(12) Patent Application Publication (10) Pub. No.: US 2013/0267471 A1 Ghatnekar Et Al
US 20130267,471 A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0267471 A1 Ghatnekar et al. (43) Pub. Date: Oct. 10, 2013
(54) FORMULATIONS AND METHODS OF USE Publication Classification FOR ALPHA CONNEXIN C-TERMINAL (ACT) PEPTIDES (51) Int. Cl. A638/17 (2006.01) (71) Applicant: FIRSTRING RESEARCH, INC., Mt. A647/48 (2006.01) Pleasant, SC (US) (52) U.S. Cl. CPC ...... A61K 38/1709 (2013.01); A61K 47/48246 (72) Inventors: Gautam Ghatnekar, Charleston, NC (2013.01) (US); Tuan Elstrom, Mt. Pleasant, SC USPC ...... 514/18.6 (US) (57) ABSTRACT This invention relates to a topical gel drug product prepara (21) Appl. No.: 13/815,723 tion containing a composition comprising an isolated polypeptide having a carboxy-terminal amino acid sequence of an alpha connexin (ACT peptide), peptide stabilizers, (22) Filed: Mar 15, 2013 excipients, buffering agents, and the like. A formulation and preparation steps are disclosed for the manufacturing of a Related U.S. Application Data stable, elegant, and pourable topical gel. The resulting formu lation possesses long term stability Suitable for aesthetic as (63) Continuation-in-part of application No. PCT/US2013/ well as therapeutic applications including the prevention of 028727, filed on Mar. 1, 2013. scaring and accelerated healing of wounds. Methods for treat (60) Provisional application No. 61/605,528, filed on Mar. ment of chronic wounds, including chronic ulcers, are also 1, 2012. provided. Patent Application Publication Oct. 10, 2013 Sheet 1 of 18 US 2013/0267471 A1
Figure 1
In a manufacturing vessel, add a major portion of purified water, EDTA, monobasic sodium phosphate, dibasic sodium nhrenhafa anri Mannitrl
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until Solution is formed. Mix
Homogenization
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In a separate beaker,
mix a portion of Clear gel (no "fish eyes") purified water with peptide until a clear Solution is obtained. Mix
Clear and homogenous gel Patent Application Publication Oct. 10, 2013 Sheet 2 of 18 US 2013/0267471 A1
Figure 2 Subject Self Assessment of Pain ITT Population (N=91)
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Figure 3 Subject Self Pain Assessment PP Population (N = 60)
:
Bose line Doy 3 week Week 2 week 3 Week 4 Week 5 week 6 week 7 week 8 week 9 Week 10 week 11 Week 12 Wisit
-- GRAN ENGE Soad ord of cott Ara a ston did of cott Patent Application Publication Oct. 10, 2013 Sheet 4 of 18 US 2013/0267471 A1
Figure 4 Kaplan-Meier Plot of Time to 100% Wound Closure ITT Population (N = 91) Product-Limit Survival Function Estimates
"...l... . . GRANEXINGEL plus SOC
D s O o n ------2us l ------> us s V
--- -o-o-m-me-ow O 5 10 5 Weeks No. of Subjects Event Censored Median Survival (90%CL) GRANEXINGEL plus SOC 45 62% (28) 38% (17) 6.00 (5.00 11.00) SOC 43 42% (18) 58% (25) 11.00 ( 9.00 18.29) Patent Application Publication Oct. 10, 2013 Sheet 5 of 18 US 2013/0267471 A1
Figure 5 Kaplan-Meier Plot of Time to 100% Wound Closure PP Population (N = 60) Product-limit Survival Function Estimates
1- - - -l r GRANEXINGEL plus soc l, ...... l------
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3. S ----- 0,4- l- -
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Logrank p=0. --r - orm O 5 10 S Weeks No. of Subjects Event Censored Median Survival (90%CL) GRANEXINGEL plus SOC 32 81% (26) 19% (6) 6,00 ( 4.00 10.86) SOC 28 54% (15) 46% (13) 14,64 (9.00 18.29) Patent Application Publication Oct. 10, 2013 Sheet 6 of 18 US 2013/0267471 A1
Figure 6 Kaplan-Meier Plot of Time to 50% Wound Closure ITT Population (N = 91)
Product-Limit Survival Function Estimates
SOC
Weeks No. of Subjects Event Censored Median Survival (90%CL) GRANEXINGEL plus SOC 45 80% (36) 20% (9) 2.00 ( 2.00 3.14) SOC 43 74% (32) 26% (11) 3.29 ( 3.00. 4,14) Patent Application Publication Oct. 10, 2013 Sheet 7 of 18 US 2013/0267471 A1
Figure 7 Kaplan-Meier Plot of Time to 50% Wound Closure PP Population (N = 60)
Product-Limit Survival Function Estimates 1 - - GRANExiN GEL plus Soc t . . . . SOC
0.8- De l O 0.6 w. C w A. - S 0.4-
V) t 0.2- --- Logrank p=0.2084 -r- 0- or m r r t O 2.5 5 7.5 10 12.5 Weeks No. of Subjects Event Censored Median Survival (90%CL) GRANEXENGEL plus SOC 32 94% (30) 6% (2) 2.00 ( 2.00 3.14) SOC 28 89% (25) 11% (3) 4.07 ( 3.00 5.00) Patent Application Publication Oct. 10, 2013 Sheet 8 of 18 US 2013/0267471 A1
Figure 8 Average Wound Closure at All Visits ITT Population (N = 91) 8
is
6
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HO-) GRANE, IN CEl so adoid of cott A a Stondard of Coe Patent Application Publication Oct. 10, 2013 Sheet 9 of 18 US 2013/0267471 A1
Figure 9 Average Wound Closure at All Visit PP Population (N = 60) O SS 90 65 80 15
65 60 SS
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CHHO CRAN IN CEL sodic of core is mi- is Sond d of core Patent Application Publication Oct. 10, 2013 Sheet 10 of 18 US 2013/0267471 A1
Figure 10 Longitudinal Response Profile of the Mean Percent Reduction of Wound Area (mm) from Baseline to Week 12 by Treatment Group - ITT Population (N=92)
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Yis it Patent Application Publication Oct. 10, 2013 Sheet 11 of 18 US 2013/0267471 A1
Figure 11 Longitudinal Response Profile of the Mean Percentage Reduction of Wound Area from Baseline to Week 12 by Treatment Group - mITT Population (N=77)
110 - -o-o-er GraNexN Gel + Stendord of Core re-e-r- standard of core
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isit Patent Application Publication Oct. 10, 2013 Sheet 12 of 18 US 2013/0267471 A1
Figue 12 Longitudinal Response Profile of the Mean Percentage Reduction of Wound Area (mm) from Baseline to Week 12 by Treatment Group - PP Population (N=68)
10 -árra-rar RANXINGE--Stodore of Cors 100 ---- Stonard of cre
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visit Patent Application Publication Oct. 10, 2013 Sheet 13 of 18 US 2013/0267471 A1
Figure 13 Kaplan-Meier Plot of Time to First 100% Wound Closure ITT Population (N = 92)
Product-Limit Survival Function Estimates
... - plus Soc SOC
0.8-
0. 5 r
4
0.2-
O- Logrank p=0.0006 O 2.5 5 7.5 10 12.5
Weeks ------.. ------No. of Subjects Event Censored Median Survival (90%CL) GRANEXINGEL plus SOC 42 67% (28) 33% (14) 6.00 ( 4.00 8.00) SOC 45 33% (15) 67% (30) 12.14 ( 10.14 NA) Patent Application Publication Oct. 10, 2013 Sheet 14 of 18 US 2013/0267471 A1
Figure 14 Kaplan-Meier Plot of Time to 100% Wound Closure - mTT Population (N=77)
Product-limit Survival Function Estimates
or t-norW - W - GRANEXINGEL plus SOC -----an - ...... SOC
0.8- umu -am--- and ------E ‘...... -: O i. U 9 - - . . . . . is a. ---...----- S 0,4- wasawwows s lTl 0.2 --
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O 25 5 7.5 10 12.5 Weeks No. of Subjects Ewert Censored Median Survival 90%C) GRAN EXINGEL plus SOC 39 72% (28) 28% (11) 6,00 ( 4.00 7.00) SOC 36 36% (13) 64% (23) NA ( 12.00 NA) Patent Application Publication Oct. 10, 2013 Sheet 15 of 18 US 2013/0267471 A1
Figure 15 Kaplan-Meier Plot of Time to First 100% Wound Closure PP Population (N = 68)
Product-Limit Survival Function Estimates
GRANEXINGEL plus SOC ...... SOC
0.8- >
O 0.6- C m A. 0.4-
s UM) 0.2m
0- Logank ps.0001 more 2.5 5. 7,5 10 12.5 Weeks No. of Subjects Event Censored Median Survival (90%CL) GRANEXINGEL plus SOC 35 80% (28) 20% (7) 6.00 ( 4.00 7,00) SOC 33 36% (12) 64% (21) NA ( 12.00 NA) Patent Application Publication Oct. 10, 2013 Sheet 16 of 18 US 2013/0267471 A1
Figure 16 Kaplan-Mcier Plot of Time to First 50% Wound Closure ITT Population (N = 92)
Product-Limit Survival Function Estimates
- GRANEXINGEL plus Soc
O 2.5 5 7.5 10 12.5 Weeks No. of Subjects Event Censored Median Survival (90%CL) GRAN EXINGEL plus SOC 42 81% (34) 19% (8) 2.857 ( 2.143 3,000) SOC 45 56% (25) 44% (20) 6.857 5,000 9.143) Patent Application Publication Oct. 10, 2013 Sheet 17 of 18 US 2013/0267471 A1
Figure 17 Kaplan-Meier Plot of Time to 50% Wound Closure - mITT Population (N=77)
Product-Limit Survival Function Estimates -- ...... GRANEXINGEL plus SOC ------+...... , SOC Wom ...... ,
>
v w w - - - - O - . a ...... , , , , al i. 2 + :-- l- ...... ---...... --- V |-
wer t 2.5 5. 7.5 10 2.5 Weeks No. of Subjects Event Censored Median Survival (90%CL) GRAN EXINGEL plus SOC 39 85% (33) 15% (6) 2.857 ( 2.000 3.000) SOC 36 64% (23) 36% (13) 8.000 (5.1439,857) Patent Application Publication Oct. 10, 2013 Sheet 18 of 18 US 2013/0267471 A1
Figure 18 Kaplan-Mcier Plot of Time to First 50% Wound Closure PP Population (N = 68)
Product-Limit Survival Function Estimates
------, ------GRANEXINGEL plus SOC
0.8-
0. 6 ---
0,4- l- --- m --- . . . . . -----
0.2m -
------Logan K p.0001 O 2.5 5 7.5 10 12.5 Weeks No. of Subjects Ewent Censored Median Survival (90%CL) GRAN EXINGEL plus SOC 35 91% (32) 9% (3) 2.857 ( 2.000 3.000) SOC 33 67% (22) 33% (11) 8,000 (5.1439,857) US 2013/0267471 A1 Oct. 10, 2013
FORMULATIONS AND METHODS OF USE cause for morbidity and can severely impair quality of life, FOR ALPHA CONNEXIN C-TERMINAL (ACT) engender high treatment costs, and serve as the most impor PEPTIDES tant risk factor for lower-extremity amputation. 0007 No single available product or procedure is adequate CROSS REFERENCE TO RELATED for the treatment of all subjects with chronic ulcers. Treat APPLICATIONS ment selection is determined by patient tolerance, patient 0001. This application is a continuation-in-part of PCT medical status, cost, availability, and physician preference. Application NO. PCT/US2013/028727, filed Mar. 1, 2013, Compression wraps are the Standard of Care (SoC) and are which claims priority to U.S. Provisional Patent Application needed to assist venous return and to address the underlying No. 61/605,528, filed Mar. 1, 2012, which is incorporated pathophysiology of venous disease and venous ulceration. herein by reference in its entirety. Compression bandages and stockings heal more ulcers com pared with no compression, but it has not been ascertained if DESCRIPTION OF THE TEXT FILE SUBMITTED intermittent pneumatic compression is beneficial compared ELECTRONICALLY with compression bandages or stockings. Occlusive (hydro colloid) dressings are no more effective than simple low 0002 The contents of the text file submitted electronically adherent dressings in people treated with compression. herewith are incorporated herein by reference in their 0008. Despite various advances in wound treatment, the entirety: A computer readable format copy of the Sequence need exists for additional effective VLU and DFU treatments Listing (filename: FIRS006 01 US SeqList ST25.txt, date that combine advanced wound care therapeutics with SoC recorded: Mar. 15, 2013, file size 32 kilobytes). compression therapy for faster healing of ulcers. Rapid heal TECHNICAL FIELD ing would result in fewer hospitalizations, decreased infec tion, less use of antibiotics, improved mobility, less pain, and 0003. The present invention relates to compositions for fewer surgical procedures for subjects with VLUs or DFUs. wound treatment comprising alpha connexin polypeptides and Stabilizing agents. Topical formulations containing alpha SUMMARY OF THE INVENTION connexin polypeptides and hydroxyethylcellulose are par ticularly stable. These compositions may be used to treat a 0009. The present invention is directed in part to a topical variety of wounds including acute Surgical wounds and gel drug product preparation containing a composition com chronic ulcers. Methods for treatment of chronic wounds are prising an isolated alpha connexin polypeptide. The topical also provided. formulation may further comprise hydroxyethylcellulose gel. which stabilizes the alpha connexin polypeptide during Stor BACKGROUND age. In certain embodiments the alpha connexin polypeptide 0004 While human tissues damaged by mechanical comprises a carboxy terminal amino acid sequence of an wounding, disease processes and other causes are capable of alpha connexin (hereinafter ACT'). The alpha connexin healing, complex tissue structure and function is rarely, if ever polypeptides of the present invention may comprise or consist wholly restored. Instead, recovery of nearly all tissues from of the carboxy-terminal most 4 to 30 contiguous amino acids injury inhumans and other higher vertebrates is dominated by of an alpha connexin protein or conservative variant thereof, the formation of scartissue. The most familiar example of this wherein said at least one alpha connexin polypeptide is linked is the discolored and fibrotic scars that linger following the at its amino terminus to a cellular internalization transporter. healing of a skin cut or graze. Less well appreciated is that 0010. It is an object of the present invention to prepare a formation of glial scar tissue following injury to the brain or formulation of a stable, elegant, and pourable topical gel spinal chord is one of the main obstacles to restoration of carrier that contains ACT for aesthetic as well as therapeutic neural function following damage to the central nervous sys applications including the prevention of scaring and acceler tem (Silver and MillerJH, 2004). There is currently no means ated healing of wounds of a Subject. The drug product may be of treating or preventing Such scarring and promoting the administered to treat acute Surgical wounds to reduce Scarring regeneration of complex tissue structure and function follow or be applied chronically to difficult-to-heal wounds includ ing injury. ing, but not limited to, Venous leg ulcers, diabetic foot ulcers, 0005. Once such treatments are discovered, there is also a pressure ulcers, and the like. The drug product possesses need to provide formulations that stabilize the active pharma physicochemical, biochemical, and rheological properties ceutical ingredients (API) which they contain. While there that enable its ability to provide a therapeutic and effective are a myriad of potential stabilizing agents, not all of them amount of ACT peptide when applied to all type of wounds. work equally well in stabilizing specific APIs. 0011. In one aspect of the invention, one or more stabilizer 0006 Venous ulcers are chronic wounds associated with is selected among many, and prepared with ACT peptide to long-standing venous hypertension of the lower extremity. stabilize the isolated polypeptide. Preferably the stabilizers The number of individuals affected by these ulcers in the are non-irritating, non-staining, and non-immunogenic. The United States is over 600,000. These indolent wounds are a stabilizers enable the long term (i.e., for 3 months, for 6 major cause of morbidity and are a major financial burden. months, for 9 months, for 12 months, for 18 months, or for 24 Diabetes affects an estimated 25.8 million people (8% of the months) storage of the drug product under a variety of tem US population) according to the Centers for Disease Control perature conditions (i.e., at about 5°C., at about 10° C., at and Prevention. People with diabetes have 12-25% chance of about 15°C., at about 20°C., at about 25°C., at about 30°C., developing foot ulcer in their lifetime. Diabetic Foot Ulcers at about 35° C., or at about 40°C.) and under a range of (DFUs) refer to wounds below the ankle of a diabetic subject relative humidities (i.e., at 0% relative humidity, at 10% rela which develops due to neuropathy (sensory, motor, or auto tive humidity, at 20% relative humidity, at 30% relative nomic deficits), ischemia or both. Foot ulcers area substantial humidity, at 40% relative humidity, at 50% relative humidity, US 2013/0267471 A1 Oct. 10, 2013
at 60% relative humidity, at 70% relative humidity, at 80% concentration of about 1.25% (w/w). In one embodiment, the relative humidity, at 90% relative humidity, or at 100% rela at least one isolated polypeptide comprises the carboxy ter tive humidity). minal-most 4 to 30 contiguous amino acids of an alpha Con 0012. In another aspect of the invention, one or more nexin, or a conservative variant thereof. In another embodi excipients are selected from the group consisting of water ment, the alpha connexin polypeptide is connexin 37. soluble porogens, polymers, ionic compounds, non-ionic connexin 40, connexin 43, or connexin 45. In one embodi compounds, and solvents. In a further embodiment, excipi ment, the alpha connexin is linked at its amino terminus to a ents act as one or more of carriers, stabilizers, and gelling cellular internalization transporter Such as, for example an agents. In one embodiment, at least one excipient is prepared Antennapedia sequence, TAT. HIV-Tat, Penetratin, Antp-3A with ACT. Preferably the excipients are non-irritating, non (Antip mutant), Buforin II, Transportan, MAP (model amphi staining, and non-immunogenic. In one embodiment the for pathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, mulation comprises at least one polymer. In a further embodi Pep-7, FIN-1, BGSC (Bis-Guanidinium-Spermidine-Choles ment, the formulation comprises a derivative of cellulose. In terol, and BGTC (BisCuanidinium-Tren-Cholesterol). In one a yet further embodiment, the derivative of cellulose is embodiment, the cellular internalization transporter is an hydroxyethylcellulose. antennapedia sequence. In a further embodiment, the formu 0013. In yet another aspect of the invention, an optimal pH lation comprises a polypeptide comprising the amino acid range for the topical gel is about pH 5 to about pH 7, and is sequence of SEQID NO: 9. maintained by the addition of one or more buffering agents. 0019. In one embodiment, the formulation for use in treat Preferably the buffering agents are non-irritating, non-stain ment of a chronic wound comprises at least one alpha con ing, and non-immunogenic. nexin polypeptide and hydroxyethylcellulose gel. In a further 0014. In yet another aspect of the invention, at least one embodiment, the hydroxycellulose gel is presentata concen excipient is a gelling agent. In one embodiment, a gelling traiton of about 2%, about 1.75%, about 1.5%, about 1.25%, agent is a carrier of ACT. In some embodiments, the gelling about 1.0%, or about 0.75%. In one embodiment, the agent also acts as a stabilizer. In some embodiments, the hydroxycellulose gel is present at a concentration of about gelling agent may be combined with at least one of the fol 1.25% (w/w). lowing: one or more buffering agent, and one or more addi 0020. In one aspect, the chronic wound is an ulcer. In a tional excipient. In one embodiment, the gelling agent is further embodiment, the chronic wound is a lower extremity hydroxyethylcellulose. ulcer. In another embodiment, the chronic wound is selected 0015. In some embodiments, the present invention from the group consisting of venous leg ulcers, diabetic foot includes a method of wound treatment comprising adminis ulcers, and pressure ulcers. In one embodiment, a method for tering to a Subject in need thereof a topical formulation com treating a chronic wound in a Subject is provided, wherein the prising at least one alpha connexin polypeptide and hydroxy method comprises administering to the Subject a topical for ethylcellulose gel. In an exemplary embodiment, the steps are mulation of at least one alpha connexin polypeptide, wherein disclosed for manufacturing of the drug product. the formulation is administered in a dosing regimen at day 0, 0016. The present invention also includes methods of day 3, week 1, week 2, week 3, week 4 week 5, week 6, week treating wounds and ulcers in a patient in need thereof, par 7, week 8, week 9, week 10, week 11, and week 12, wherein ticularly wounds and ulcers that are chronic in nature. The the symptoms of the chronic wound are reduced. In a further present inventors have found that the peptides and formula embodiment, the formulation does not induce excessive lev tions of the present invention provide faster healing of ulcers els of side effects. In another embodiment, the chronic wound when combined with compression therapy than is achieved is improved in the absence of clinically significant abnormali by compression therapy alone. Accordingly, the present ties. In another embodiment, the method reduces the time to invention includes methods of treating a chronic wound in a 100% wound closure, as compared to the time to 100% Subject, comprising administering to the Subject a topical wound closure when the standard of care treatment is used. In formulation comprising at least one alpha connexin polypep other embodiments, the method reduces the time to 100% tide in addition to standard of care compression therapy, wound closure when administered in conjunction with stan wherein the chronic wound is healed at a faster rate and/or dard of care, as compared to treatment with either standard of increased frequency than achieved with standard of care com care alone. pression therapy alone. 0021 Standard of care treatments are known to those of 0017. In one aspect a method of treating a chronic wound skill in the art and include, for example, cleaning the wound, in a subject comprising administering to the Subject a topical applying dressing to the wound, and applying a pressure formulation comprising at least one alpha connexin polypep bandage to the wound. In one embodiment, the Subject treated tide is provided. In a further embodiment, the formulation is with the methods and formulations provided herein has a administered daily or weekly or a combination thereof. In higher percent wound closure as compared to a subject another embodiment, the formulation is administered accord receiving standard of care therapy, at 12 weeks, 11 weeks, 10 ing to any dosing regiment that is effective in the treatment of weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 the chronic wound. In a further embodiment, the formulation weeks, 3 weeks, or 2 weeks. In one embodiment, the percent is administered in a dosing regimen at day 0, day 3, week 1, wound closure at 12 weeks is higher in subjects treated with week 2, week 3, week 4, week 5, week 6, week 7, week 8, the methods and formulations disclosed herein, as compared week 9, week 10, week 11, and week 12. to the percent wound closure at 12 weeks in Subjects receiving 0018. In one embodiment a method of treating a chronic standard of care treatment. In one embodiment, the method wound in a Subject comprising administering to the Subject a reduces the time to 50% wound closure, as compared to the topical formulation comprising at least one alpha connexin time to 50% wound closure when the standard of care treat polypeptide and hydroxyethylcellulose is provided. In a fur ment is used. In another embodiment, the percent wound ther embodiment, the hydroxyethylcellulose is present at a closure at 4 weeks is higher in subjects treated with the US 2013/0267471 A1 Oct. 10, 2013
