended and/or sustained release systems using ingredients that someone skilled in the art can formulate.

These formulations include but are not limited to microencapsulation processes, application of the use of nanoparticles, or transdermal patches. These systems can include an anesthetic (e.g., lidocaine) as part of the formulation meant to provide temporary relief of pain. The same technology can be applied if formulated as a foam to provide occlusion and physical protection of the wound while providing disinfection. In addition, the delivery system can include liquid systems for irrigation such as flushing a wound to remove dead and necrotic tissue while at the same time providing immediate disinfection of the affected area. It can also be used during surgical procedures to maintain a clean wound and prevent surgical site infections.

Typical formulations can include excipients for a combination of the POH or derivative alone, or with anesthetic (e.g., lidocaine), or POH or derivative plus alcohol (e.g., IPA, ethanol), or POH or derivative plus alcohol plus anesthetic in gel, cream, ointment, foam, time-release, microencapsulation, nanoparticles. These formulations are meant to deliver the antimicrobial prophylactic and therapeutic effect of the POH or derivatives alone or in combination with an alcohol (e.g., IPA, ethanol), with anesthetic, or with anesthetic and alcohol to the affected area including disinfection of the intact skin, as well as open wounds, in the presence of organics (e.g., bodily fluids), inorganic (e.g., dirt, soil), blood, or soap. For the purpose of the invention, the formulation is administered to the skin region of a subject needing disinfection, for either prophylactic reasons (e.g., disinfecting an intact skin region prior to a surgical procedure, disinfecting a nail area or as a hand disinfectant) or therapeutic reasons (e.g., disinfecting an open wound or a cut). Thus, for the purpose of the invention, the term “in need thereof” includes both situations, the need to prevent possible infection on a skin region and the need to reduce or eliminate infection on a skin region already infected. Moreover, one of ordinary skill in the art would understand that an effective amount of the formulation is determined at least on the size of the skin region, the depth of a wound or cut on the skin and level of infection on the skin region. among other considerations.

For formulation in gels can include gelling agents, stabilizers, dispersing agents, penetration enhancers, buffers, and preservatives, as well as excipients that provide solvent action and humectant properties. They can also be prepared in cream form by addition of thickeners, emulsifying agents, preservatives, antioxidants, and buffer agents, and those ingredients as included in the current version of the United States Pharmacopeia, Europe Pharmacopoeia, Japan and other international standards such as the International Pharmaceutical Excipients Council (IPEC) selected and formulated to provide the desired viscosity and can be easily spread.

Gel formulations according to the present invention can be prepared using gel bases according to U.S. Pat. No. 4,883,660 A to Blackman et al., incorporated by reference herein in its entirety, where glycol solvents, specifically propylene glycol and polyethylene glycols having average molecular weights of from about 200 to about 800, can be gelled through the addition of small quantities of ethoxylated saturated fatty alcohols, i.e., fatty alcohols having chain lengths of from 16 to 22 carbon atoms which are ethoxylated with 2 to 30 moles of ethylene oxide. While the ethoxylated alcohols are known primarily as nonionic surfactants, they also form very elegant anhydrous gels with glycol solvents, producing gel bases for pharmaceutical compositions that have excellent drug solubilizing, penetration and release characteristics because of the high percentage of glycol solvent in the gels. In addition, the ethoxylated alcohols act as penetration enhancers for the base. The term “gel’ is used herein in the general sense of a semi-solid, apparently homogeneous substance that may be elastic and jelly-like (as gelatin) or more or less rigid. The gel bases can act in themselves as vehicles for active pharmaceutical agents suitable for topical administration in order to achieve a local therapeutic effect, topical administration in order to achieve transdermal or transmucosal penetration and a systemic therapeutic effect, or oral administration. Alternatively, the gel bases can be combined with other ingredients, e.g., diluents, opacifiers, penetrants, coupling agents, fragrances, coloring agents, humectants, moisturizers and the like to provide a total pharmaceutical composition. In addition, for purposes of oral administration, the gels can be incorporated into an oral dosage unit such as a conventional gelatin or other hard capsule, hollow tablet or caplet to provide sustained release from the gel of a pharmaceutical agent upon dissolution of the dosage unit in the gastric juices. Other topical, transdermal, transmucosal and oral dosage forms incorporating the gels of the present invention would be readily apparent to those skilled in the medical and pharmaceutical arts.