methods and formulations disclosed herein, as compared to 0034 FIG. 10 shows the Longitudinal Response Profile of the percent wound closure at 4 weeks in subjects treated with the Mean Percent Reduction of Wound Area (mm) from standard of care treatment. Baseline to Week 12 by Treatment Group—ITT Population 0022. In another embodiment, the method results in a (N=92) reduction in pain levels in the subject. In a further embodi 0035 FIG. 11 Longitudinal Response Profile of the Mean ment, the pain level is determined through patient self-assess Percentage Reduction of Wound Area from Baseline to Week ment. In one embodiment, the method increases the average 12 by Treatment Group mITT Population (N=77) percent of wound closure at 12 weeks, 11 weeks, 10 weeks, 9 0036 FIG. 12 shows the Longitudinal Response Profile of weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 the Mean Percentage Reduction of Wound Area (mm) from weeks, or 2 weeks as compared to standard of care treatment. Baseline to Week 12 by Treatment Group PP Population In one embodiment, the method increases the average percent (N=68) of wound closure at 12 weeks. In one embodiment, the 0037 FIG. 13 shows the Kaplan-Meier Plot of Time to method decreases the wound area as compared to the wound First 100% Wound Closure ITT Population (N=92) area in Subjects that are treated with standard of care therapy. 0038 FIG. 14 shows the Kaplan-Meier Plot of Time to In one embodiment, the method does not induce the produc First 100% Wound Closure ITT Population (N=92) tion of anti-alpha connexin polypeptide antibodies in the 0039 FIG. 15 shows Kaplan-Meier Plot of Time to First Subject. 100% Wound Closure PP Population (N=68) 0023. In another embodiment, the method increases the 0040 FIG. 16 shows the Kaplan-Meier Plot of Time to incidence or frequency of 100% complete wound closure First 50% Wound Closure ITT Population (N=92) compared to standard of care treatments for wound healing. 004.1 FIG. 17 shows the Kaplan-Meier Plot of Time to In another embodiment, the method is used to treat an ulcer 50% Wound Closure mITT Population (N=77) lacking Sufficient wound size reduction within one, four, 12. 0042 FIG. 18 shows the Kaplan-Meier Plot of Time to 24, 36, or more weeks of standard of care. In one embodi First 50% Wound Closure PP Population (N=68) ment, the method is used to treat an ulcer with less than 50% wound closure within one four, 12, 24, 36, or more weeks of DETAILED DESCRIPTION standard of care. In one embodiment, the method is used to 0043. Before the present compositions and methods are treat an ulcer lacking Sufficient wound size reduction within described, it is to be understood that this invention is not one, four, 12, 24, 36, or more weeks of standard of care. In one limited to the particular processes, compositions, or method embodiment, the method is used to treat an ulcer lacking ologies described, as these may vary. The terminology used in sufficient wound size reduction within one, four, 12, 24, 36, or the description is for the purpose of describing the particular more weeks of Standard of care. In one embodiment, the versions or embodiments only, and it is not intended to limit method is used to treat a patient with an ulcer possessing the scope of the present invention which will be limited only wound area and duration characteristics of a chronic wound. by the appended claims. Unless defined otherwise, all tech In one embodiment, the method is used to treat a patient with nical and Scientific terms used herein have the same meanings an ulcer possessing a wound with a non-healing woundtra as commonly understood by one of ordinary skill in the art. jectory. In one embodiment, the method is used to treat an All publications mentioned herein are incorporated by refer ulcer possessing wound area and time duration characteristics ence in their entirety. Nothing herein is to be construed as an of a chronic wound. admission that the invention is not entitled to antedate Such disclosure by virtue of prior invention. DESCRIPTION OF DRAWINGS 0044. It must also be noted that as used herein and in the 0024 For a fuller understanding of the nature and advan appended claims, the singular forms “a”, “an', and “the tages of the present invention, reference should be had to the include plural reference unless the context clearly dictates following detailed description taken in connection with the otherwise. Thus, for example, reference to an “element' is a accompanying drawings, in which: reference to one or more elements and equivalents thereof 0025 FIG. 1 illustrates the manufacturing process flow known to those skilled in the art, and so forth. chart. 0045. The term “topical refers to administration of inven 0026 FIG. 2 shows the subject self assessment of pain in tive drug product at, or immediately beneath, the point of the intent-to-treat population in the Diabetic Foot Ulcer application. As used herein “topical application” refers to application onto one or more Surfaces(s) including keratinous (DFU) study. tissue, i.e., “topically applying.” Topical application or “topi 0027 FIG. 3 shows the subject self assessment of pain in cally applying may involve direct application to the area of the per-protocol (PP) population in the DFU study. the desired Substrate. The topical preparation and/or compo 0028 FIG. 4 shows the time to 100% wound closure in the sition may be applied by pouring, dropping, or spraying, if a intent-to-treat (ITT) population in the DFU study. liquid; rubbing on, if an ointment, lotion, cream, gel, or the 0029 FIG. 5 shows the time to 100% wound closure in the like; dusting, if a powder, spraying, if a liquid or aerosol PP population in the DFU study. composition; or by any other appropriate means. 0030 FIG. 6 shows the time to 50% wound closure in the 0046. As used herein, the phrase "gelling agents’ refers to ITT population in the DFU study. agents that make a topical preparation denser or more viscous 0031 FIG. 7 shows the time to 50% wound closure in the in consistency. Gelling agents may be water-based or oil PP population in the DFU study. based. Gelling agents may also be referred to as “thickening 0032 FIG. 8 shows the average wound closure at each agents.” “Excipients, as used herein, are inactive ingredients visit in the ITT population in the DFU study. that serve as carriers of the active pharmaceutical ingredient 0033 FIG. 9 shows the average wound closure at each (API). Excipients may also serve as gelling or thickening visit in the PP population in the DFU study. agents, or stabilizers. US 2013/0267471 A1 Oct. 10, 2013
0047. A “subject' or a “mammal’ includes a human or a the progression of the condition, disorder or disease; amelio non-human mammal. Non-human mammals include, but not ration of the condition, disorder or disease state; and remis limited to, livestock and pets, such as ovine, bovine, porcine, sion (whether partial or total), whether detectable or unde canine, feline and murine mammals. In one embodiment, the tectable, or enhancement or improvement of the condition, Subject or mammal is human. disorder or disease. Treatment includes eliciting a clinically 0048 “Administering when used in conjunction with a significant response without excessive levels of side effects. therapeutic means to administer a therapeutic directly into or In some embodiments, treatment may include preventing or onto a target tissue or to administer a therapeutic to a patient reducing Scarring and/or promoting the regeneration of com whereby the therapeutic positively impacts the tissue to plex tissue structure and function following injury. which it is targeted. Thus, as used herein, the term “adminis 0054 As used herein, the terms “peptide.” “polypeptide.” tering, when used in conjunction with the drug product, can or “protein’ are used interchangeably, and refer to a com include, but is not limited to, providing the drug product and pound comprised of amino acid residues covalently linked by the ACT peptide into or onto the target tissue. Administer peptide bonds. A protein or peptide must contain at least two ing a composition may be accomplished by injection, topical amino acids, and no limitation is placed on the maximum administration, or by any method in combination with other number of amino acids that can comprise the sequence of a known techniques. protein or peptide. Polypeptides include any peptide or pro 0049. Unless otherwise indicated, the term “skin' means tein comprising two or more amino acids joined to each other that outer integument or covering of the body, consisting of by peptide bonds. As used herein, the term refers to both short the dermis and the epidermis and resting upon Subcutaneous chains, which also commonly are referred to in the art as tissue. peptides, oligopeptides and oligomers, for example, and to 0050. As used herein, the term “therapeutic' means an longer chains, which generally are referred to in the art as agent utilized to treat, combat, ameliorate, prevent or improve proteins, of which there are many types. “Polypeptides’ an unwanted condition or disease of a patient. In part, include, for example, biologically active fragments, Substan embodiments of the present invention are directed to the tially homologous polypeptides, oligopeptides, homodimers, treatment of Scarring and delayed wound healing. heterodimers, variants of polypeptides, modified polypep 0051. As used herein, the phrase “effective amount’ or tides, derivatives, analogs and fusion proteins, among others. “therapeutically effective amount” refers to a nontoxic but The polypeptides include natural peptides, recombinant pep sufficient amount of the ACT polypeptides used in the prac tides, synthetic peptides or a combination thereof. A peptide tice of the invention that is effective to achieve the desired that is not cyclic will have an N-terminal and a C-terminal. effect, i.e., to treat acute and chronic wounds. The activity The N-terminal will have an amino group, which may be free contemplated by the present methods includes both medical (i.e., as a NH2 group) or appropriately protected (for therapeutic and/or prophylactic treatment, as appropriate, example, with a BOC or aFmoc group). The C-terminal will including, for example, a reduction and/or alleviation of the have a carboxylic group, which may be free (i.e., as a COOH signs, symptoms, or causes of a disease or disorder, or any group) or appropriately protected (for example, as a benzyl or other desired alteration of a biological system. The specific a methyl ester). A cyclic peptide does not have free N- or dose of a compound administered according to this invention C-terminal, since the ends are covalently bonded through an to obtain therapeutic and/or prophylactic effects will, of amide bond to form the cyclic structure. Amino acids may be course, be determined by the particular circumstances Sur represented by their full names (for example, leucine), 3-let rounding the case, including, for example, the specific ter abbreviations (for example, Leu) and 1-letter abbrevia polypeptide administered and the condition being treated. A tions (for example, L). The structure of amino acids and their therapeutically effective amount of compound of this inven abbreviations can be found in the chemical literature, such as tion is typically an amount Such that when it is administered in Stryer, “Biochemistry”, 3rd Ed., W. H. Freeman and Co., in a physiologically tolerable excipient composition, it is New York, 1988. sufficient to achieve an effective intracellular concentration 0055 Excipients for use in topical gels are well-known in and local concentration in the tissue. the art and examples may be found in the Handbook of Phar 0052 Generally speaking, the term “tissue' refers to any maceutical Excipients (Rowe, R. C. etal, APhA Publications: aggregation of similarly specialized cells which are united in 5" ed., 2005). Exemplary excipients may include waxes, vari the performance of a particular function. As used herein, ous Sugars and types of starch, polymers, gels, emollients, “tissue’, unless otherwise indicated, refers to tissue which thickening agents, rheology modifiers, humectants, glycerol, includes elastin as part of its necessary structure and/or func organic basic compounds, cellulose derivatives, gelatin, Veg tion. etable oils, polyethylene glycols and solvents. Examples of 0053. The terms “treat,” “treated,” or “treating as used rheology modifiers include Carbopol, hydroxypropyl cellu herein refers to both therapeutic treatment and prophylactic lose, C-2s alkyl dimethicone, C-2s alkyl methicone, or preventative measures, wherein the object is to prevent, polyphenylsisquioxane, trimethylsiloxysilicate, crosspoly ameliorate the effects of or slow down (lessen) an undesired mers of cyclopentasiloxane and dimethicone/vinyltrimethyl physiological condition, disorder or disease, or to obtain ben siloxysilicate, fumed silica (e.g. Cab-O-Sil M5P), and mix eficial or desired clinical results. For the purposes of this tures thereof. Examples of emollients include glycerine, invention, beneficial or desired clinical results include, but pentylene glycol, Sodium pyrrolidone carboxylic acid, lano are not limited to, alleviation of symptoms; diminishment of lin, saccharide isomerate, Stearoxy dimethicone, Stearyl the extent of the condition, disorder or disease; reducing the dimethicone, and mixtures thereof. Emollients may be useful severity of a symptom of a condition, disease or disorder; to prevent stratum corneum dehydration occurring due to the reducing the frequency of a symptom of a condition, disease use of anhydrous solvents in the formulation. Examples of or disorder, stabilization (i.e., not worsening) of the state of organic bases include 2-amino-2-methyl propanol, niacina the condition, disorder or disease; delay in onset or slowing of mide, methanolamines, triethanolamines, Trisamino, AMP US 2013/0267471 A1 Oct. 10, 2013
95, AmP-Ultra PC 2000, triisopropanolamine, diisopropano over a wide area, and may serve to maintain the alpha con lamine, Neutrol TE, Ethomeen, and mixtures thereof. The nexin polypeptides on the affected area of the skin. organic base may render the pH of the medicament basic or 0062 Other excipients may include various ionic or non neutral. ionic compounds to maintain stability of the formulation, 0056. Other exemplary excipients include water-soluble thereby protecting from the de-emulsification, settling, porogens. A water-soluble porogen is an additive that may agglomeration or degradation of the formulation constituents facilitate water uptake and diffusion into the gel. Any suitable that may reduce its therapeutic or aesthetic value. porogen may be used, but in Some embodiments, the porogen 0063 Examples of ionic compounds may include salts may include sodium chloride, potassium chloride. Sucrose, Such as Sodium chloride, potassium chloride; cationic, glucose, lactose, Sorbitol, Xylitol, polyethylene glycol, poly anionic or Zwitterionic Surfactants such as sodium dodecyl vinylpyrrollidone, polyvinyl alcohol or mixtures thereof. sulfate (SDS), perfluorooctanoate (PFOA), perfluorooctane 0057 Polymers may also act as excipients in topical gels. sulfonate (PFOS), ammonium lauryl sulfate (ALS), sodium Exemplary polymers include hydrophilic polyurethanes, lauryl ether sulfate (SLES), alkyl benzene sulfonate, cetyl hydrophilic polyacrylates, co-polymers of carboxymethyl trimethylammonium bromide (CTAB), cetylpyridinium cellulose and acrylic acid, N-Vinylpyrrolidone, poly(hydroxy chloride (CPC), polyethoxylated tallow amine (POEA), ben acids), polyanhydrides, polyorthoesters, polyamides, poly Zalkonium chloride (BAC), benzethonium chloride, dodecyl carbonates, polyalkylenes (e.g., polyethylene and polypropy betaine, cocamidopropyl betaine and cocoamphoglycinate. lene), polyalkylene glycols (e.g., poly(ethylene glycol)), 0064. Examples of non-ionic compounds that may act as polyalkylene oxides (e.g., polyethylene oxide), polyalkylene excipients include non-ionic Surfactants such as Pluronic, terephthalates (e.g., polyethylene terephthalate), polyvinyl Tween, AMP, and Brij family of surfactants; and surfactants alcohols, polyvinyl ethers, polylvinyl esters, polyvinyl derived from biological sources, e.g., natural or semi-syn halides (e.g., poly(Vinyl chloride)), polyvinylpyrrolidone, thetic Surfactants, such as oleic acid, Sorbitan trioleate, Sorbi polysiloxanes, poly(vinyl acetates), polystyrenes, polyure tan monooleate, lecithin, cocamide MEA, cocamide DEA thane copolymers, cellulose, derivatized celluloses (e.g., and cocamidopropyl betaine. Surfactants (both ionic and non hydroxyethyl cellulose, hydroxypropyl cellulose, hydrox ionic) may reduce the interfacial Surface energy and may ypropyl methyl cellulose, methylcellulose, ethylcellulose, facilitate spreading of the topical formulation over a wider carboxymethyl cellulose, or cellulose acetate), alginates, aca. poly(acrylic acid), poly(acrylic acid) derivatives, acrylic acid 0065. In some embodiments of the invention, solvent copolymers, methacrylic acid, methacrylic acid derivatives, excipients may be used as a carrier vehicle for the alpha methacrylic acid copolymers, poly(butyric acid), poly(va connexin polypeptides and other excipients. The polymer leric acid), poly(lactide-co-caprolactone), copolymers chains may interact with the solvent and undergo Swelling to thereof and blends thereof. form a network that may impart visco-elastic properties to the 0058. In some embodiments of the invention, the poly topical formulation. In some embodiments of the topical for mers may be superabsorbent polymers (SAPs). A polymer is mulation, the solvent may evaporate upon application, leav considered superabsorbent, as defined per IUPAC, as a poly ing a residual film of the polymer along with the entrapped mer that can absorb and retain extremely large amounts of alpha connexin polypeptides. water relative to its own mass. SAPs may absorb water up to 0.066 Exemplary solvent excipients that may be useful in 500 times their own weight and may swell up to 1000-times hydrophilic formulations may include dimethyl isosorbide, their original volume. Particular SAPs of interest include propylene glycol, glycerol, isopropanol, ethanol, benzyl alco sodium polyacrylate, the polyurethane Tecophilic TG-2000, hol, ethylene glycol, polyethylene glycol, ethoxydiglycol or and polymers prepared by the use of polyacrylamide copoly mixtures thereof. Exemplary solvent excipients that may be mer, ethylene maleic anhydride copolymer, cross-linked car useful in hydrophobic formulations may include capric/ca boxy-methyl-cellulose, polyvinyl alcohol copolymers, poly prylic triglycerides, isopropyl myristate, mineral oil, isod odecane, isodecyl neopentanoate, butylene glycol, pentylene vinylpyrrolindone and cross-linked polyethylene oxide. glycol, hexylene glycol, methoxypolyethyleneglycol, cyclo 0059. In some embodiments of the invention, polymers pentasiloxane, cyclotetrasiloxane, dimethicone, caprylyl that are relatively hydrophobic may be used. Any suitable methicone or mixtures thereof. hydrophobic polymer may be used. However, exemplary 0067. In addition to the alpha connexin polypeptides and polymers that are relatively hydrophobic include aromatic excipients, the topical formulation may also include at least polyurethanes, silicone rubber, polysiloxanes, polycaprolac one additional therapeutic agent Such as antimicrobial agents, tone, polycarbonate, polyvinylchloride, polyethylene, poly anti-acne agents, anti-inflammatory agents, analgesic agents, L-lactide, poly-DL-glycolide, polyetheretherketone (PEEK), anesthetic agents, antihistamine agents, antiseptic agents, polyamide, polyimide and polyvinyl acetate. In addition, a immunosuppressants, antihemorrhagic agents, vasodilators, hydrophobic gel-base and/or rheology modifier may be used. wound healing agents, anti-biofilm agents and mixtures 0060. In some embodiments of the invention, the poly thereof. mers may act as thickening agents in the medicaments. Spe 0068 Examples of antimicrobial agents include penicil cifically, the polymeric portion of the gel may act as a visco lins and related drugs, carbapenems, cephalosporins and elastic Substance and may retain the gel at the site of related drugs, erythromycin, aminoglycosides, bacitracin, application, along with the alpha connexin polypeptides dis gramicidin, mupirocin, chloramphenicol, thiamphenicol, persed therein. fusidate sodium, lincomycin, clindamycin, macrollides, novo 0061. In some other embodiments, a gel that includes a biocin, polymyxins, rifamycins, spectinomysin, tetracy polymer may have spreadability such that it forms a thin film clines, Vanomycin, teicoplanin, streptogramins, anti-folate when applied on the skin surface. This film may enable the agents including Sulfonamides, trimethoprim and its combi application of the contained alpha connexin polypeptides nations and pyrimethamine, synthetic antibacterials includ US 2013/0267471 A1 Oct. 10, 2013
ing nitrofurans, methenamine mandelate and methenamine others known in the art, predonisolone, dexamethasone, fluo hippurate, nitroimidazoles, quinolones, fluoroquinolones, cinolone acetonide, hydrocortisone acetate, predonisolone isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acetate, methylpredonisolone, dexamethasone acetate, acid (PAS), cycloserine, capreomycin, ethionamide, pro betamethasone, betamethasone Valerate, flumetaSone, fluo thionamide, thiacetazone, viomycin, eveninomycin, glyco rometholone, beclomethasone diproprionate, fluocinonide, peptide, glyclyclycline, ketolides, oxazolidinone; imipenen, topical corticosteroids, and may be one of the lower potency amikacin, netilmicin, fosfomycin, gentamycin, ceftriaxone, corticosteroids Such as hydrocortisone, hydrocortisone-21 Ziracin, Linezolid, Synercid, Aztreonam, and Metronidazole, monoesters (e.g., hydrocortisone-21-acetate, hydrocorti Epiroprim, Sanfetrinem Sodium, Biapenem, Dynemicin, Cef Sone-21-butyrate, hydrocortisone-21-propionate, hydrocorti luprenam, Cefoselis, Sanfetrinem celexetil, Ce?pirome, Sone-21-valerate, etc.), hydrocortisone-17,21-diesters (e.g., Mersacidin, Rifalazil, Kosan, Lenapenem, Veneprim, hydrocortisone-17,21-diacetate, hydrocortisone-17-acetate Sulopenem, ritipenam acoxyl, Cyclothialidine, micacocidin 21-butyrate, hydrocortisone-17,21-dibutyrate, etc.), alclom A, carumonam, CefoZopran and Cefetamet pivoXil. etaSone, dexamethasone, flumethasone, prednisolone, or 0069. Examples of topical anti-acne agents include ada methylprednisolone, or may be a higher potency corticoster palene, azelaic acid, benzoyl peroxide, clindamycin and clin oid Such as clobetasol propionate, betamethasone benzoate, damycin phosphate, doxycycline, erythromycin, keratolytics betamethasone dipropionate, diflorasone diacetate, fluocino Such as Salicylic acid and retinoic acid (Retin-A), norgesti nide, mometasone furoate, triamcinolone acetonide. mate, organic peroxides, retinoids such as isotretinoin and 0073. Examples of analgesic agents include alfentanil, tretinoin, Sulfacetamide Sodium, and tazarotene. Particular benzocaine, buprenorphine, butorphanol, butamben, capsai anti-acneagents include adapalene, azelaic acid, benzoyl per cin, clonidine, codeine, dibucaine, enkephalin, fentanyl. oxide, clindamycin {e.g., clindamycin phosphate), doxycy hydrocodone, hydromorphone, indomethacin, lidocaine, cline {e.g., doxycycline monohydrate), erythromycin, isotre levorphanol, meperidine, methadone, morphine, nicomor tinoin, norgestimate, Sulfacetamide Sodium, tazarotene, phine, opium, oxybuprocaine, oxycodone, oxymorphone, etretinate and acetretin. pentazocine, pramoxine, proparacaine, propoxyphene, 0070. Examples of antihistamine agents include diphen proxymetacaine, Sufentanil, tetracaine and tramadol. hydramine hydrochloride, diphenhydramine Salicylate, 0074 Examples of anesthetic agents include alcohols diphenhydramine, chlorpheniramine hydrochloride, chlor Such as phenol; benzyl benzoate; calamine; chloroxylenol; pheniramine maleate isothipendyl hydrochloride, tripelen dyclonine; ketamine; menthol; pramoxine; resorcinol; tro namine hydrochloride, promethazine hydrochloride, methdi closan; procaine drugs such as benzocaine, bupivacaine, lazine hydrochloride, and the like. Examples of local chloroprocaine; cinchocaine; cocaine; dexivacaine; anesthetic agents include dibucaine hydrochloride, diamocaine; dibucaine; etidocaine; hexylcaine; levobupiv dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, acaine; lidocaine; mepivacaine; oxethazaine; prilocalne; p-buthylaminobenzoic acid 2-(die-ethylamino) ethyl ester procaine; proparacaine; propoxycaine; pyrrocaine; risocaine; hydrochloride, procaine hydrochloride, tetracaine, tetracaine rodocaine; ropivacaine; tetracaine; and derivatives, such as hydrochloride, chloroprocaine hydrochloride, oxyprocaine pharmaceutically acceptable salts and esters including bupi hydrochloride, mepivacaine, cocaine hydrochloride, piper vacaine HCl, chloroprocaine HCl, diamocaine cyclamate, ocaine hydrochloride, dyclonine and dyclonine hydrochlo dibucaine HCl, dyclonine HCl, etidocaine HCl, levobupiv ride. acaine HCl, lidocaine HCl, mepivacaine HCl, pramoxine 0071 Examples of antiseptic agents include alcohols, HCl, prilocalne HCl, procaine HCl, proparacaine HCl, pro quaternary ammonium compounds, boric acid, chlorhexidine poxycaine HCl, ropivacaine HCl, and tetracaine HC1. and chlorhexidine derivatives, iodine, phenols, terpenes, bac 0075 Examples of antihemorrhagic agents include throm tericides, disinfectants including thimerosal, phenol, thymol. bin, phytonadione, protamine Sulfate, aminocaproic acid, benzalkonium chloride, benzethonium chloride, chlorhexi tranexamic acid, carbazochrome, carbaxochrome sodium dine, povidone iode, cetylpyridinium chloride, eugenol and Sulfanate, rutin and hesperidin. trimethylammonium bromide. 0076 Beside the bioactive polypeptide component, the 0072 Examples of anti-inflammatory agents include non instant invention may also contain other active agents such as steroidal antiinflammatory agents (NSAIDs); propionic acid niacinamide, phytantriol, farnesol, bisabolol and salicylic derivatives such as ibuprofen and naproxen; acetic acid acid. It is expected that certain additional active agents will derivatives Such as indomethacin; enolic acid derivatives Such act synergistically with the bioactive peptide component, or as meloxicam, acetaminophen; methyl salicylate; monogly will enhance the shelf-life of the formulation. col salicylate; aspirin, mefenamic acid; flufenamic acid; 0077. Examples of wound treatments that may be used indomethacin; diclofenac; alclofenac; diclofenac sodium; together with the drug product of the present invention ibuprofen, ketoprofen; naproxen; pranoprofen; fenoprofen; include fibrinolytic enzymes, such as fibrinolysin, deoxyri Sulindac, fenclofenac; clidanac; flurbiprofen; fentiazac, bonuclease, Streptokinase, and Streptodornase, necrotomy bufexamac, piroXicam, phenylbutaZone; oxyphenbutaZone; tissue agents containing lysozyme chloride, antimicrobial clofeZone; pentazocine; mepirizole; tiaramide hydrochlo agents containing gentamicin Sulfate, Sulfadiazine silver, ride; steroids such as clobetasol propionate, bethamethasone bacitracin, and fradiomycin Sulfate, incarnant agents contain dipropionate, halbetasol proprionate, diflorasone diacetate, ing trafermin, bucladesine Sodium, tretinoin tocoferil (to fluocinonide, halcinonide, amcinonide, desoximetasone, tri coretinate), alprostadil alfadex, Solcoseryl (extract from amcinolone acetonide, mometasone furoate, fluticaSone hemolysed blood of young cattle), and alcloxa, iodine prepa proprionate, betamethasone diproprionate, triamcinolone rations containing white soft Sugar, povidone iodine, and acetonide, fluticaSone propionate, desonide, fluocinolone iodine, and preparations containing bendazac, dimethyl iso acetonide, hydrocortisone Vlaerate, prednicarbate, triamcino propylaZulene (guaiaZulene), and epinephrine as active ingre lone acetonide, fluocinolone acetonide, hydrocortisone and dients. US 2013/0267471 A1 Oct. 10, 2013
0078. In addition to the alpha connexin polypeptides, selected from the group consisting of SEQID NOs: 1, 2, 3, 4, excipients, and other therapeutic agents, the gels may also and 5, or a conservative variant thereof. In a further embodi include other compounds that improve the organoleptic prop ment of the present invention, the alpha connexin polypeptide erties of the topical formulation. comprises the amino acid sequence of SEQ ID NO: 2. In 0079. Examples of such compounds include perfumes, another embodiment of the present invention, the polypeptide dyes and colorants; chelating agents including but not limited comprises an alpha connexin polypeptide and a cellular inter to edetate disodium (EDTA), EGTA, CP94, citric acid; pre nalization transporter. In a further embodiment, the alpha servatives including but not limited to quaternary ammonium connexin polypeptide is linked at is amino terminus to the compounds, such as benzalkonium chloride, benzethonium cellular internalization transporter. In a yet further embodi chloride, cetrimide, dequalinium chloride, and cetylpyri ment, the amino acid sequence of the alpha connexin dinium chloride; mercurial agents, such as phenylmercuric polypeptide linked to the cellular internalization transporter nitrate, phenylmercuric acetate, and thimerosal; alcoholic is selected from the group consisting of SEQID NO: 8, 9, 10. agents, for example, chlorobutanol, phenylethyl alcohol, and 11, and 12, or a conservative variant thereof. In one embodi benzyl alcohol; antibacterial esters, for example, esters of ment, the polypeptide consists of or comprises the amino acid parahydroxybenzoic acid; and other anti-microbial agents sequence of SEQ ID NO: 9, namely, RQPKIWFPNRRKP Such as chlorhexidine, chlorocresol, benzoic acid and poly WKKRPRPDDLEI. myxin. I0084. When specific proteins are referred to herein, vari 0080 Alpha connexin polypeptides that may be used in ants, derivatives, and fragments are contemplated. Protein the formulation of the present invention are described in U.S. variants and derivatives are well understood to those of skill in Pat. No. 7,786,074, which is hereby incorporated by refer the art and can involve amino acid sequence modifications. ence. In some embodiments, the alpha connexin polypeptide For example, amino acid sequence modifications typically is a full length alpha connexin protein Such as connexin 37. fall into one or more of three classes: substitutional, inser connexin 40, connexin 43, or connexin 45. In some embodi tional or deletional variants. Insertions include amino and/or ments, the polypeptide does not comprise the full-length con carboxyl terminal fusions as well as intrasequence insertions nexin protein. of single or multiple amino acid residues. Insertions ordi 0081 Peptides useful in the formulations of the present narily will be smaller insertions than those of amino or car invention include alpha connexin polypeptides. The alpha boxyl terminal fusions, for example, on the order of one to connexin polypeptides useful in the formulations of the four residues. Deletions are characterized by the removal of present invention may comprise or consist of the carboxy one or more amino acid residues from the protein sequence. terminal most 4 to 30 contiguous amino acids of an alpha These variants ordinarily are prepared by site specific connexin protein or conservative variant thereof. Thus, the mutagenesis of nucleotides in the DNA encoding the protein, polypeptides useful in the formulations can comprise the thereby producing DNA encoding the variant, and thereafter c-terminal-most 4 to 30 amino acids of the alpha Connexin, expressing the DNA in recombinant cell culture. Techniques including the c-terminal most 4,5,6,7,8,9, 10, 11, 12, 13, 14, for making Substitution mutations at predetermined sites in 15, 16, 17, 18, 19, 20, 21, 25 22, 23, 24, 25, 26, 27, 28, 29, 30 DNA having a known sequence are well known and include, amino acids of the alpha Connexin. In some aspects, the for example, M13 primer mutagenesis and PCR mutagenesis. provided polypeptide further comprises a deletion of one or Amino acid Substitutions are typically of single residues, but more amino acids of the c-terminal-most 4 to 30 amino acids can occurata number of different locations at once; insertions of the alpha Connexin, including a deletion of 1, 2, 3, 4, 5, 6, usually will be on the order of about from 1 to 10 amino acid 7,8,9, or 10amino acids of the c-terminal-most 4 to 30 amino residues. Deletions or insertions may be made in adjacent acids of the alpha Connexin. For example, in some aspects, pairs, i.e., a deletion of 2 residues or insertion of 2 residues. the polypeptides useful in the formulations of the present Substitutions, deletions, insertions or any combination invention do not comprise the c-terminal-most 1, 2, or 3 thereof may be combined to arrive at a final construct. The amino acids of the alpha Connexin. For example, the mutations must not place the sequence out of reading frame polypeptides can consist essentially of the amino acid and preferably will not create complementary regions that sequence SEQ ID NO:92, or a carboxy terminal fragment could produce secondary mRNA structure unless Such a thereof of at least 4, 5, 6, 7, 8, 9, 10 amino acids in length. change in secondary structure of the mRNA is desired. Sub 0082 In certain embodiments, the alpha connexin stitutional variants are those in which at least one residue has polypeptide of the present invention is linked at its amino or been removed and a different residue inserted in its place. carboxy terminus to a cellular internalization transporter. Such substitutions generally are referred to as conservative Separate embodiments include the alpha connexin polypep Substitutions. For example, the replacement of one amino tide without any cellular internalization transporter attached. acid residue with another that is biologically and/or chemi The cellular internalization transporter linked to the alpha cally similar is known to those skilled in the art as a conser connexin polypeptides may be any internalization sequence vative substitution. For example, a conservative substitution known or newly discovered in the art, or conservative variants would be replacing one hydrophobic residue for another, or thereof. Non-limiting examples of cellular internalization one polar residue for another. The substitutions Conserva transporters and sequences include Antennapedia sequences, tively substituted variations of each explicitly disclosed TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, sequence are included within the polypeptides provided Transportan, MAP (model amphipathic peptide), K-FGF, herein. Typically, conservative substitutions have little to no Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC impact on the biological activity of a resulting polypeptide. In (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (Bis a particular example, a conservative Substitution is an amino Guanidinium-Tren-Cholesterol). acid substitution in a peptide that does not substantially affect 0083. In one embodiment of the present invention, the the biological function of the peptide. A peptide can include amino acid sequence of the alpha connexin polypeptide is one or more amino acid Substitutions, for example 2-10 con US 2013/0267471 A1 Oct. 10, 2013