The gel bases for pharmaceutical compositions comprise (a) from about 0.5 to about 10.0% by weight CH₃(CH₂)₂₁(OCH₂CH₂)_nOH and from about 90 to about 99.5% of a glycol solvent, or (b) from about 2.5 to about 10.0% by weight CH₃(CH₂)_x(OCH₂CH₂)_nOH and from about 90 to about 97.5% of a glycol solvent, wherein n is an integer from 2 to 30 and x is an integer from 15 to 20, the bases being substantially free of any added gelling agents. Preferred concentration ranges (by weight) for the components of the gels are from about 0.5 to about 2.5% of ethoxylated behenyl (C₂₂) alcohol and from about 97.5 to about 99.5% of a glycol solvent, or from about 2.5 to about 5% of a C₁₆-C₂₁ethoxylated alcohol and from about 95 to about 97.5% of a glycol solvent. The preferred range for moles of ethoxylation for all the C₆-C₂₂alcohols is from 2 to 10. Preferred glycol solvents include propylene glycol and polyethylene glycol having an average molecular weight of about 400. Within the range of C₁₆-C₂₁ethoxylated alcohols, those alcohols with the highest molecular weight prior to ethoxylation (e.g., behenyl alcohol) and the lowest degree of ethoxylation (e.g., 2 to 5 moles) formed gels with glycols readily even in concentrations as low as 0.5-1.0%. Hence, the most preferred ethoxylated alcohol surfactants for use where low concentration of surfactant in the gel base is desired, for example to minimize irritation to the mucous membranes, are behenyl alcohol ethoxylates (2 to 5 moles of ethoxylation). In addition, these preferred surfactants are highly lipophilic and cause better adhesion of the gel to the phospholipid layers of the mucosa in the nasal and oral cavities. Pharmaceutical agents suitable for incorporation into the gel bases or into pharmaceutical compositions incorporating those gel bases include, but are not limited to, analgesics, decongestants, bronchodilators and other antiasthmatic agents, beta blockers, antihistamines, anesthetics, antifungals, antinauseants, antiemetics, antibacterial agents, antifungal agents, corticosteroids and anticonvulsants. The concentration of the active ingredient in the gel base is, of course, dependent on the identity of the active agent, the condition and patient being treated and the potency desired. For purely topical treatment, for example, treatment of a skin area, compositions with excellent release and penetration characteristics can be formed utilizing the gel bases of the present invention and antibacterials, antifungals, local anesthetics, corticosteroids and similar agents, particularly those which are highly soluble in glycol solvents, and which are suitable for use with anhydrous vehicles. Compositions intended for systemic administration via the mucosa, e.g., intranasally, buccally, or sublingually, can be prepared with the novel gel bases and suitable analgesics, decongestants, bronchodilators, antiasthmatics, antinauseants, anticonvulsants, and other agents where rapid blood level peaks and onset of action are desirable to quickly alleviate the symptoms of the disorder being treated. By virtue of the high glycol solvent content and low surfactant content in the novel gel vehicles, rapid percutaneous and transmucosal absorption are facilitated, and therapeutic blood levels of many agents in these categories can be quickly achieved. Moreover, even at these low concentrations, the selected surfactants augment percutaneous absorption of the active drug ingredient.

The gel bases may be prepared by any conventional method suitable for combining the ethoxylated alcohol surfactant component with the glycol solvent. By one preferred method, the glycol, for example propylene glycol or a polyethylene glycol having a molecular weight of about 200 to 800, is heated to about 80° C., and the surfactant is then stirred in with the mixture being immediately removed from heat. The mixture is allowed to cool to approximately 60°−65° C., at which point an active pharmaceutical ingredient may be stirred into the gel base until a homogeneous mixture, solution or suspension is achieved. In the case of certain active drug ingredients which, because of their solubility characteristics, form precipitates or grainy aggregates when added to the gel bases according to the foregoing method, a modified method for combining the active ingredient with the gel is utilized. The gel base is formulated as described above and allowed to cool to about 60°−65° C., at which point the active ingredient is added, but the gel is then reheated to 70°−80° C. until the active ingredient is well-dissolved in the gel. The gel is then allowed to cool slowly to avoid precipitation of the active ingredient. This method overcomes solubility problems and enables the formation of elegant gels in the case of most pharmaceutical agents soluble in glycols. When the gel bases are formulated with ethoxylated behenyl (C₂₂) alcohol, they have a remarkable capability for retaining substantial quantities of ethyl or isopropyl alcohol without losing their gel-like consistency. Indeed, gel compositions comprising about 50% by weight of a gel base consisting of ethoxylated behenyl alcohol and propylene or polyethylene glycol, and up to 50% alcohol by weight, have been successfully formulated. These gels with high alcohol content are of great utility in solubilizing active ingredients which are poorly soluble in glycol but highly soluble in alcohol. Moreover, the alcohol contained in the gels exerts a bactericidal and bacteriostatic effect on skin areas to which the gels are applied, and provides a cooling counter-balance to the glycol solvent which may sometimes create a warming sensation when applied to the skin. Gel compositions containing high concentrations of ethyl or isopropyl alcohol may not be suitable, however, for application to the mucosa. The gel bases provide unique advantages as vehicles or components of vehicles for pharmaceutical compositions. They comprise a nonionic surfactant-containing system which provides for stable solutions of a wide variety of many pharmaceutical agents with little reactivity of surfactant with drug. Moreover, the non-ionic character of the surfactant and the high glycol concentration aids in the penetration of the gel bases with even high molecular weight active ingredients through the skin and mucous membranes.