servative substitutions, 2-5 conservative substitutions, 4-9 mer), 1.00% gelling agent (polymer), 1.25% gelling agent conservative substitutions, such as 2, 5 or 10 conservative (polymer), 1.50% gelling agent (polymer), 1.75% gelling Substitutions. A polypeptide can be produced to contain one agent (polymer), 2.00% gelling agent (polymer), 2.25% gel or more conservative Substitutions by manipulating the nucle ling agent (polymer), or 2.50% gelling agent (polymer). The otide sequence that encodes that polypeptide using, for ACT peptide may be well preserved and adequately buffered example, standard procedures such as site-directed mutagen to pH 6. In one embodiment of the topical formulation, the esis or PCR. Alternatively, a polypeptide can be produced to qualitative and quantitative composition is that listed in Table contain one or more conservative Substitutions by using stan 1. dard peptide synthesis methods. An alanine scan can be used to identify which amino acid residues in a protein can tolerate TABLE 1 an amino acid Substitution. In one example, the biological activity of the protein is not decreased by more than 25%, for Drug Product Gel Qualitative & Quantitative Composition example not more than 20%, for example not more than 10%, Concentration when an alanine, or other conservative amino acid (such as Ingredients Grade Function (% w/w) those listed below), is substituted for one or more native amino acids. Further information about conservative substi Peptide 328967 Active 0.0072; 0.018 (ACT peptide) 0.036; 0.072 tutions can be found in, among other locations, in Ben-Bassat Methylparaben NF Preservative O.17 et al., (J. Bacterial. 169:751-7, 1987), O'Regan et al., (Gene Propylparaben NF Preservative O.O2 Glycerin USP Solvent S.O 77:237-51, 1989), Sahin-Toth et al., (Protein Sci. 3:240-7, Sodium Phosphate USP Buffer Agent O.263 1994), Hochuliet al., (Bio/Technology 6:1321-5, 1988) and in Monobasic standard textbooks of genetics and molecular biology. Sodium Phosphate Dibasic USP Buffer Agent O.O44 Propylene Glycol USP Solvent 3.0 0085. As used herein, “analytical methods’ includes the Edetate Disodium (EDTA) USP Chelating Agent O.OS following techniques that are useful for determining the iden D-Mannitol USP Stabilizer O.OS tity and quantity of the ACT peptide in a particular formula Hydroxyethylcellulose, NF Gelling Agent 1.25% tion: NMR, HPLC, Amino Acid Composition and Molecular 2SOHHX Weight Determinations, and Specific Optical Rotation. Purified Water, qsad USP Solvent 100% I0086. The present invention includes a topical formulation comprising at least one alpha connexin polypeptide and hydroxyethylcellulose gel, wherein the hydroxyethylcellu I0088. The ACT1 peptide sequence is listed in Table 2 lose gel stabilizes the alpha connexin polypeptide. In certain below in which AhX refers to L-2-aminohexanoic acid, also embodiments, the hydroxyethylcellulose gel stabilizes the known as 6-aminohexanoic acid: alpha connexin polypeptide so that after 3 months of storage at 5° C. at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, TABLE 2 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Peptide 328967 (Antp/ACT 1) sequence. 98%, 99%, or 100% of the alpha connexin polypeptide is detectable by analytical methods. In some embodiments, the Biotin-Ah:x-Arg-Gln-Pro-Lys-Ile-Trp-Phe-Pro-Asn alpha connexin polypeptide is present in the formulation at a Arg-Arg-Lys-Pro-Trp-Lys-Lys-Arg-Pro-Arg-Pro-Asp concentration of about 0.0025% (w/w), of about 0.005% (w/w), of about 0.0075% (w/w), of about 0.010% (w/w), of Asp-Leu-Glu-Ile-OH about 0.015% (w/w), of about 0.020% (w/w), of about 0.025% (w/w), of about 0.030% (w/w), of about 0.035% (w/w), of about 0.040% (w/w), of about 0.045% (w/w), of I0089. The general properties of the Peptide 328967 are about 0.050% (w/w), of about 0.055% (w/w), of about listed in Table 3. 0.060% (w/w), of about 0.065% (w/w), of about 0.070% (w/w), of about 0.075% (w/w), of about 0.080% (w/w), of TABLE 3 about 0.085% (w/w), of about 0.090% (w/w), of about General Physical Properties of Peptide 328967 0.095% (w/w), of about 0.100% (w/w), of about 0.150% (w/w), of about 0.200% (w/w), of about 0.250% (w/w), of Physical Appearance White to off-white powder Molecular Weight 3597.33 - 2.0 amu about 0.500% (w/w), of about 0.750% (w/w), of about 1.00% Counter-Ion AcOH (w/w), of about 1.50% (w/w), of about 2.00% (w/w), of about Solubility Soluble in water at room temperature 2.50% (w/w), or of about 5.00 (w/w). In one embodiment, the alpha connexin polypeptide is present in the formulation at a concentration of between about 0.005% (w/w) and about 0090. In one aspect of the invention, the excipients used in 1.00% (w/w). the drug product topical preparation are selected from the 0087. In other embodiments, the drug product of the group consisting of or are one or more of the following: invention is a clear colorless gel which contains 0.0072% (0091 Methylparaben (w/w) (20 uM) of the ACT peptide, 0.018% (w/w) (50 uM) of the ACT peptide, 0.036% (w/w) (100M) of the ACT peptide, 0092 Propylparaben or 0.072% (w/w) (200 uM) of the ACT peptide. The ACT peptide may be dissolved in a semisolid dosage form that 0093 Glycerin contains >0% water, >10% water, >20% water, >30% water, (0094 Sodium Phosphate Monobasic >40% water, >50% water, >60% water, >70% water, >80% water, or >90% water and 0.25% gelling agent (polymer), (0095 Sodium Phosphate Dibasic 0.55% gelling agent (polymer), 0.75% gelling agent (poly (0096 Propylene Glycol US 2013/0267471 A1 Oct. 10, 2013
0097 Edetate Disodium (EDTA) (w/w), or 0.01% (w/w) to 0.0.05% (w/w). In one embodi 0098. D-Mannitol ment, the Mannitol is present in the formulation at about 0099 Hydroxyethylcellulose, 250 HHX 0.05% (w/w). 0100 Purified Water 0104. In another aspect of the present invention, a buffer 0101. In one embodiment, the drug product topical prepa ing agentis included in the topical formulation to maintain the ration comprises a peptide, D-mannitol, hydroxyethylcellu pH in a certain range. Suitable buffering agents can include, lose, and purified water. Said preparation may further com but are not limited to, acetate buffers, citrate buffers, phos prise one or more of methylparaben, propylparaben, glycerin, phate buffers, lactic acid buffers, malic acid buffers, succinic Sodium phosphate monobasic, sodium phosphate dibasic, acid buffers, borate buffers, sodium hydroxide, potassium propylene glycol, and edetate disodium (EDTA). In a further hydroxide, and ammonium hydroxide. Phosphate salts such embodiment, the hydroxyethylcellulose is 250 HHX. In a as monosodium phosphate (NaH2PO4; also known as further embodiment, the peptide is an alpha connexin monobasic sodium phosphate), disodium hydrogen phos polypeptide. phate (NaHPO, also known as dibasic sodium phosphate), 0102. In such embodiments, the drug product topical monopotassium phosphate (KHPO), dipotassium phos preparation comprises a peptide at a concentration between phate (KHPO), and mixtures thereof can also be used. In about 0.001% (w/w) and about 0.5% (w/w) (for example, at one embodiment, phosphate buffer provides superior stability about 0.0072%, 0.018%, 0.036%, or 0.072% (w/w)); meth compared to citrate buffer. A buffer capacity at 25 mM was ylparaben at a concentration between about 0.10% (w/w) and found to be adequate for the drug product. Buffer is needed to about 0.25% (w/w) (for example, about 0.17% (w/w)); pro control pH of the gel system and to maintain stability of the pylparaben at a concentration between about 0.01% (w/w) peptide drug. The pH range of the topical formulation may be and about 0.03% (w/w) (for example, about 0.02% (w/w)); pH 2 to pH 12, pH 4 to pH 10, or pH 6 to pH 8. In one glycerin at a concentration between about 1% (w/w) and embodiment, the proper pH range is in the range of 5 to 7. In about 10% w/w) (for example, about 5% (w/w)): sodium some embodiments, the pH of the topical formulation of the phosphate monobasic at a concentration between about 0.1% present invention is 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. (w/w) and about 0.5% (w/w) (for example, about 0.263% In yet another aspect, propylene glycol in the amount of 3% (w/w)): sodium phosphate dibasic at a concentration between provides better solubilization of parabens in the aqueous sys about 0.02% and about 0.06% (for example, about 0.044% tem. (w/w)); propylene glycol at a concentration between about 0105. In one embodiment, the formulation with an ACT 1% (w.fw) and about 5% (w.fw) (for example, about 3% peptide incorporated into hydroxyethylcellulose enables (w/w)); EDTA at a concentration between about 0.01% and large scale manufacturing of a product with the characteris about 0.1% (for example, about 0.05% (w/w)); D-mannitol at tics that make it practical for clinical treatments as well as a concentration between about 0.01% (w/w) and about 0.1% meeting desired storage and stability requirements. While a (w/w) (for example, about 0.05% (w/w)); hydroxyethylcel formulation for ACT1 using Pluronic gel may require a rela lulose at a concentration between about 0.5% and about 2.5% tively long incorporation time of approximately 2.5 hours and (for example, about 1.25% (w/w)), and purified water at a only yield 50 gram batches, a formulation with hydroxyeth concentration of about 0.1% to about 10% (for example, ylcellulose provides allows for significantly faster incorpora about 1%). In a further embodiment, the peptide is an alpha tion of the ACT peptide into the gel and yields much larger connexin polypeptide. In a yet further embodiment, the pep batches. For example, when using Pluronic F 127 gel in the tide comprises an amino acid sequence selected from the topical formulation it takes over an hour to incorporate the group consisting of SEQID NO: 1,2,3,4,5,8,9, 10, 11, and polymer, and the formulation needs to be placed in a water 12. In one embodiment, the peptide comprises an amino acid bath to help with incorporation. In contrast, hydroxyethlycel sequence according to SEQID NO: 9. lulose (e.g., HEC 250 HHX) is easily incorporated at room 0103) Derived through in vitro and in vivo studies, these temperature and hydrates within 30 minutes. Thus, the use of stabilizers and excipients are incorporated into the drug prod hydroxyethylcellulose may facilitate large scale manufactur uct since they are non-irritating, non-staining, and non-im ing. The manufacturing process with Pluronic gel may munogenic. Stability studies showed that the ACT1 peptide is require a cold bath to bring its viscosity into a desired range more stable in the gelling agent Hydroxyethylcellulose, 250 and require more energy than the manufacturing process with HHX (1.25%) compared to Pluronic gels. The ACT peptide hydroxyethylcellulose. In addition, the final formulation in within the drug product containing 1.25% hydroxyethylcel Pluronic gel may be very thin at a storage condition of 5 lulose only dropped to 98% of the label claim (i.e., initial degrees C. concentration) when stored at 5° C. for three months and to 0106. It has been discovered that hydroxyethylcellulose 84% of the label claim when stored at 25° C. for the same (HEC) is a suitable gelling agent and acceptable carrier of the duration. In one aspect of the invention, edetate disodium drug product of the present invention. In one embodiment, the (EDTA) and Mannitol are incorporated within the drug prod gelling agent is Hydroxyethylcellulose (HEC), 250 HHX. In uct to provide stability to the ACT1 peptide. In some embodi one embodiment, the percent (w/w) of HEC is in the range of ments of the invention, the Mannitol is present in the formu 1-5%. In a further embodiment, the percent (w/w) of HEC is lation at 0.01% (w/w) to 1.6% (w/w), 0.01% (w/w) to 1.5% 1.25%. In the manufacture of HEC, a purified cellulose is (w/w), 0.01% (w/w) to 1.4% (w/w), 0.01% (w/w) to 1.3% reacted with sodium hydroxide to produce a swollen alkali (w/w), 0.01% (w/w) to 1.2% (w/w), 0.01% (w/w) to 1.1% cellulose. The alkali-treated cellulose is more chemically (w/w), 0.01% (w/w) to 1.0% (w/w), 0.01% (w/w) to 0.9% reactive than cellulose. By reacting the alkali cellulose with (w/w), 0.01% (w/w) to 0.8% (w/w), 0.01% (w/w) to 0.7% ethylene oxide, a series of hydroxyethylcellulose ethers is (w/w), 0.01% (w/w) to 0.6% (w/w), 0.01% (w/w) to 0.5% produced. In this reaction, the hydrogen atoms in the (w/w), 0.01% (w/w) to 0.4% (w/w), 0.01% (w/w) to 0.3% hydroxyl groups of cellulose are replaced by hydroxyethyl (w/w), 0.01% (w/w) to 0.2% (w/w), 0.01% (w/w) to 0.1% groups, which confer water solubility to the gel. It is contem US 2013/0267471 A1 Oct. 10, 2013
plated in this invention that a single HEC ether may be used, tering to a subject in need thereof a topical formulation com or a mixture of HEC ethers of difference molecular weight prising at least one alpha connexin polypeptide and hydroxy and structure may be used. Suitable grades of HEC for phar ethylcellulose gel, wherein the hydroxyethylcellulose gel maceutical purposes are well known and full described in the stabilizes the alpha connexin polypeptide. The wound treated pharmaceutical literature. Suitable commercially available may be an acute Surgical wound or a chronic, non-infected, brands of HEC include but are not limited to Fuji HEC-HP; full-thickness lower extremity ulcer. Fuji HEC-AG 15; NATRO-SOL 250HR; NATROSOL 250 0109. In a certain embodiment, the drug product of the MH; NATROSOL 250G; CELLOSIZE QP 30000: TYLOSE present invention may be used to mitigate excessive scar H SERIES: NATROSOL 180L NATROSOL 300H; formation associated with acute Surgical wounds. In this TYLOSE P-X; NATROSOL 250M; CELLOSIZEWP4400; embodiment, the drug product of the present invention may CELLOSIZE UT 40; NATROSOL 250H4R; Tylose H2OP: be applied at the time of Surgical incision closure, 1 hour after NATROSOL LR; TYLOSE MHB; NATROSOL 250HHP; Surgical incision closure, 2 hours after Surgical incision clo HERCULES N 100; CELLOSIZE WP 300; TYLOSE P-Z Sure, 3 hours after Surgical incision closure, 4 hours after SERIES: NATROSOL 250H; TYLOSE PS-X: Cellobond Surgical incision closure, 5 hours after Surgical incision clo HEC 400; CELLOSIZE QP; CELLOSIZE QP 1500; Sure, 6 hours after Surgical incision closure, 7 hours after NATRO-SOL 250; HYDROXYETHYL CELLULOSE Surgical incision closure, 8 hours after Surgical incision clo ETHER: HESPAN: TYLOSE MHB-Y: NATROSOL 240JR; sure, 9 hours after surgical incision closure, 10 hours after HYDROXYETHYL STARCH; CELLOSIZE WP; CELLO Surgical incision closure, 11 hours after Surgical incision clo SIZE WP 300H; 2-HYDROXYETHYL CELLULOSE Sure, 12 hours after Surgical incision closure, 13 hours after ETHER: BL 15; CELLOSIZE QP 4400; CELLOSIZE QP3; Surgical incision closure, 14 hours after Surgical incision clo TYLOSE MB; CELLULOSE HYDROXY-ETHYLATE; Sure, 15 hours after Surgical incision closure, 16 hours after CELLOSIZEWPO 9H17; CELLOSIZE 4400H16; CELLU Surgical incision closure, 17 hours after Surgical incision clo LOSE HYDROXYETHYL ETHER: Hydroxyethyl Cellu sure, 18 hours after surgical incision closure, 19 hours after lose; Hydroxyl Ethyl Cellulose (HEC); Hydroxyethyl Cellu Surgical incision closure, 20 hours after Surgical incision clo lose 100H (celocell 100h): TYLOSE MH-XP; NATROSOL Sure, 21 hours after Surgical incision closure, 22 hours after 250HX: Natrosol; Daicel EP 500; HEC-Unicel; HEC (Hy Surgical incision closure, 23 hours after Surgical incision clo droxyethyl cellulose); Cellosize: HEC-Al 5000; Fuji HEC Sure, 24 hours after Surgical incision closure, 48 hours after AL 15: HEC-Unicel QP 09L: Cellulose, ethers, 2-hydroxy Surgical incision closure, 72 hours after Surgical incision clo ethyl ether; Unicel QP 52000H; HEC-QP 4400; SP 250 sure, or thereafter. (cellulose); Hetastarch; Cellulose, ethers, 2-hydroxyethyl 0110. In another embodiment, the drug product of the ether: Glutofix 600; FL 52: Fuji HEC-AX 15F. Tylose H present invention may be used to treat chronic ulcers. For 300P; HEC-Unicel QP 300H; Tylose H 300: Daicel SP 550; example, ulcers may include diabetic foot ulcers, venous leg Daicel SE 600: Unicel QP 15000; HEC-QP 100 MH; HEC ulcers, and pressure ulcers. These ulcers may be chronic, QP 9H; OETs; Daicel EP 850; H. E. Cellulose; Cellobond non-infected, full-thickness lower extremity ulcers. In one 25T: Unicel QP 100 MH; Tylose H 4000; SE850K; Tylomer embodiment, the drug product of the present invention may H20: Daicel SE 850K; Tylose H30000YP: Unicel QP 4400; be applied to a chronic ulcer in a daily regimen, a regimen of SP 407; Tylose H 100000: Daicel SP 200; Culminal HEC every other day, a regimen of once a week, or in various other 5000PR; Tylopur H 300: Daicel SP 750; Sanhec; BL 15 regimens until healing of the chronic ulcer is apparent. In (cellulose derivative); Unicel QP 300H; Tylomer H 200; J another embodiment, the drug product of the present inven 164: Tylose H 10; Tylose H20: AH 15: Daicel SP600: Daicel tion may be applied to a chronic ulcer in a regimen at day 0, SE900; HEC-Unicel QP 4400H; AX 15; Daicel SP800; Fuji 3, 7, 14, 21, and 28. In another embodiment, the drug product HEC-AW 15F; HEC-SE 850; HEC-A 5-25CF; Metolose of the present invention may be applied to a chronic ulcer in 90SEW: AW 15 (polysaccharide); Cellobond HEC 5000; a regimen at day 0, day 3, week 1, week 2, week 3, week 4, HEC-QP 100M; Cellobond HEC 15A; Tylose H 15000YP2: week 5, week 6, week 7, week 8, week 9, week 10, week 11, Walocel HT 6.000 PFV; 2-Hydroxyethyl cellulose (Natrosol and week 12. In another aspect of the present invention, the Type 250HRCS); Fuji HEC-BL 20; Fuji HEC-SY 25F; Tel drug product is manufactured with the following steps: hec; HEC-SP 200; HEC-AH 15; HEC-Unicel QP 30000H; see: HEC 10A; Daicel SP 400; Admiral 3089FS: Fuji HEC-A 0111 Step 1: In a suitable size of beaker, add propylene 5000F; HEC-SP 400; Hydroxyethyl Methyl Cellulose glycol, glycerin, methylparaben and propylparaben. Mix (HEMC), HYDROXYETHYL CELLULOSE (HEC); with a propeller until the parabens are completely dissolved. Hydroxyethyl Starch (CAS No:9004-62-0); Hydroxy Ethyl 0112 Step 2: In a manufacturing vessel, add purified water Cellulose: “Natrosol Aqualon; HEC: 2-HYDROXY (part I), EDTA, monobasic sodium phosphate, dibasic ETHYLCELLULOSE; NATROSOL 150LTYLOSEMHB sodium phosphate and D-mannitol. Mix with a propeller until YP: HYDROXYETHYL ETHER CELLULOSE; NATRO a clear Solution is obtained. SOL 250L, CELLOSIZE WP 400H; TYLOSE P. 0113 Step 3: Add the solution from step 1 to the manu CELLULOSE, 2-HYDROXYETHYL ETHER: TYLOSE facturing vessel. Rinse the beaker with purified water (part II, MH-K; NATROSOL 250HHR. divided into approximately 3 equal portions) and add the rinse 0107. In another embodiment, the drug product of the back to the vessel. Continue with propeller mixing until the present invention is packaged in 20 mL vial (USP Type I, Solution is visually homogeneous. Borosilicate clear Scintillation glass with poly-seal cone urea 0114 Step 4: With homogenization mixing, add hydroxy screw cap). A mixture of methylparaben at 0.17% (w/w) and ethyl cellulose into the manufacturing vessel from Step 3. propylparaben 0.02% (w/w) is used as a preservative. Mix until the polymer is fully dispersed. 0108. In some embodiments, the present invention 0115 Step 5: In a separate beaker, add purified water (part includes a method of wound treatment comprising adminis III) and an alpha connexin polypeptide (e.g., Peptide 328967 US 2013/0267471 A1 Oct. 10, 2013 11
ACT1 peptide). Mix with a stir bar or propeller mixer until the - Continued peptide is completely dissolved and a gel is formed. 0116 Step 6: With continuous propeller mixing, add the SEQ ID NO: 22 (Ku70) drug solution from step 5 to the manufacturing vessel. Rinse the beaker with purified water (part IV, divided into approxi SEQ ID NO: 23 (Prion) mately 3 equal portions) and add the rinse back to the vessel. MANLGYWLLALFWTMWTDWGLCICICRPKP Mix until the gel is homogeneous. SEQ ID NO: 24 (pVEC) 0117 The manufacturing process flow chart is provided in LLIILRRRIRKOAHAHSK FIG. 1 Manufacturing Process Flow Chart. SEQ ID NO: 25 (Pep-1) 0118 Sequences KETWWETWWTEWSOPKKKRKV
SEQ ID NO: 26 (SynB1) SEO ID NO : 1 (ACT 2) RGGRLSYSRRRFSTSTGR PSSRASSRASSRPRPDDLEI SEQ ID NO: 27 (Pep-7) SEO ID NO: 2 (ACT 1) SDLWEMMMVSLACOY RPRPDDLEI SEQ ID NO: 28 (HN-1) SEO ID NO : 3 (ACT 3) TSPLNIHNGOKL RPRPDDLEW SEQ ID NO: 29 (Chick alpha CX43 ACT) SEO ID NO: 4 (ACT 4) PSRASSRASSRPRPDDLEI RPRPDDWPW SEQ ID NO: 30 (Human alpha CX45 SEO ID NO: 5 (ACT 5) GSNKSTASSKSPDPKNSWWI KARSDDLSW SEQ ID NO: 31 (Chick alpha CX45 SEO ID NO: 6 GSNKSSASSKSGDGKNSWWI aga cct cqg cct gat gac Ctg gag att SEQ ID: 32 (Human alpha CX46) SEQ ID NO: 7 (Antp) GRASKASRASSGRARPEDIAI ROPKIWFPNRRKPWKK SEQ ID: 33 (Human alpha CX46.6) SEQ ID NO: 8 (Antp/ACT 2) GSASSRDGKTWWI ROPKIWFPNRRKPWKKPSSRASSRASSRPRPDDLEI SEQ ID NO: 34 (Chimp alpha Cx36 SEQ ID NO: 9 (Antp/ACT 1) PRVSVPNFGRTOSSDSAYV ROPKIWFPNRRKPWKKRPRPDDLEI SEQ ID NO: 35 (Chick alpha Cx36 SEQ ID NO: 10 (Antp/ACT 3) PRMSMPNFGRTOSSDSAYV ROPKIWFPNRRKPWKKRPRPDDLEV SEQ ID NO: 36 (Human alpha CX47 SEQ ID NO: 11 (Antp/ACT 4) PRAGSEKGSASSRDGKTTWWI ROPKIWFPNRRKPWKKRPRPDDVPV SEQ ID NO: 37 (Human alpha CX40 SEQ ID NO: 12 (Antp/ACT 5) GYHSDKRRLSKASSKARSDDLSW ROPKIWFPNRRKPWKKKARSDDLSV SEQ ID NO: 38 (Human alpha Cx50 SEQ ID NO: 13 (encodes polypeptide of SEQ ID NO 9) PLSRLSKASSRARSDDLTW cgg cag ccc aag atc t t t c ccc aac cqg cgg aag SEQ ID NO: 39 (Human alpha Cx59 CCC tig aag aag C9g C cc ggc ccg acg acc tig aga tic SEO ID NO: 14 (HIV-Tat) SEQ ID NO: 40 (Rat alpha Cx33) GRKKRRORPPO PSCVSSSAVLTTICSSDOVWPWGLSSFYM SEO ID NO: 5 (Penetratin) SEQ ID NO: 41 (Sheep alpha CX44) ROIKIWFONRRMKWKK GRSSKASKSSGGRARAADLAI SEQ ID NO: 16 (Antip-3A) SEQ ID NO: 42 (Human beta CX26) ROIAIWFONRRMKWAA LCYLLI RYCSGKSKKPW SEO ID NO: 17 (Tat) SEQ ID: 43 (Human alpha CX37) RKKRRORRR GOKPPS RPSSSASKKO'KYW SEO ID NO: 8 (Buforin II) 44: (conservative CX43 variant) TRSSRAGLOFPWGRVHRLLRK RASSRPRPDDLEW SEO ID NO: 9 (Transportan) 45: (conservative CX43 variant) GWTLNSAGYLLGKINKALAALAKKIL EI SEQ ID NO: 2O (model amphipathic peptide) 46; (conservative CX43 variant) KLALKLALKALKAAIKLIA RASSRPKPDDLEI,
SEO ID NO: 21 (K-FGF) 47: (conservative CX43 variant) AAVAILPAWLLALLAP LDI
US 2013/0267471 A1 Oct. 10, 2013 13
- Continued Phosphate buffers at pH 5, 6, and 7 and two gelling agents, SEQ ID NO: 85 (Antp/ACT 1) Pluronic and Hydroxyethylcellulose (HEC) were evaluated. CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCG The quantity of API required for the formulations was calcu GCCCCGGCCCGACGACCTGGAGATC lated as follows: I0123 Batch size for each formulation=12x5 g=60 g or 70 SEQ ID NO: 86 (Antp/ACT 3) 9. CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCG (0.124 API quantity for 6 formulations=6x70x0.072%/82. GCCCCGGCCCGACGACCTGGAGGTG 4%–0.37g (0.125 API purity value=82.4% SEQ ID NO: 87 (Antp/ACT 4) 0.126 Five formulations, formulae 2414-10 to -14, were CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCG gelled with Pluronic 127, which was assumed to be the pre GCCCCGGCCCGACGACGTGCCCGTG ferred gelling agent. Only one formulation, 2414-15, was gelled with HEC as a back-up. The available stability data SEQ ID NO: 88 (Antp/ACT 5) indicated the following conclusions. CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGAA I0127 Buffer was needed to control the pH of the gel GGCCCGGTCCGACGACCTGTCCGTG system and to maintain stability of the peptide drug. Proper pH range seemed to be between 5 and 7. Phosphate buffer was SEQ ID NO: 89 (Zebrafish alpha CX43) PCSRASSRMSSRARPDDLDW adequate. I0128 Surprisingly, the API, ACT1, was more stable in SEQ ID NO: 90 (Chick alpha Cx36) HEC gel than Pluronic gels. With Pluronic gels, the API PRVSVPNFGRTOSSDSAYV dropped to an average of 85% of the label claim (LC) from SEQ ID NO: 91 (Zebrafish alpha Cx36) initial when gels were stored at 5° C. for one month; and to PRMSMPNFGRTOSSDSAYV 79% when stored at 25°C. for one month. With HEC gel, the API dropped to 98% of label claim from initial when stored at SEO ID NO: 92 (Cx43 isoleucine deletion) 5° C. for 3 months and 84% when Stored at 25° C. for three ROPKIWFPNRRICPWICKRASSRASSRPRPDDLE months. I0129 HEC appeared to be a better choice of gelling agent EXAMPLES than Pluronic when comparing formula 2414-14 to 2414-15. Both formulations were exactly same except that formula Example 1 2414-14 was gelled with Pluronic and formula 2414-15 was 0119 The objective was to develop a stable, elegant and gelled with HEC. The API in formula 2414-14 dropped to pourable gel in which the Active Pharmaceutical Ingredient 68% of the label claim from the initial concentration when (API) is in solution for wound healing purposes. stored at 5° C. for one month while the API in formula 0120. The major challenges encountered during formula 2414-15 only dropped to 98% of the label claim from the tion development were the selection of suitable stabilizers for initial concentration when stored at 5° C. for 3 months. the API (ACT 1), and other excipients that are non-irritating 0.130 EDTA and Mannitol appear to provide some stabil for wound healing application. Studies were conducted to ity to the peptide drug. evaluate prototype formulations. I0131 The formulations did not appear to be stable at 25° 0121 R&D Study Per Protocol #1062 C. The final product might require refrigeration, which is 0122) The first study was conducted per protocol #1062. typical for products containing peptides and proteins. During this study, 6 prototype formulations were evaluated as I0132) Formula 2414-15 with HEC as the gelling agent is outlined in Table 4. The purposes of the study were to select the most stable formulation compared to the rest of the for a proper pH range for the product and also to select a proper mulations tested. It was selected for further development. gelling agent. The API concentration used for this study was Stability data of formulations 2414-14 and 2414-15 are sum 0.072% which was the initial targeted product concentration. marized in Tables 5 and 6, respectively. TABLE 4
Wound Healing Gel Formulation Compositions for Stability Testing per Study #1062
Components Formula #: Formula ii: Formulati: Formulati: Formulati: Formulaii: (% WW) 2414-10 2414-11 2414-12 2414-13 2414-14 2414-15
API O.O72 O.O72 O.O72 O.O72 O.O72 O.O72 Propylene glycol 1.O 1.O 1.O 1.O 1.O 1.O Glycerin S.O S.O S.O S.O S.O S.O Monobasic Sodium O.300 O.263 O.120 O.263 O.263 Phosphate Dibasic Sodium O.OO36 O.044 O.217 O.O44 O.044 Phosphate Methylparaben O.17 O.17 O.17 O.17 O.17 O.17 Propylparaben O.O2 O.O2 O.O2 O.O2 O.O2 O.O2 Pluronic 127 2O.O (Poloxmer 407) US 2013/0267471 A1 Oct. 10, 2013
TABLE 4-continued Wound Healing Gel Formulation Compositions for Stability Testing per Study #1062 Components Formulati: Formulati: Formulati: Formula if: Formulati: Formula if: (% WW) 2414-10 2414-11 2414-12 2414-13 2414-14 2414-15 EDTA Disodium O.OS O.OS Mannitol O.OS O.OS Hydroxyethylcellulose 1.25 2SOHHX Purified water q.S. 100 q.S. 100 q.S. 100 q.S. 100 q.S. 100 q.S. 100 No Buffer Buffer pH 5 Buffer pH 6 Buffer pH 7 Buffer pH 6 Buffer pH 6
TABLE 5 TABLE 6-continued Stability Results of Formula 2414-14 Containing Pluronic 127 per Stability Results of Formula 2414-15 Containing HEC per Study #1062 Study #1062 Physical Time Physical Chemical ASSay (90, LC Time Properties Chemical ASSay (%, LC Storage Point Properties and Propyl Storage Point and Methyl Condition (Month) Appearance API Methylparaben paraben Condition (Month) Appearance API paraben Propylparaben Initial Clear and 98.8 92.0 96.6 25o C. /2 Conforms 91.6 99.7 106.7 colorless gel 1 Conforms 93.4 96.8 103 Freeze. Thaw Conforms 89.8 886 94.0 3 Conforms 85.7 92.8 96.5 50 C. 1 Conforms 846 93.0 97.9 25o C. /2 Conforms 90.6 93.6 98.6 1 Conforms 82.8 88.8 94.0 Example 2 R&D Study Per Protocol #1074 I0133 Based on the results from study #1062, additional TABLE 6 prototype formulations based on formula 2414-15 were designed and evaluated per protocol #1074. During this study, Stability Results of Formula 2414-15 Containing HEC per Study #1062 7 prototype formulations were prepared. The purposes of the Physical study were to select a proper buffering system and antioxi Time Properties Chemical ASSay (%, LC dants for the formulation. Five (5) antioxidants were evalu Storage Point and Methyl ated: Mannitol, Methionine, Acetylcysteine, Lysine and His Condition (Month) Appearance API paraben Propylparaben tidine. Two buffer systems were used: phosphate buffer at pH 6 and citrate buffer at pH 5. Glycerin (5%) was introduced Initial Clear 101.9 97 103.1 into the new prototype formulations to provide some humec colorless gel Freeze. Thaw Conforms 98.8 97.1 103.9 tant property to the wound treatment topical gel. PEG 200 was 50 C. 1 Conforms 96 98.1 104.6 also evaluated for its potential stabilizer effect. The compo 3 Conforms 99.7 98.3 101.8 sitions of the prototypes evaluated per study #1074 are listed in Table 7. TABLE 7 Composition of Prototype Formulations per Study #1074 Formula # 2414
Component (% w/w) 18 19 2O 21 22 23 26 Peptide 328967 (ACT1) O.O36 0.036 O.O36 O.O36 O.036 O.036 O.036 Propylene Glycol 1.O 1.O 1.O 1.O 1.O PEG 200 S.O S.O Glycerin S.O S.O S.O S.O S.O S.O S.O Monobasic Sodium Phosphate 0.263 O.263 O.263 O.263 O.S26 Dibasic Sodium Phosphate O.044 O.044 O.O44 O.O44 O.O88 Citric Acid, anhydrous O60 O.6O Sodium Citrate, anhydrous 1.73 1.73 Methylparaben O.17 O.17 O.17 O.17 O.17 O.17 O.17 Propylparaben O.O2 O.O2 O.O2 O.O2 O.O2 O.O2 O.O2 EDTA disodium O.OS O.OS O.OS O.OS O.OS O.OS O.OS Mannitol O.OS O.OS Methionine O.S O.S O.S O.S Acetylcysteine O.2 Lysine O.1 O.1 Histidine O.1 Hydroxyethylcellulose 1.25 1.25 1.25 1.25 1.25 1.25 1.25 US 2013/0267471 A1 Oct. 10, 2013 15
TABLE 7-continued Composition of Prototype Formulations per Study #1074 Formulai 2414
Component (% w/w) 18 19 2O 21 22 23 26 Purified Water, qsad 1OO 100 1OO 100 100 100 1OO 25 mM 25 nM 25 mM 25 nM 50 mM 100 mM 100 mM Buffer Buffer Buffer Buffer Buffer Buffer Buffer pH 6 pH 6 pH 6 pH 6 pH 6 pH 5 pH 5
0134. Due to API availability limitations, 0.036% of API TABLE 8-continued concentration was used for this study. Formula 2414-18 is identical to 2414-15 except that the API concentration was Stability Results of Formulas 2414-18, 2414-19, 2414-20, reduced from 0.072% to 0.036%. The available stability data - 22, 222, 2t2, and 220 on Study 1974 from study #1074 found in Table 8 indicated the following Chemical Assay conclusions. Time Physical 20, LC 0135 Phosphate buffer seemed to provide better stability Storage Point Properties Methyl- Propyl to the peptide when compared to the citrate buffer. The buffer Sample Condition (Month) Appearance API paraben paraben capacity at 25 mM is adequate for the system. - 0 25o C. Conforms 103.3 94.6 117.4 0136. Mannitol seemed to provide adequatestability to the 2 Conforms NA NA NA peptide. 3 C OOS NA NA NA Formula Initial Clear and 107.0 96.9 99.9 0137 The effect of other antioxidants was not clear due to 2414-20 Colorless the variability in the chemical data. Gel 50 C. Conforms 106.8 96.7 98.4 0138 Based on the study results mainly from study #1062 2 Conforms NA NA NA and partially from #1074, a final formulation was selected for 3 Conforms' NA NA NA 97.9 94.2 95.6 further development. The final formulation contained 25 mM 25° C. Conforms NA NA NA phosphate p buffer- to maintain the ppH of the formulation,s man- 32 Conforms'Conforms NA NA NA nitol as a stabilizer for the API, and HEC as a gelling agent. Formula Initial Clear and 104.3 95.1 10.4 The amount of propylene glycol was increased from 1% to 2414-21 Colorless 3% in prototype formulations to allow for better solubiliza- g Gel ion ofth bens inth tem. F tential API 50 C. Conforms 96.7 91.5 O4.2 tion of theparabens in the aqueous system. Four potentia 2 Conforms NA NA NA concentrations were selected: 0.0072%, 0.018%, 0.036% and 3 Conforms NA NA NA 0.072%. The final formula is Summarized in Table 9. The 25o C. Conforms 96.4 95.0 O8.4 stability of the final selected formula containing different 2 Con orms, NA NA NA levels of API was also evaluated with analytical methods. In 3 Conforms NA NA NA eve s O - y Formula Initial Clear and 112.2 96.1 O9.4 addition, the overall stability was assessed by visual Inspec- 2414-22 Colorless tion for white powder or precipitate and determination of Gel Water COIntent. 50 C. Conforms 1114 94.9 O6.1 2 Conforms NA NA NA 3 Conforms' NA NA NA TABLE 8 25o C. Conforms 96.S 89.5 OO.O NA NA NA Stability Results of Formulas 2414-18, 2414-19, 2414-20, an orms, NA NA NA 2414-21. 2414-22, 2414-23, and 2414-26 from Study #1074 Formula Initial Clearoniorms and 87.8 94.7 O16 Chemical Assay 2414-23 Colorless Time Physical 20, LC Gel 50 C. Conforms 88.3 92.9 97.0 Storage Point Properties Methyl- Propyl- 2 Conforms NA NA NA Sample Condition (Month) Appearance API paraben paraben 3 Conforms' NA NA NA 25o C. Conforms 74.4 84.4 88.4 Formula Initial Clear and 106.4 94.6 102.2 2 Conforms NA NA NA 2414-18 Colorless 3 Conforms' NA NA NA g Gel Formula Initial Clear and 83.O 95.5 105.9 50 C. 1 Conforms 90.1. 834 88.2 2414-26 Colorless 2 Conforms NA NA NA Gel 3 Conforms' NA NA NA 5o C Conforms 78.S 91.0 98.5 25o C. 1 Conforms 94.3 90.4 95.5 2 C 2 Conforms NA NA NA onforms, NA NA NA 3 Conforms' NA NA NA g 3 Conforms NA NA NA Formula Initial Clear and 109.3 94.2 119.7 25o C. Conforms 77.4 93.3 100.7 2414-19 Colorless 2 Conforms NA NA NA Gel 3 Conforms' NA NA NA 50 C. 1 Conforms 101.1 90.4 112.6 2 Conforms NA NA NA 'Particles at the bottom of vial; 3 Conforms NA NA NA NA = data not available US 2013/0267471 A1 Oct. 10, 2013
0.139. In one embodiment, a batch of the drug product may 0155 Exclusion Criteria: be prepared by following the steps of the manufacturing pro 0156 1. A subject was not eligible for this study if he/she cess flow chart and using the composition of Table 9. met any of the following criteria: (O157 2. Decrease or increase in the ulcer size by 30% or Example 3 more during 7 day Screening period 0158. 3. Cannot tolerate the off-loading method or comply Treatment of Diabetic Foot Ulcer with SoC 0159. 4. An ulcer which showed signs of severe clinical 0140 A Phase II, randomized, prospective, double blind, infection, defined as pus oozing from the ulcer site parallel group, multi-center study was conducted to assess the 0160 5. The ulcer to be treated required operative debri safety and efficacy of ACT1 peptide in the treatment of dia dement betic foot ulcer. 0.161 6. An ulcer positive for B-hemolytic streptococcus 0141. Subjects in Group A received 100 uM of the ACT1 upon culture peptide formulation (i.e., GranexinTM Gel) in addition to stan 0162 7. Requirement for total contact casts dard-of-care treatment (SoC) compared to SoC alone in 0163 8. The ulcer had more than 50% slough, significant Group B. The total study duration for efficacy assessments necrotic tissue, bone, tendon, or capsule exposure was 12 weeks with additional 12 weeks follow up for safety 0164. 9. Highly exuding wounds (wounds that require a evaluations. The study procedures were divided into three daily dressing change) ABPI<0.7 or >1.3 or ankle systolic phases: Screening Procedures (Study Days: -7 and 0), Treat pressure <70 mm H ment Procedures (Study Day 0 through Study Week 12), and 0.165. 10. glad systemic infections Follow-up Procedures (Study Months 4 through 6). 0166 11. Met one of the following (only 1 out of 3 tests was required): 0142. Subjects enrolled in the study may have had mul 0.167 a. Doppler waveform analysis on the dorsalis pedis tiple ulcers but only one ulcer was considered as the target and posterior tibial arteries a monophasic or biphasic flow ulcer which was within the range of 4-1250 mm and was (with loss of reverse flow) in either foot artery, or separated from other ulcers by at least 20 mm. According to (0168 b. A toe: brachial index <0.7 or >1.3, or the randomization schedule, subjects had their ulcer site 0169 c. Transcutaneous oxygen pressure <40 mm Hg treated with either GranexinTM Gel and SOC or SoC alone. 0170 12. Presence of an active systemic or local cancer or Each subject in Group A was treated with up to 14 applica tumor of any kind (with the exception of non-melanoma tions of GranexinTM Gelover the initial 12 weeks of the study skin cancer) period. Other than the application of GranexinTM Gel, man (0171 13. Congestive Heart Failure New York Heart Asso agement of the wound sites was equivalent in both the groups. ciation class II-IV or coronary heart disease with ST seg Each subject was assessed weekly for the first 12 weeks of ment elevation myocardial infarction or coronary artery treatment. Statistical analysis of Subject data up to 12 weeks bypass graft or percutaneous transluminal coronary angio was done for efficacy and safety analyses. Wound photo plasty within the last 6 months graphs were used to assess wound closure for both control and 0172 14. Active osteomyelitis of the study foot test sites. Safety was determined by Treatment Emergent (0173 15. Active connective tissue disease Adverse Events (TEAEs). 0.174 16. Acute Charcot's neuro-arthropathy as deter 0143 Inclusion Criteria: mined by clinical and/or radiographic examination 0144. A subject was enrolled in this study if he/she met the 0.175 17. Active treatment with systemic corticosteroids following criteria: 0176 18. Previous or current radiation therapy to the distal (0145 1. Male or female aged 18 years and older lower extremity or likelihood to receive this therapy during 0146 2a. Female subjects were post-menopausal or sur study participation gically sterilized, or 0177. 19. Pregnant or nursing subjects (0178. 20. Uncontrolled anemia (Hb<10 g/dL in females 0147 2b. Female of child-bearing potential must have had and <12 g/dL in males) a negative pregnancy test at Screening, and agreed to use 0179. 21. Estimated glomerular filtration rate <25 mL/min hormonal contraceptive or intra-uterine device or dia 0180 22. Poor nutritional status defined as an albumin <25 phragm with spermicide or condom with spermicide or g/L abstinence throughout the study 0181. 23. Significant peripheral edema as per Investiga 0148 3. Diabetes mellitus (type I or II) with an tor's discretion HbA1c310.0% 0182 24. Known prior inability or unavailability to com 0149 4. Diagnosis of neuropathic foot ulcer plete required study visits during study participation 0150 5. Cutaneous, full thickness (University of Texas 0183 25. A psychiatric condition (e.g. suicidal ideation) grade A1), below ankle surface ulcer between 0.5 cm and or chronic alcohol or drug abuse problem, determined from 40 cm post debridement the subject's medical history, which, in the opinion of the 0151. 6. A viable, granulating wound as per Investigator's Investigator, may pose a threat to Subject compliance discretion 0.184 26. Use of a platelet-derived growth factor within 28 days prior to Screening 0152 7. Ulcer present for at least 4 weeks prior to Screen 0185 27. Use of any investigational drug ortherapy within ing 28 days prior to Screening 0153 8. An Ankle Brachial Pressure Index (ABPI) 0186 28. Any other factor which may have, in the opinion between 0.70 and 1.3 measured at Screening of the Investigator, compromised participation and follow 0154) 9. Signed informed consent form up in this study US 2013/0267471 A1 Oct. 10, 2013