Gel pharmaceutical compositions including the gel bases and active pharmaceutical agents may be administered by a variety of methods, depending on the activity of the pharmaceutical agent or agents incorporated in the gel and the condition being treated. Such methods of administration include squeezing the drug-containing gel directly from a tube or other container onto an affected tissue area and spreading a thin film of the gel over that tissue area for topical treatment; inserting the tip of a tube or other suitable container into the nostrils and administering an effective dosage amount of the gel into the nasal passages for rapid dissolution and transmucosal absorption; similar direct application of the gel for transmucosal absorption on other mucous membrane areas, including buccal and sublingual administration; and oral administration of an effective dosage amount of the gel composition directly or within a soluble, orally ingestible outer shell, such as in capsules, tablets or caplets. Other suitable uses and routes of administration for the subject gel bases and pharmaceutical compositions including the same will be apparent to those skilled in the medical and pharmaceutical arts. Among the therapeutic advantages provided by the gel bases and gel compositions containing the same are more rapid and more complete percutaneous absorption of topically active pharmaceutical agents, such as corticosteroids, in comparison with conventional topically applied bases. It is believed that various advantageous features of the novel gel bases, including their high glycol content, their nonionic and anhydrous character, and the absence of any added or extrinsic gelling agents, promote more rapid absorption of topical agents through the outer skin layers and the mucosa. It has also been discovered that certain drugs conventionally utilized for systemic therapy only in the form of salts or esters, not free bases, can be effectively administered transmucosally in free form when incorporated into the gel bases.

Formulations in ointment can include hydrocarbons (e.g., petrolatum), mineral oils, lanolin, and polymers. The polymers can be polyvinyl alcohols (e.g., glycols or fatty alcohols), Carbopol and cellulose derivatives such as methyl cellulose in a combination to provide the correct characteristics of viscosity.

Other formulations meant to cover wounds and provide therapeutic effect contain foaming agents such as sodium laurate sulfate, delivered through a pressurized system that emit a liquid or semisolid dispersion in a gaseous medium. Other natural foaming agents can be used such as those derived from the soap bark tree, decyl glucoside, cocamidopropyl betaine, coco glucoside, and sodium cocoamphoacetate, as well as other well-known surfactants.

Another delivery method would be through modified release formulations and as defined by the United States Pharmacopeia, designed to provide timed-release of the POH or derivatives via a combination of excipients, via nanoparticles, or microencapsulation. The use of excipients in the proper combination to produce the slow release of the POH or derivatives over a 12-24 hr. period such as hydrogels, cellulose derivatives (e.g., hydroxypropyl methyl cellulose, carbomer, carboxypolymethylene), emulsifiers or polymeric emulsifiers, the use of thickening and gel forming agents, and other agents that can provide adequate drug dispersion and provide thermal stability.

Preparation of nanoparticles can be achieved by any individual skilled in the art that can combine excipients such as synthetic polymers (e.g., polyvinyl alcohol, poly-L-lactic acid, polyethylene glycol, and poly (lactic-co-glycolic acid), and natural polymers (e.g., alginate and chitosan) to fabricate nanoparticles. Additionally, micelles, and phospholipids can be used, as well as available compact lipid nanostructures, and the use of green nanoparticulate systems.

Specific compositions for POH and derivatives are presented below:

A preferred cream formulation includes POH or perillartine as an active ingredient (Active), and octadecanol, propanediol, hexadecanol, oleic acid, and mineral oil, as inactive ingredients. The cream formulation acts as a bactericide, fungicide, and disinfectant.

TABLE 20

Order
Reagent
%

1
Active
1.019

2
Propanediol
72.994

3
Hexadecanol
11.273

4
Oleic Acid
0.314

5
Mineral Oil
0.308

6
Octadecanol
14.091

TOTAL
100.00%

A preferred ointment formulation includes POH or perillartine as an active ingredient (Active), and bee's wax, mineral oil, and hexadecanol, as inactive ingredients. The ointment formulation is suitable to treat skin and nail fungal infections.

TABLE 21

Order
Reagent
%

1
Active
1.193

2
Mineral Oil
85.179

3
Hexadecanol
1.947

4
Bee's Wax
11.68

(Cera Alba)

TOTAL
100.00%

A preferred gel formulation includes POH or perillartine as an active ingredient (Active), and hydroxypropyl cellulose, tween 60 (polyoxyethylene sorbitan monostearate), DI water, carbopol 940 (polyacrylic acid), sodium bicarbonate, and isopropanol 70%, as inactive ingredients. The gel formulation is suitable to treat skin and nail fungal infections.

TABLE 22

Order
Reagent
%

1
Active
0.5-3.0

2
polyoxyethylene
3.635

sorbitan

monostearate

(Tween 60)

3
DI water
QS

4
polyacrylic acid
0.908

(Carbopol 940)

5
Sodium
0.454

bicarbonate

6
Isopropanol
3.572

70%

7
Hydroxypropyl
0.545

cellulose