0187 GranexinTM Gel (100 uMACT1 peptide, in the for 0204 The mean percent wound closure at 4 weeks com mulation described in Table 1 of this application) was applied pared to Baseline was analyzed as described in the primary topically on the wound on Day 0, Day 3, Week 1, 2, 3, 4, 5, 6, efficacy analysis. Subject assessment of intensity of pain at 7, 8, 9, 10, 11, and 12. Week 12 was analyzed by the Wilcoxon Mann-Whitney U 0188 Total duration of the study was 12 months, including teSt. 6 months enrollment period. Individual Subject participation 0205 For time-to-event endpoints, the distribution for for the period of interim analysis was approximately 12 each treatment group was estimated by the Kaplan-Meier weeks. method and compared by the log-rank test. The median time (0189 Efficacy: along with 90% confidence limits was presented for each Efficacy was evaluated by assessment of the following treatment group. Simultaneous confidence bounds for the parameters between study Day 0 to Week 12: Kaplan-Meier curve were computed for both the treatment (0190. Wound photographs groups. Further, Cox proportional hazard regression model (0191 Wound tracings analysis was conducted to compare the event-time distribu 0.192 Wound closure assessment tion function between the treatment groups. The incidence of 0193 Wound characteristics (clinical assessment)** 100% study wound closure was a time-to-event analysis com puted using Kaplan-Meier methods where all scheduled visits (0194 Wound treatment response from Day 0 though Week 12 were included in the analysis. (0195 Safety: This was the primary analysis of the secondary efficacy end The safety was evaluated by assessing the following param point. Summary statistics, including the median for incidence eters: of 100% study wound closure was presented. The statistical (0196) TEAEs (each visit from Day 0 up to Week 12)* significance of the difference in the incidence of 100% wound (0197) Vitals (Day -7 and Week 12 or Last Subject Last closure was evaluated using the log-rank test. Cox's propor Visit ILSLV)** tional hazard regression analysis was performed to evaluate 0198 Laboratory assessments (Day -7 and Week 12 or the individual and joint effects of several covariates on the LSLV)** incidence of 100% wound closure. * These evaluations were considered for the interim analysis 0206 Incidence of 100% wound closure (closed or not * These evaluations were not considered for the interim analysis closed) by or on Week 12 was analyzed overall and by center. (0199 Sample Size Calculations Statistical significance in frequency between treatment (0200. The sample size was calculated with reference to the groups were assessed by Fisher's Exact test (two-tailed) to primary endpoint (comparing the reduction in wound size as determine the overall wound healing frequencies. This was measured by the mean percentage decrease from Baseline to followed by the Cochran-Mantel-Haenszel test which com the last visit). Assuming a 25% difference in favor of subjects pared the 2 treatment groups with respect to the incidence of treated with GranexinTM Gel, power of 80%, significance 100% wound closure after adjusting for pooled center. The level of 95% (two-sided) and Standard Deviation (SD) of Breslow-Day test was used in conjunction with the Cochran 40%, 40 subjects were calculated to be required in each Mantel-Haenszel test to determine the statistical significance treatment group. Adjusting for 15% dropout rate, 92 Subjects of a treatment by-pooled-center interaction were required to be enrolled in the study. 0207 Results 0201 All statistical tests were carried out as two-sided on 5% level of significance unless otherwise stated. Summary 0208. Mean Percent Wound Closure from Baseline to tables and descriptive statistics were done for demographics, Week 12 efficacy, and safety variables. To adjust the main efficacy 0209. The primary efficacy endpoint of the study was to analyses for measured covariates at Baseline, Analysis of evaluate mean percent wound closure from Baseline to Week Co-Variance (ANCOVA) and survival analysis were per 12. The non-parametric Wilcoxon Mann-Whitney Utest was formed. The descriptive statistics for continuous variables used to analyze mean percent reduction of wound area from were presented with number (N) of non-missing observa Baseline to Week 12 in both the treatment groups since the tions, mean, SD, median, minimum and maximum or range. data did not follow a normal distribution, as confirmed by a For categorical data, descriptive statistics were presented Shapiro-Wilk p-value of <0.0001 and a non-linear Q-Q plot. with number of exposed subjects and number (N) with per The addition of the ranks of both groups obtained after sorting centage of observations in the various categories of the end the data of wound size reduction in ascending order in the PP point, where percentage was based on the exposed Subjects. population showed that the actual sum of scores (1036.00) 0202 Primary efficacy analysis was performed on Intent was higher in Group A Subjects than the expected Sum of to-Treat (ITT) and Per Protocol (PP) populations. Primary scores i.e. sum of scores when there is no difference in 2 efficacy data was calculated using sample number (n), mean, groups, under the null hypothesis (899.00) and in Group B, the actual sum of scores (617.00) was found to be lower than SD, median, minimum, maximum, and 95% Confidence the expected sum of the scores under the null hypothesis Interval (CI). (754.00). Therefore, in the PP population, subjects treated 0203 ANCOVA with repeated measures was performed with GranexinTM Gel plus SoC (Group A) had a higher per for comparison of mean percent reduction of wound area cent wound closure from Baseline to Week 12 compared to from Baseline compared to Week 12 between the two treat subjects treated with SoC alone (Group B), which was statis ment groups. Mean percent reduction of wound area from tically significantly (p-value=0.0069). Baseline to Week 12 was considered as response variable adjusted for the strata, wound duration, viable tissue, exudate 0210 Mean Percent Wound Closure from Baseline to level, and the Body Mass Index (BMI) as covariates and Week 4 treatment group, treatment by visit interaction, and visit as 0211 Similarly non-parametric analysis for secondary factor included in ANCOVA model. endpoint showed that in the PP population, the mean percent US 2013/0267471 A1 Oct. 10, 2013
reduction of wound area from Baseline to Week 4 was statis 100% and 50% epithelialization occurred at a higher fre tically significantly higher in Group A as compared to Group quency in wounds of subjects treated with GranexinTM Gel B (p-value=0.0128). than subjects treated with SoC only. 0212 Subject Self Assessment of Change in Intensity of 0220 Safety: Pain from Baseline to Week 12 0221. Overall, 21 subjects reported 26 AEs of which 18 0213. The intensity of pain experienced by the subjects (69.2%) AEs were mild in intensity. A total of 4 moderate and was recorded on a Visual Analogue Scale of 1 to 10 where 1 4 severe AEs were reported in this study. In Group A, death indicated “no pain' and 10 indicated “extreme pain'. The and foot fracture were the severe AEs and myocardial infarc intensity of pain as assessed by the subjects of the PP popu tion and inadequate control of diabetes mellitus were severe lation at treatment Week 12 of the study showed using the AES reported in Group B. None of the AES reported in this Wilcoxon Mann-Whitney Utest that there was no statistically study were considered probably or possibly related to significant difference in Subject self assessment of intensity GranexinTM Gel or the SoC treatment. Of the 26 events, 18 of pain at Week 12 between the 2 treatment groups (Group A, (69.2%) recovered and 3 (11.5%) events recovered with 0.4+1.22; Group B, 0.3+0.81; p-value=0.9683). sequelae. No action was taken against 22 AES. None of the 0214 Time-to-Wound Closure Analysis subjects withdrew due to an AE. Five subjects reported 5 0215. The time taken for complete wound closure in the Serious Adverse Events (SAEs). In Group A, 1 death was PP population was statistically significantly lower in subjects reported due to an unknown cause and 1 death in Group B in Group A as compared to subjects in Group B (p-value=0. occurred due to myocardial infarction. None of the SAEs, 0057). Of the 32 subjects in Group A, 81.3% subjects had including the deaths, were related to GranexinTM Gel. complete (100%) wound closure and 18.8% subjects who did 0222. This phase II study was a double blind, randomized, not, were censored. The median duration of 100% wound prospective, parallel group, multi-center study conducted at 8 closure in Group A was 6 weeks. Of the 28 subjects in Group centers in India to evaluate the efficacy and safety of B, 53.6% subjects had complete wound closure and 46.4% GranexinTM Gel in the treatment of DFU. The total duration of subjects not having 100% wound closure, were censored and the study was 12 months, including 6 months enrollment the median duration of time to complete 100% wound closure period and 6 months for study procedures (3 months each for was 14.64 weeks. efficacy and safety analyses). Hence, the study duration for an 0216. The time taken to achieve 50% wound closure by individual subject was approximately 6 months, including 18 Week 12 in the PP population was not statistically signifi visits. For interim analysis, data up to 12 weeks (Visit 15) was cantly lower in subjects in Group A as compared to the time considered. A total of 92 subjects were randomized in the taken by subjects in Group B (p-value=0.2084). The analysis study to receive Granexin TM Gel plus SoC or only SoC in the of time taken for complete 50% wound closure by Week 12 treatment of DFU. Of these subjects, 76 (82.6%) completed showed that of the 32 subjects in Group A, 93.8% subjects the study treatment visits up to Visit 15 and are now in the completed 50% wound closure and 6.3% subjects did not follow-up stage of the study. complete 50% wound closure and were censored. In compari 0223) The primary efficacy endpoint of the study was to son to that, out of 28 subjects in Group B, 89.3% subjects had evaluate mean percent wound reduction from Baseline to 50% wound closure and 10.7% subjects without at least 50% Week 12. The non-parametric Wilcoxon Mann-Whitney U wound closure during the study period were censored. The test was used to analyze mean percent reduction of wound median duration of 50% wound closure was 2 weeks in Group area from Baseline to Week 12. In the PP population, subjects A and 4.07 weeks in Group B. treated with GranexinTM Gel plus SoC (Group A) showed 0217 Cox Proportional Hazard Regression Analysis for statistically significantly higher percent wound closure from time to 100% wound closure in the PP population showed that Baseline to Week 12 compared to subjects treated with SoC though treatment was not a statistically significant factor alone (Group B) (p-value=0.0069), suggesting that at Week affecting the wound closure (p-value=0.0856), the subjects in 12 wounds treated with GranexinTM Gel plus SoC healed Group A treated with GranexinTM Gel plus SoC would have better than the wounds treated with SoC alone. wound closure 2.339 times more than the subjects in Group B 0224. The secondary endpoints of the study were to assess treated with SoC only. Similarly, baseline wound depth and mean percent wound closure at Week 4. Subject assessment of BMI were not statistically significant factors (baseline wound pain, and time to complete wound closure. At Week 4. Group depth: p-value=0.4554, BMI: p-value=0.4435) but would A subjects in the PP population showed statistically signifi affect wound closure by 1.715 times and 1.050 times, respec cantly higher percent reduction of wound area from Baseline tively. All other parameters, including wound size, wound compared to Group B subjects in the PP population. There duration, and use of dressings did not have a statistically was no statistically significant difference in intensity of pain significant effect on the rate of wound closure for both the as assessed by the subject from Baseline to Week 12 between treatmentS. the 2 treatment groups. Hence, GranexinTM Gel did not affect 0218 Incidence of 100% Wound Closure by or on Week the intensity of pain experienced by the Subjects as recorded 12 on a Visual Analogue Scale of 1 to 10. 0219. The categorical analysis of incidence of 100% and 0225 Complete wound closure was defined as 100% epi 50% wound closure by or on Week 12 in the PP population thelialization of the wound and the absence of drainage from was done using the Cochran-Mantel-Haenszel analysis with the wound. In the PP population, subjects in Group A took Breslow-Day test and the Chi-square test. The Chi-square test significantly less time to achieve 100% wound closure than performed by combining the data from all the study centers those in Group B. However, the time taken for 50% wound showed that there were statistically significantly higher num closure was not significantly lowerin Group A as compared to ber of responders in Group A than in Group B (100% wound Group B. The categorical analysis of incidence of 100% and closure: p-value=0.01.04: 50% wound closure: p-value=0. 50% wound closure at Week 12 was conducted and the sub 0063). Therefore it can be concluded that the frequency of jects who had 100% wound closure were considered as US 2013/0267471 A1 Oct. 10, 2013 responders. The number of responders in Group A was sig of the study at Day 0, then the subject was not eligible for nificantly higher than in Group B, indicating that the wounds participation in the study. The following procedures were treated with GranexinTM Gel plus SoC have higher incidences performed at Baseline: of 100% and 50% epithelialization than wounds treated with Inclusion and exclusion criteria checked to confirm that no SoC alone. change had occurred since Screening 0226 Cox Proportional Hazard Regression Model was used to compare the event-time distribution function between Randomization the treatment groups. Although treatment was not a signifi 0232 Photography of the ulcer cant factor affecting time to 100% wound closure, in the PP Wound tracing population Group A Subjects demonstrated 2.3 times higher Clinical assessment wound closure than Group B subjects. In addition, baseline Compliance checked wound depth and BMI were not significant factors but Wound closure assessment affected wound closure by 1.715 times and 1.050 times, Wound bed preparation respectively. All other parameters, including wound size, Treatment with GranexinTM Gel (if applicable) wound duration, and use of dressings did not have a statisti Dressing changed cally significant difference in affecting wound closure for Pain assessment both the treatments. Cox Proportional Hazard Regression Recorded changes in concomitant medication Analysis for time to 50% wound closure showed that none of the covariates (treatment group, wound size, wound duration, Recorded Adverse Events (AEs), if any baseline wound depth, and BMI) were significantly associ ated with time to 50% wound closure. However, Group A 0233. Visit 3-14: Treatment Visits (Day 3 through Week subjects had wound closure 1.186 times more than Group B 11) Subjects. On Treatment Visits were conducted at Day 3 followed by 0227. In terms of safety, out of 92 subjects, 21 (22.8%) Week 1, 2,3,4,5,6,7,8,9, 10, and 11 after Baseline Visitil subjects reported 26 AEs. There was no significant difference day. The following procedures were performed at these visits: in the number of subjects with at least 1 AE between the 2 Photography of the ulcer treatment groups (Group A, 11: Group B, 10; p-value=0. Wound tracing 8038). A total of 4 AEs were of severe intensity. None of the Clinical assessment AES were related to the GranexinTM Gel or SOC. None of the Compliance checked subjects withdrew due to an AE. A total 5 (5.4%) subjects Wound closure assessment reported 5 SAEs where 2 SAEs were observed in Group A Wound bed preparation subjects and 3 SAEs were in Group B subjects. In Group A, 1 Treatment with GranexinTM Gel (if applicable) death was reported due to an unknown cause and 1 death in Dressing changed Group B occurred due to myocardial infarction. None of the Pain assessment SAEs were related to GranexinTM Gel or SOC. Recorded changes in concomitant medication 0228. In conclusion, GranexinTM Gel along with SoC has shown to accelerate wound healing in subjects with DFU, Recorded AEs, if any making it an efficacious, safe, and well tolerated therapeutic 0234 Visit 15: End of Treatment Visit (Week 12) option for the treatment of DFU. The End of Treatment Visit was conducted at Visit 15 (Week 0229. The description of 18 visits along with premature 12+1 day). The following procedures were performed at this study discontinuation and unscheduled visits is Summarized visit: below: Laboratory tests 0230 Visit 1: Screening (Day -7) Immunology tests The Screening Visit was held a maximum of 7 days prior to Photography of the ulcer the Baseline Visit. The following procedures were performed Wound tracing at the Screening Visit: Clinical assessment Screening Procedures: informed consent, medical history, Compliance checked physical examination including neuro-vascular examination, Wound closure assessment Vitals, ulcer identification and history, assessment of wound Wound bed preparation infection, X-rays, Electrocardiogram (ECG), and serum Treatment with GranexinTM Gel (if applicable) pregnancy test Dressing changed Inclusion and exclusion criteria checked Pain assessment Laboratory tests Recorded changes in concomitant medication Additional end of treatment visit evaluations (assessment of Immunology tests ulcer response to treatment, comparison of current treatment Photography of the ulcer to previous treatment, incidence of ulcer recurrence within Wound debridement the 12-week treatment period, and vital signs) Wound tracing Wound bed preparation Recorded AEs, if any Recorded the use of concomitant medication 0235 Visit 16-18: Follow-up Visits, including End of Recorded the presence of concomitant illness(es) Study Visit (Month 4 through 6) The Follow-up visits would 0231. Visit 2: Baseline (Day 0) be conducted at Month 4-3 days, Month 5+3 days, and Month The Baseline Visit was held a maximum of 7 days after 6+3 days (End of Study EOS visit). The following proce Screening. If ulcers healed by >30% within 7 days of the start dures would be performed at these visits: US 2013/0267471 A1 Oct. 10, 2013 20
Photography of the ulcer healing and reduced scarring. In addition, the low molecular Pain assessment weight (25 amino acid) peptide is expected to reduce the risk Recorded changes in concomitant medication of an immunogenic response. Follow-up procedures (evaluation of ulcer recurrence, care of 0246 GranexinTM Gel is also designed to provide a local unhealed ulcers, assessment of healed wounds, and Subject ized protective barrier against microbial colonization and a assessment of pain) moist environment to promote natural autolytic debridement of necrotic tissue surrounding the wound. The lot number of Recorded AEs, if any GranexinTM Gel used in this study was DP 1493. 0236 EOS procedures (only at Visit 18) 0247 Subjects were randomized 1:1 to either Group A 0237 GranexinTM Gel is a topical hydrogel that contains (GranexinTM Gel plus SoC) or Group B (SoC only). Interac the active ingredient ACT1 peptide (100 uM). GranexinTM tive Web Response System (IWRS) was used for randomiza Gel has greater than 90% water, which provides a barrier to tion of the eligible subjects to the treatment group. The ran infection or external elements while maintaining a moist domization was further stratified by wound size (<10 cm and environment for the wound. In Group A, the ulcer was >10 cm) to avoid bias of undue number of subjects with cleansed with Sterile saline and if any bleeding existed, it was Small or large wounds going into one arm of the study. The stopped before applying the GranexinTM Gel on Days 0 and 3, subjectID, initials and study Day 0 date were written on the and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The randomization code sheet and filed with the subjects CRF. GranexinTM Gel was dispensed directly from its 5 ml glass Randomization lists were prepared centrally by the MNI data vial(s) to completely cover the wound bed. With a clean management center using a validated computer program (Sta sterile cotton Swab, Sterile tongue depressor, or similar appli tistical Analysis Software SAS 9.1.3). cation aid, the GranexinTM Gel was evenly spread over the 0248. Two studies, including a preclinical and clinical wound Surface. The treatment site was then dressed according study tested the efficacy and safety of 100 uM concentration to the following regimen: of ACT1 formulated in GranexinTM Gel. In the preclinical 0238 Used adequate off-loading other than TCC as per study, the efficacy of GranexinTM Gel in wound healing was the Investigator's judgment assessed on 5 mm diameter full thickness excisional wounds 0239 Applied a non-adherent dressing over the ulcer in adult mice and pigs. Wounds treated with 100 uM of ACT1 and GranexinTM Gelwhich extended 0.5 inch beyond the peptide closed faster, appeared less Swollen and inflamed, and ulcer perimeter and inflamed skin margins healed with a smoother and less discolored appearance as 0240 Applied a non-occlusive dressing such as fine compared to the wounds applied with the vehicle control 18. mesh gauze which was either folded or rolled as a bolster Subsequently, a phase I, double blind, single-center, con 0241 Standard of Care (Control) Treatment trolled study was conducted to evaluate the safety and toler In the control group, the ulcer was cleansed with sterile saline ability of 20, 50, 100, and 200 uM concentrations of ACT1 and if any bleeding existed, it was stopped before dressing. formulated in GranexinTM Gel versus placebo in 48 healthy The ulcer was dressed according to the following regimen: subjects following punch biopsy. The results showed the 0242 Used adequate off-loading other than TCC as per GranexinTM Gel (ACT 1) at each concentration to be safe and the Investigator's judgment well tolerated with no evidence of localized or systemic AEs 0243 Applied a non-adherent dressing such as the 4 or Serious Adverse Events (SAEs). Both preclinical and clini layer dressing over the ulcer with either Soframycin, cal studies concluded 100 uM concentration of the ACT1 Lyseal ointment or hydroheal AM gel which extended peptide to be optimum in safety and effectiveness. Hence, the 0.5 inch beyond the ulcer perimeter and inflamed skin dose of 100 uMACT1 of GranexinTM Gel was selected in the margins treatment of DFU in this study. 0244. Applied a non-occlusive dressing such as pres 0249 For each subject, GranexinTM Gel (100 uMACT1) Sure bandage or fine mesh gauze which was either folded was applied at Days 0 and 3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, or rolled as a bolster 9, 10, 11, and 12 0245 GranexinTM Gel is a topical gel with the active drug (0250) Efficacy and Safety Variables: substance ACT1 peptide developed at FirstString Research 0251. The primary efficacy variable was mean percent for the management of wounds. ACT1 is a synthetic peptide wound closure from Baseline to Week 12 (25 amino acid) designed to mimic the Carboxy (C)-terminus of the ubiquitous transmembrane gap junction protein Con (0252) Secondary Efficacy Variables nexin 43 (CX43), with high binding specificity to Zona 0253 Mean percent wound closure at 4 weeks occludens, a cytoplasmic tight junction protein. While the 0254 Subject self-assessment of intensity of pain (till mechanism of action of ACT1 has not been completely elu Week 12) cidated, the peptide is soluble and engineered to directly 0255 Time to 50% wound closure translocate within cells. ACT1 peptide interacts with a known 0256 Time to complete wound closure with 100% epi binding partner of CX43—the Postsynaptic Density 95/Discs thelialization of the wound Large/Zonula Occludens (PDZ)-2 domain of Zonula Occludens-1 (ZO-1). The binding of the peptide to specific 0257 Safety Variable: CX43 C-terminus interaction domains such as PDZ-2 on 0258. The safety variable was incidence of treatment ZO-1 serves to competitively inhibit its association with the related Adverse Events (AEs) (till Week 12). CX43 C-terminus. Connexins are gap junction proteins where 0259 An AE was defined as “any untoward medical occur as ZO-1 is a tight junction associated protein. The binding of rence in a Subject or clinical investigation Subject adminis the 25 amino acid ACT1 peptide to the PDZ-2 domain of tered a pharmaceutical product that did not necessarily have a ZO-1 serves to stabilize gap junctions as well as tight junc causal relationship with this treatment” (ICHE6:1.2). An AE, tions. This junctional stability has been associated with faster therefore, was any unfavorable and unintended sign, Symp US 2013/0267471 A1 Oct. 10, 2013
tom, or disease temporally associated with the use of a TABLE 9-continued medicinal product, whether or not considered related to the medicinal product. Disposition of Subjects 0260 A clinically significant abnormal laboratory finding was also regarded as an AE and it was the responsibility of the Treatment Group Investigator to review the laboratory test results and deter mine whether an abnormal laboratory value was of clinical Group A Group B Overall significance. In general, clinically significant laboratory val Category (N = 46) (N = 46) (N = 92) ues which Suggested disease progression and/or required active management were considered as AES. Total number of subjects not 9 (19.6) 7 (15.2) 16 (17.4) 0261 Worsening of a pre-existing medical condition (e.g. completed the study (Visit 15) diabetes, migraine, headaches, and gout) was considered as an AE if there was either an increase in severity, frequency, Note: duration of the condition, or an association with significantly Percentage was calculated taking respective column header group count as denominator, worse outcomes. Pre-planned procedures and pre-existing conditions that did not worsen were not considered AEs. 0272. The following were the reasons, which led to the 0262 A Serious Adverse Event (SAE) was defined as an exclusion of 32 subjects from the PP population: Consent AE that: withdrawal (13 subjects); Lost to follow up (2 subjects): 0263 Was fatal 0264 Was life threatening (places the subject at imme Death (2 subjects); Drop outs (1 subject); Protocol deviation diate risk of death) (s): Decrease or increase in the ulcer size by 30% or more 0265 Required in-subject hospitalization or prolonga during 7 day Screening period (14 Subjects) tion of existing hospitalization 0273 ITT Population: 0266 Resulted in persistent or significant disability/ incapacity 0274 All subjects randomized to any treatment group, 0267 Was a congenital anomaly/birth defect received at least one dose of study medication or reference 0268 Met other significant medical hazard treatment and performed study assessments within and/or 0269. An AE necessitating hospitalization met the regula outside the time window period specified in the protocol. The tory definition for “serious” if the in-subject hospital admis assessments performed outside time window period were sion included a minimum of an overnight stay in a health care re-assigned to the closest nominal time point of actual sample facility. Any AE that did not meet one of the definitions of collection serious (e.g. an AE requiring an emergency room visit, out 0275 PP Analysis Population: Subject Surgery, or requiring urgent investigation) was con sidered by the Investigator to meet the “other significant 0276 All subjects who completed study assessments medical hazard criterion for classification as an SAE. within the protocol defined window period for the assigned 0270. If the above interventions were performed as SoC randomized time point and also completed all study assess and not associated with an AE, the health issue for which the ments until Week 12. This included subjects who came for all intervention was performed was not considered as an SAE. If the visits within or outside the window period, including there was a complication as a result of the procedure and the subjects whose wounds had healed before Week 12. Subjects complication met at least 1 seriousness criterion, then that who had a major protocol deviation were excluded from the complication was reported as an SAE. PP population. This included subjects who had an increase or (0271 Study Subjects: A total of 98 subjects with DFU decrease of more than 30% in wound size during the screen were screened. Of these, 6 were screen failures. The remain ing period of 7 days (per exclusion criteria no. 1 in the pro ing 92 subjects were randomized in the study. Of these sub tocol) jects, 76 (82.6%) completed the study treatment visits up to Visit 15 and are now in follow-up. The remaining 16 subjects (0277 Safety Population: did not complete the study treatment visits up to Visit 15 0278 All subjects who were randomized in the study to (Table 9). either of the treatment groups were used for the safety analy sis TABLE 9 (0279. The ITT population included 91 subjects; the PP Disposition of Subiects population included 60 Subjects; and the safety population Treatment Group included 92 subjects. (0280 Primary Efficacy Endpoint: Group A Group B Overall Category (N = 46) (N = 46) (N = 92) (0281 Mean Percent Wound Closure from Baseline to Population, n (%)"l Week 12
Screened 98 0282 Parametric Analysis: Table 10 Summarizes the para Randomized 46 (100.0) 46 (100.0) 92 (100.0) metric analysis of the mean percent reduction of wound area Intent-to-treat 46 (100.0) 45 (97.8) 91 (98.9) Per-Protocol 32 (69.6) 28 (60.9) 60 (65.2) from Baseline to Week 12 in the ITT population. Of 91 Total number of subjects completed 37 (80.4) 39 (84.8) 76 (82.6) subjects in the ITT Population, non-missing observations for the study (Visit 15) change from Baseline to Week 12 for percent reduction of wound area were available for 63 subjects. The mean US 2013/0267471 A1 Oct. 10, 2013 22 percent-SD reduction of wound area from Baseline to Week TABLE 11-continued 12 was higher in Group A than Group B (Group A: 72.12%+128.52: Group B: 57.09%+80.88) but was not sta tistically significant (p-value=0.6567) as per parametric Mean Percent Reduction of Wound Area from Baseline to Week 12 analysis. (Parametric analysis) PP Population (N = 60)
TABLE 10 Treatment Group
Mean Percent Reduction of Wound Area from Baseline to Week 12 Group A Group B Parametric analysis) ITT Population (N = 91 Statistics (N = 32) (N = 28) Treatment Group 95% CI (L.:U.) (6.18:71.54) Group A Group B p-valuell-ll O.O2O2 Statistics (N = 46) (N = 45)
Total number of subjects evaluated N = 631 Note: Percentreduction of wound area from Baseline to Week 12 IkTotal no. of subjects evaluated” represents subjects having non-missing observations for change from Baseline to Week 12 for percent reduction of wound area l 34 29 PDifference estimate for Granexin TM Gel plus SoC indicated (Granexin TM Gel plus SoC) Mean 72.121 57.086 - SoC). SD 128.5222 80.8795 PlSE of Granexin TM Gel plus Standard of Care indicated Standard Error of Differences Median 1OO.OOO 1OOOOO (Granexin TM Gel plus SoC) - SoC). Range (Min.:Max.) (-648.91:100.00) (-271.50:100.00) 4 p-value for GranexinTM Gel plus SoC indicated significance of treatment differences LS Mean Estimate SS.490 44.O32 (Granexin TM Gel plus SoC) - (SoC). ANCOVA mixed model with repeated measures was used to compare average percent Difference Estimatell 114575 reduction in wound area with treatment, visit and treatment visit as factors and strata, SEl 25.6968 wound duration, viable tissue, exudate level and the body mass index as covariates using 95% CI (L.:U.) (-39.53:62.44) PROC Mixed procedure of SAS software. p-valuell-ll O.6567 General Note: Group A indicates Granexin TM Gel plus SoC and Group B indicates SoC only IkTotal no. of subjects evaluated” represents subjects having non-missing observations for change from Baseline to Week 12 for percent reduction of wound area SD: Standard Deviation, PDifference estimate for Granexin TM Gel plus SoC indicated (Granexin TM Gel plus SoC) Min: Minimum, - SoC). PlSE of Granexin TM Gel plus Standard of Care indicated Standard Error of Differences Max: Maximum, (Granexin TM Gel plus SoC) - SoC). p-value for Granexin TM Gel plus SoC indicated significance of treatment differences CI: Confidence Interval, (Granexin TM Gel plus SoC) - (SoC). ANCOVA mixed model with repeated measures was used to compare average percent L: Lower Limit, reduction in wound area with treatment, visit and treatment visit as factors and strata, wound duration, viable tissue, exuda to leveland the BMI as covariates using PROC Mixed U: Upper Limit procedure of SAS software. Missing data was accounted by using repeated measures analysis, 0283. The parametric analysis of the mean percent reduc Mean percent reduction from Baseline = ((Baseline - week X); Baseline) * 100 tion of wound area from Baseline to Week 12 in the PP population is summarized in Table 11. Of the 60 subjects in 0284. To confirm the applicability of parametric statistical the PP population, non-missing observations for change from inference methodology in the wound reduction analysis, it Baseline to Week 12 for reduction of wound area were avail was tested whether the data followed normal distribution. able for 57 subjects. The mean percent-SD reduction of Using PROCUNIVARIATE of SASR (version 9.1.3) Sha wound area from Baseline to Week 12 was statistically sig piro-Wilk W statistic was calculated. The Shapiro-Wilk nificantly higher in Group A than in Group B (Group A: p-value <0.0001 and this indicated that the data did not follow 93.58%+17.67: Group B: 52.14%+84.14; p-value=0.0202). the normal distribution. Therefore, the hypothesis of normal distribution of the data in the sample was rejected and non TABLE 11 parametric analysis was also conducted.
Mean Percent Reduction of Wound Area from Baseline to Week 12 0285 Table 12 Summarizes the non-parametric analysis Parametric analysis) PP Population (N = 60 for mean percent reduction of wound area from Baseline to Week 12 in the ITT population. The Wilcoxon Mann-Whit Treatment Group ney Utest is a non-parametric test which determines whether Group A Group B there is a difference in two samples of independent observa Statistics (N = 32) (N = 28) tions. The actual sum of scores (1215.00) obtained by adding Total number of subjects evaluated N57ll the ranks of both groups after Sorting the data of wound size Percentreduction of wound area from Baseline to Week 12 reduction in ascending order, was found higher in Group A Subjects than the expected Sum of Scores i.e. sum of Scores l 31 26 Mean 93.582 52.135 when there is no difference in two groups, under the null SD 17.6708 84.1427 hypothesis (1088.00) and in Group B, the actual sum of scores Median 1OO.OOO 1OOOOO (801.00) was found lower than the expected sum of the scores Range (Min.:Max.) (27.61:100.00) (-271.50:100.00) LS Mean Estimate 69.038 30.178 under the null hypothesis (928.00). Subjects in Group A had Difference Estimate 38.86OS statistically significantly higher percent reduction of wound SEI3] 16.4998 area from Baseline to Week 12 than Group B subjects (p-value=0.0287). US 2013/0267471 A1 Oct. 10, 2013
TABLE 12 TABLE 1.4
Mean Percent Reduction of Wound Area from Baseline to Week 12 Mean Percent Reduction of Wound Area from Baseline to Week 4 (Non-parametric analysis) IIT Population (N = 91) (Parametric analysis) ITT Population (N = 91) Treatment Group Treatment Group Group A Group B Statistics (N = 46) (N = 45) Group A Group B Statistics (N = 46) (N = 45) Percentreduction of wound area from Baseline to Week 12 Total no. of subjects evaluated N = 6Ol Sum of Score 1215.OO 801.00 Percent reduction of wound area from Baseline to week 4 Expected Sum of Scores under H. 1088.00 928.00 p-value O.O287 N 33 27
Note: Mean 53.306 52.920 p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between SD 126.2293 42.6OO1 treatment group, Median 81.414 61484 General Note: Mean percent reduction from Baseline = ((Baseline - week X). Baseline) * 100 Range (Min.:Max.) (-626.65:100.00) (-72.94:100.00) H: Null Hypothesis LS Mean Estimate 46.931 41.098 Difference Estimatell 5.8329 0286 Table 13 summarizes the non-parametric analysis SEl 26.072O for mean percent reduction of wound area from Baseline to 95% CI (L.:U.) (-45.87:57.54) Week 12 in the PP population. The addition of the ranks of p-valuell-l O.8234 both groups obtained after sorting the data of wound size Note: reduction in ascending order gave the actual sum of scores as Total no. of subjects evaluated” represents subjects having non-missing observations for 1036.00 which was found to be higher in Group A subjects all the dependent and covariate variables *Difference estimate for Granexin TM Gel plus SoC indicated (Granexin TM Gel plus SoC) than the expected sum of scores (899.00) i.e. sum of scores - SoC). when there is no difference in the two groups, under the null lSE ofGranexin TMGel plus SoC indicated Standard Error of Differences (Granexin TMGel hypothesis and in Group B the actual sum of scores (617.00) plus SoC) - SoC). "p-value for GranexinTM Gel plus SoC indicated significance of treatment differences was found to be lower than the expected sum of the scores (Granexin TM Gel plus SoC) - (SoC). under the null hypothesis (754.00). Subjects in Group A had ANCOVA mixed model with repeated measures was used to compare average percent reduction in wound area with treatment, visit and treatment visit as factors and strata, statistically significant higher percentage of reduction of wound duration, viable tissue, exudate level and the body mass index as covariates using wound area from Baseline to Week 12 than Group B subjects PROC Mixed procedure of SAS software. (p-value=0.0069). 0288 The parametric analysis of mean percent reduction TABLE 13 of wound area from Baseline to Week 4 in the PP population is summarized in Table 15. Of the 60 subjects in the PP Mean Percent Reduction of Wound Area from Baseline to Week 12 population, non-missing observations from Baseline to Week Non-parametric analysis) PP Population (N = 60 4 were available for a total of 49 subjects. The mean Treatment Group percent-SD reduction of wound area from Baseline to Week 4 was 73.49%+35.52 in Group A subjects and 47.43%+43.61 Group A Group B in Group B subjects; but the reduction was not statistically Statistics (N = 32) (N = 28) significantly higher in Group A Subjects than Group B Percentreduction of wound area from Baseline to Week 12 (p-value=0.0871). Sum of Score 1036.OO 617.00 Expected Sum of Scores under H. 899.OO 754.00 TABLE 1.5 p-value O.OO69 Mean Percent Reduction of Wound Area from Baseline to Week 4 p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between Parametric analysis) PP Population (N = 60 treatment group, General Note: Mean percent Reduction from Baseline = ((Baseline - week X). Baseline) * 100 Treatment Group H: Null Hypothesis Group A Group B Statistics (N = 32) (N = 28) 0287. The parametric evaluation of mean percent wound Total no. of subjects evaluated N = 491 closure from Baseline to Week 4 in the ITT population is Percent reduction of wound area from Baseline to week 4 summarized in Table 14. Of 91 subjects in the ITT population, N 26 23 non-missing observations from Baseline to Week 4 for per Mean 73.487 47.430 cent reduction of wound area were available for 60 subjects. SD 35.5187 43.6110 The mean percent-SD reduction of wound area from Base Median 88.167 56.931 Range (Min.:Max.) (-40.60:100.00) (-72.94:100.00) line to Week 4 was 53.30%+126.23 in Group A subjects and LS Mean Estimate 53.184 24.019 52.92%-42.60 in Group B subjects; but the reduction in Difference Estimate 29.1649 Group A was not statistically significant compared to Group SEI3] 16.9134 B (p-value=0.8234). US 2013/0267471 A1 Oct. 10, 2013 24
TABLE 15-continued TABLE 17
Mean Percent Reduction of Wound Area from Baseline to Week 4 Parametric analysis) PP Population (N = 60 Mean Percent Reduction of Wound Area from Baseline to Week 4 (Non-parametric analysis) PP Population (N = 60) Treatment Group Treatment Group Group A Group B Statistics (N = 32) (N = 28) Group A Group B 95% CI (L.:U.) (-4.31:62.64) Statistics (N = 32) (N = 28) p-valuePll O.O871
Note: Percent reduction of wound area from Baseline to week 4 IkTotal no. of subjects evaluated” represents subjects having non-missing observations for all the dependent and covariate variables PDifference estimate for Granexin TM Gel plus SoC indicated (Granexin TM Gel plus SoC) Sum of Score 773.00 452.OO SoC), Expected under H. 6SO.OO 575.00 PlSE of Granexin TM Gel plus SoCindicated Standard Error of Differences (Granexin TMGel Bl SoC) - SoC). p-value O.O128 "p-value for Granexin TM Gel plus SoC indicated significance of treatment differences Granexin TM Gel plus SoC) - (SoC). ANCOVA mixed model with repeated measures was used to compare average percent Note: reduction in wound area with treatment, visit and treatment visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using p-value was evaluated by using non-parametric WilcoxonMann-Whitney Utest between PROC Mixed procedure of SAS software. treatment group, General Note: 0289 Table 16 summarizes the non-parametric analysis Mean percent Reduction from Baseline = ((Baseline - week X). Baseline) * 100 for mean percent reduction of wound area from Baseline to H: Null Hypothesis Week 4 in the ITT population. In Group A, the expected sum of Score under the null hypothesis i.e. Sum of scores when 0291. The intensity of pain experienced by the subjects there is no difference in two groups was 1006.50 and the was recorded on a Visual Analogue Scale of 1 to 10 where 1 actual sum of score was 1148.00 obtained by adding the ranks indicated “no pain' and 10 indicated “extreme pain'. The of both groups after sorting data of wound size reduction in Summary of Subject self assessment of intensity of pain at all ascending order, and for Group B, it was 823.50 and 682.00 visits from Baseline to Week 12 for the ITT population have for expected and actual, respectively. Group A subjects had a been given in FIG. 2. statistically significantly higher percent reduction of wound 0292 Table 18 summarizes the intensity of pain as area than Group B subjects (p-value=0.0341). assessed by the subject at treatment Week 12 of the study. There was no statistically significant difference in the mean TABLE 16 intensity of pain from Baseline to Week 12 between the 2 Mean Percent Reduction of Wound Area from Baseline to Week 4 treatment groups (Group A, 0.5-1.46; Group B, 0.3+0.93; Non-parametric analysis) ITT Population (N = 91 p-value=0.8484).
Treatment Group TABLE 1.8 Group A Group B Analysis of Subject Self Assessment of Intensity of Pain at Week 12 ITT Statistics (N = 46) (N = 45) Population (N = 91 Percentreduction of wound area from Baseline to Week 4 Treatment Group Sum of Score 1148.00 682.OO Expected Sum of Score under Ho 1OO6.50 823.50 Group A Group B p-valuel O.O341 Statistics (N = 46) (N = 45)
Note: Intensity of pain at Week 12 p-value was evaluated by using non-parametric WilcoxonMann-Whitney Utest between treatment group, N 36 38 General Note: Mean O.S O.3 Mean percent reduction from Baseline = ((baseline - week X); baseline) * 100 SD 1.46 O.93 H: Null hypothesis Median O.O O.O Range (Min.:Max.) (0:6) (0:4) Missing 1 O 0290 The non-parametric analysis for mean percent p-valuel O8484 reduction of wound area from Baseline to Week 4 in the PP Note: population is summarized in Table 17 The expected sum of p-value was evaluated by using non-parametric WilcoxonMann-Whitney Utest between scores, which is the sum of scores when there is no difference treatment group, in two groups, under the null hypothesis was 650.00 and the actual sum of Scores, obtained by adding the ranks of both 0293 Table 19 summarizes the intensity of pain as groups after Sorting data of wound size reductioninascending assessed by the subject at treatment Week 12 of the study for order, was higher with 773.00 for subjects treated with the PP population. There was no statistically significant dif GranexinTM Gel plus SoC and in subjects treated with SoC ference in the mean intensity of pain at Week 12 between the only, the expected sum of score was 575.00 and the actual 2 treatment groups (Group A, 0.4+1.22; Group B, 0.3+0.81; score was lower with 452.00. The percent reduction of wound p-value 0.9683). The summary of subject self assessment of area in Group A was statistically significantly higher than intensity of pain at all visits from Baseline to Week 12 for the Group B subjects (p-value=0.0128). PP population is given in FIG. 3. US 2013/0267471 A1 Oct. 10, 2013 25
TABLE 19 TABLE 20-continued
Analysis of Subject Self Assessment of Intensity of Pain at Week 12 PP Summary of Time to Complete (100%) Wound Closure by Week 12 ITT Population (N = 60) Population (N = 91 Treatment Group Treatment Group Group A Group B Group A Group B Statistics) (N = 46) (N = 45) Statistics (N = 32) (N = 28) Median duration (weeks) of 6.00 (5.00; 11.00) 11.00 (9.00; 18.29) complete wound Intensity of pain at Week 12 closure (90% CI) p-valuel O.O.306 l 30 27 Note: Mean 0.4 O.3 Percentage was calculated taking count of corresponding treatment groups as denomina SD 122 O.81 tor. The median duration of complete wound closure was estimated by Kaplan-Meier method Median O.O O.O and also 90% confidence interwal is calculated for the median duration of 100% wound Range (Min.:Max.) (0:4) (0:4) closure, Plp-value is calculated using Log-rank test for treatment groups using Proc Lifetest proce Missing 1 O dure of SAS software p-value O.9683 General Note: Time to first complete wound closure where complete wound closure is defined as 100% epithelialization of the wound and the absence of drainage from the wound. Note: Censoring: Subjects without occurrence of complete wound closure during study period, drop outs and lost-to-follow up subjects is censored at the time of last follow-up visit. p-value was evaluated by using non-parametric WilcoxonMann-Whitney Utest between The data of 1 subjectin Group A and 2 subjects in Group B were considered as missing. They treatment group, had missing data after Screening or had missing visit date, 0294 The time taken for 100% wound closure for the ITT 0296. The time taken to complete (100%) wound closure population is Summarized in FIG. 4. Complete wound closure in the PP population is summarized in Table 21 and FIG. 5 was defined as 100% epithelialization of the wound and the displays the Kaplan-Meier plot for time to 100% wound absence of drainage from the wound. One Subject in Group A closure. Of the 32 subjects in Group A, 26 (81.3%) subjects had complete (100%) wound closure and 6 (18.8%) subjects and 2 subjects in Group B had the visit date missing and were were censored. Subjects who dropped out or were lost to not included in the analysis (Error! Reference source not follow-up were already excluded from the PP population and found. 4). Seventeen (37.0%) subjects in Group A and 25 Subjects who did not have complete wound closure during the (55.6%) subjects in Group B were censored. Of these, 2 study period were censored. The duration of wound closure (4.8%) subjects in Group B were lost-to-follow-up, 1 (2.4%) was in the range of 4 weeks to 10.86 weeks and the median subject in Group A dropped out, 9 (21.4%) subjects (Group A: duration was 6 weeks. Of the 28 subjects in Group B, 15 7: Group B: 2) were withdrawn, and 31 (73.8%) subjects (53.6%) subjects had complete wound closure and 13 (46. (Group A: 10; Group B: 21) did not have complete wound 4%) subjects were censored. The duration of wound closure closure during the study period. One patient withdrew from was in the range of 9 weeks to 18.29 weeks. The median the study but had complete wound closure and was therefore duration of time to complete wound closure along with 90% considered in the ITT population, but excluded from the PP CI was 14.64 weeks. The time taken for complete wound population. closure was lower in Group A as compared to Group B which was statistically significant (p-value=0.0057). 0295). In Group A, 28 (60.9%) subjects and 18 (40.0%) subjects in Group B had 100% wound closure. The median duration of 100% wound closure in Group A was 6 weeks TABLE 21 (90% CI was 5 to 11 weeks) and 11 weeks (90%CI:9 to 18.29 Summary of Time to Complete (100%) Wound Closure by Week 12 PP weeks) in Group B. In Group B, for 1 subject the assessments Population (N = 60 were performed outside the window period; the assessment Treatment Group was reassigned to the closest nominal time point which was Visit 15 at 12 weeks. Statistically significantly less time was Group A Group B taken by subjects in Group A to achieve 100% wound closure Statistics) (N = 32) (N = 28) than Group B subjects (p-value=0.0306). Number (%) of subjects with 26 (81.3%) 15 (53.6%) complete (100%) wound closure Number (%) of censored 6 (18.8%) 13 (46.4%) TABLE 20 Subjects Median duration (weeks) of 6.00 (4.00; 10.86) 14.64 (9.00; 18.29) Summary of Time to Complete (100%) Wound Closure by Week 12 ITT complete wound closure Population (N =91 (90% CI)(2) p-valuel 0.0057 Treatment Group Note: Group A Group B Percentage was calculated taking count of corresponding treatment groups as denomina Statistics (N = 46) (N = 45) tor Thee median duration of completecompl woundd closurecl was estimatedimated by Kaplan-Meier methomethod and also 90% confidence interwal was calculated for the median duration of 100% wound Number (%) of subjects with 28 (60.9%) 18 (40.0%) closure, complete (100%) p-value was calculated using Log-rank test for treatment groups using Proc Lifetestt wound closure procedure of SAS software Number (%) of censored 17 (37.0%) 25 (55.6%) Subjects 0297. The time taken to complete 50% wound closure by Week 12 in the ITT population is summarized in Table 22 and US 2013/0267471 A1 Oct. 10, 2013 26
FIG. 6 displays the Kaplan-Meier plot for time to 50% wound TABLE 23 closure in the ITT population. One subject in Group A and 2 Subjects in Group B had the visit date missing and were not Summary of Time to Complete (50%) Wound Closure by Week 12 PP included in the analysis (FIG. 6). In Group A, 9 (19.6%) Population (N = 60 subjects and 11 (24.4%) subjects in Group B were censored. Treatment Group Overall, of the 20 censored subjects, 2 (10.0%) subjects were Group A Group B lost-to-follow-up, 1 (5.0%) subject dropped out, 9 (45.0%) Statistics) (N = 32) (N = 28) subjects were withdrawn, and 8 (40.0%) subjects did not have Number (%) of subjects with 30 (93.8%) 25 (89.3%) 50% wound closure during the study period. 50% wound closure 0298. Thirty six (78.3%) subjects in Group A and 32 (71. Number (%) of censored subjects 2 (6.3%) 3 (10.7%) Median duration (weeks) of 2.00 (2.00; 3.14) 4.07 (3.00; 5.00) 1%) subjects in Group Bhad 50% wound closure. The median complete wound closure duration of 50% wound closure was 2 weeks (90% CI was 2 (90% CI)12 to 3.14 weeks) in Group A and 3.29 weeks (90% CI was 3 to 4.14 weeks) in Group B. The time taken for 50% wound Note: closure was not statistically significantly lower in Group A Percentage was calculated taking count of corresponding treatment groups as denomina tor than Group B (p-value=0.5433). The median duration of complete wound closure is estimated by Kaplan-Meier method and also 90% confidence interwal is calculated for the median duration of 100% wound closure, TABLE 22 Plp-value is calculated using Log-rank test for treatment groups using Proc Lifetest proce dure of SAS software Summary of Time to Complete 50% Wound Closure by Week 12 (0300. Additional to the Kaplan-Meier Model, Cox Pro ITT Population (N = 91) portional Hazard Regression Model was used to compare the event-time distribution function between the two treatment Treatment Group groups. This analysis was conducted to evaluate if one or more covariates were associated with the time to complete 100% or 50% wound closure. Table 24 Summarizes the Cox Group A Group B Proportional Hazard Regression Analysis of time to complete Statistics) (N = 46) (N = 45) wound closure in the ITT population. The effect of covariates estimated by the proportional hazard model is reported as Number (%) of subjects with 36 (78.3%) 32 (71.1%) hazard ratios. Though treatment was not a significant factor 50% wound closure affecting wound closure (p-value=0.2903), the subjects Number (%) of censored 9 (19.6%) 11 (24.4%) treated with GranexinTM Gel plus SoC would have wound Subjects closure 1.568 times more than the subjects treated with SoC Median duration (weeks) of 2.00 (2.00; 3.14) 3.29 (3.00; 4.14) only. All other parameters, including wound size, wound complete wound closure duration, baseline wound depth, and use of dressings did not (90% CI)12 affect wound closure in both the treatment groups. p-valuel O.S433 TABLE 24 Note: Analysis of Time to Complete (100%) Wound Closure Percentage was calculated taking count of corresponding treatment groups as denomina ITT Population (N =91 tor The median duration of complete wound closure was estimated by Kaplan-Meier method 95% CI for Hazard and also 90% confidence interwal is calculated for the median duration of 50% wound Hazard Ratio closure, Plp-value was calculated using Log-rank test for treatment groups using Proc Lifetestt Statistics) Ratio) Lower Upper p-value procedure of SAS software Treatment group 1.568 O.681 3.607 O.2903 Wound size O.999 O.997 1.OOO O.1265 0299. The time taken to complete 50% wound closure in Wound duration O.995 O.976 1.013 0.5754 the PP population is summarized in Table 23 and FIG. 7 Baseline wound depth O.782 O.239 2.558 O.6839 displays the Kaplan-Meier plot for time to 50% wound clo BMI O966 O.861 1.084 0.5592 sure in the PP population. Of the 32 subjects in Group A, 30 Use of dressings (93.8%) subjects completed 50% wound closure and 2 (6.3%) Note: subjects were censored. In Group A, the duration of wound lcox Proportional Hazard ratio with 95% confidence interval and p-value was calculated closure was in the range of 2 weeks to 3.14 weeks and the using PROCPHREG procedure. median duration was 2 weeks. Of the 28 subjects in Group B, 0301 Cox Proportional Hazard Regression Analysis for 25 (89.3%) subjects had 50% wound closure and 3 (10.7%) time to 100% wound closure in the PP population (Table 25) subjects who did not have at least 50% wound closure during shows that though treatment was not a significant factor the study period were censored. The duration of wound clo affecting wound closure (p-value=0.0856), the subjects sure was in the range of 3 weeks to 5 weeks. The median treated with GranexinTM Gel plus SoC would have wound duration of time to complete 50% wound closure was 4.07 closure 2.339 times more than the subjects treated with SoC weeks. The time taken for complete wound closure was not only. Similarly, baseline wound depth and BMI were not statistically significantly lower in Subjects in Group A as significant factors (baseline wound depth: p-value=0.4554, compared to the time taken by subjects in Group B BMI: p-value=0.4435) but would affect wound closure by (p-value=0.2084). 1.715 times and 1.050 times, respectively. All other param US 2013/0267471 A1 Oct. 10, 2013 27 eters, including wound size, wound duration, and use of TABLE 26 dressings did not affect wound closure in both the treatment groups. Overview of Adverse Events-Safety Population (N = 92 Treatment Group TABLE 25 Group A Group B Overall Analysis of Time to Complete (100%) Wound Closure (Cox's Proportional Statistics (N = 46) (N = 46) (N = 92) Hazards Regression Analysis) PP Population (N = 60 Total number of AEs 13 13 26 95% CI for Hazard reported Hazard Ratio Subjects reporting 11 (23.9%) 10 (21.7%) 21 (22.8%) Any AEs Statistics) Ratio) Lower Upper p-value Subjects reporting 1 AE 9 (19.6%) 7 (15.2) 16 (17.4%) Subjects reporting >1 AE 2 (4.3%) 3 (6.5%) 5 (5.4%) Treatment group 2.339 O.888 6.161 O.O856 Subjects reporting 35 (76.1%) 36 (78.3%) 71 (77.2%) Wound size O.999 O.997 1.OOO O.1703 No AEsl Wound duration O.992 O.974 1.011 O4177 Number of AEs with severity of? Baseline wound depth 1715 O416 7.071 O4554 BMI 1.OSO O.927 1.190 O4435 Mild 9 (69.2%) 9 (69.2%) 18 (69.2%) Use of dressings Moderate 2 (15.4%) 2 (15.4%) 4 (15.4%) Severe 2 (15.4%) 2 (15.4%) 4 (15.4%) Note: Number of AEs with relationship of: lcox Proportional Hazard ratio with 95% confidence interval and p-value was calculated using PROCPHREG procedure. Not Related 10 (76.9%) 11 (84.6%) 21 (80.8%) Unlikely 3 (23.1%) 2 (15.4%) 5 (19.2%) 0302) None of the covariates such as treatment group, Possible 0 (0.0%) 0 (0.0%) 0 (0.0%) Probable 0 (0.0%) 0 (0.0%) 0 (0.0%) wound size, wound duration, baseline wound depth, and BMI Definite 0 (0.0%) 0 (0.0%) 0 (0.0%) were significantly associated with time to 50% wound clo Number of AEs by outcome:Pl sure. However, in the PP population, subjects treated with Recovered 7 (53.8%) 11 (84.6%) 18 (69.2%) GranexinTM Gel plus SoC would have wound closure 1.186 Recovered with sequelae 2 (15.4%) 1 (7.7%) 3 (11.5%) times more than subjects in Group B. Ongoing when subject 3 (23.1%) 0 (0.0%) 3 (11.5%) completed the study 0303 FIG.8 and FIG.9 display the average woundclosure Death 1 (7.7%) 1 (7.7%) 2 (7.7%) in the ITT and PP populations in the study, respectively. Unknown O O O Number of AEs by action taken: 0304 Safety evaluation: Brief Summary of Adverse Events None 11 (84.6%) 11 (84.6%) 22 (84.6%) Discontinued study drug 2 (15.4%) 2 (15.4%) 4 (15.4%) 0305 Of 92 subjects, 71 (77.2%) subjects did not report Subjects reporting AES O O O eading to withdrawal any AEs and 21 (22.8%) subjects reported 26 AEs. Out of 26 Subjects Reporting 2 (4.3%) 3 (6.5%) 5 (5.4%) AEs, 18 (69.2%) were mild and 2 (15.4%) events in each SAEsil treatment group were moderate and severe, respectively. A Subject reporting death 1 (2.2%) 1 (2.2%) 2 (2.2%) total of 21 (80.8%) AEs were not related and 5 (19.2%) AEs Note: were unlikely related to the study drug or treatment. None of Percentage was calculated by taking respective column headergroup countas denomina tor. the AEs were related to the study drug. Of the 26 events, 18 PlPercentage was calculated by taking count of Total Number of AEs Reported in corre (69.2%) recovered and 3 (11.5%) events recovered with sponding treatment group as denominator, sequelae. No action was taken against 22 AES. None of the 0307 The AEs by MedDRA system organ class and pre subjects withdrew due to an AE. ferred term are summarized in Table 27. There was no statis (0306 A total of 5 subjects, of which 2 (4.3%) subjects tically significant difference in the number of subjects with at were in Group A and 3 (6.5%) subjects in Group B, reported least 1 AE between the 2 treatment groups (p-value=0.8038). 5 SAEs. One subject in Group A died due to an unknown In Group A, more than 5% study population reported wound cause and 1 subject in Group B died due to myocardial inf complication (Group A: 5 (10.9% subjects) and headache arction (Table 26). (Group A: 48.7% subjects) as AEs in this study. TABLE 27 Summary of Adverse Events by MedDRA System Organ Class and Preferred Term Safety Population (N = 92 Treatment Group, n (% l Group A Group B Overall System Organ Class/Preferred Term (N = 46) (N = 46) (N = 92) p-value Total number of subjects with at 11 (23.9%) 10 (21.7%) 21 (22.8%) O.8038 least one AEs Total number of AES reported 13 13 26 Cardiac disorders O 1 (2.2%) 1 (1.1%) Myocardial infarction O (O) 1 (2.2%) 1 1 (1.1%) 1 Gastrointestinal disorders O 1 (2.2%) 1 (1.1%) Toothache O (O) 1 (2.2%) 1) 1 (1.1%) 1 US 2013/0267471 A1 Oct. 10, 2013 28
TABLE 27-continued Summary of Adverse Events by MedDRA System Organ Class and Preferred Term Safety Population (N = 92 Treatment Group, in (% l Group A Group B Overall System Organ Class/Preferred Term (N = 46) (N = 46) (N = 92) p-value? General disorders and 1 (2.2%) O 1 (1.1%) administration site conditions Death 1 (2.2%) 1) O (O) 1 (1.1%) 1 infections and infestations O 2 (4.3%) 2 (2.2%) Cellulitis O (O) 1 (2.2%) 1 1 (1.1%) 1 Respiratory tract infection O (O) 1 (2.2%) 1 1 (1.1%) 1 njury, poisoning and procedural 6 (13.0%) 2 (4.3%) 8 (8.7%) complications Wound complication 5 (10.9%) 2 (4.3%) 3 7 (7.6%) 9 6 Footfracture 1 (2.2%) 1) O (O) (1.1%) 1 Metabolism and nutrition disorders O 1 (2.2%) (1.1) Diabetes mellitus inadequate O (O) 1 (2.2%) 1 (1.1%) 1 control Musculoskeletal and connective O (6.5%) 3 (3.3%) issue disorders Back pain O (O) 1 (2.2%) 1 (1.1%) 1 Pain in extremity O (O) 2 (4.3%)2 2 (2.2%) Nervous system disorders 4 (8.7%) 1 (2.2%) 5 (5.4%) Headache 4 (8.7%) 4 1 (2.2%) 1 5 (5.4%) 5 Respiratory, thoracic and 1 (2.2%) O (1.1%) mediastinal disorders Pneumonitis 1 (2.2%) 1) O (O) (1.1%) 1 Skin and Subcutaneous tissue O 1 (2.2) (1.1%) disorders Blister O LO 1 (2.2%) 1 (1.1%) 1
Note: Percentage was calculated by taking respective column headergroup count as denominator, 'p-value was calculated by comparing two treatment group using Chi-square test, Myocardial infarction led to death of the subject
0308 Out of 26 AEs, 2 events each in both the treatment infarction (Subject 827), diabetes mellitus inadequate control groups were considered severe AEs. In both Group A and B, (Subject 201), and cellulitis (Subject 524) were reported by 9 (69.2%) AEs each were mild, 2 (15.4%) AEs each were Group B subjects. moderate and 2 (15.4%) AES each were severe. The AEs with severe intensity were death and foot fracture in Group A and TABLE 28 myocardial infarction and inadequate control of diabetes mel litus in Group B. None of the severe AEs were related to the Summary of Serious Adverse Events by MedDRA System Organ Class study drug or study treatment and Preferred Tern Safety Population (N = 92 0309 Overall, 10 (76.9%) AEs in Group A and 11 (84.6%) Treatment AEs in Group B were not related, 3 (23.1%) AEs in Group A Group, n (%) and 2 (15.4%) AEs in Group B were unlikely related. None of Group A Group B Overall the AEs were related to GranexinTM Gel or study treatment. System Organ Class/Preferred Term (N = 46) (N = 46) (N = 92) 0310. Two AEs of death (Subject 903) and foot fracture Total number of subjects with at 2 (4.3) 3 (6.5) 5 (5.4) least one SAE (Subject 504) in Group Aled to study drug discontinuation. In Total number of SAEs 2 3 5 Group B, two events of cellulitis (Subject 524) and wound Cardiac disorders 0 (0.0) 1 (2.2) 1 (1.1) complication (Subject 806 with increase in size of ulcer) led Myocardial infarction 0 (0.0) (O. 1 (2.2) 1 1 (1.1) 1 to discontinuation of study treatment. General disorders and administration 1 (2.2) 0 (0.0) 1 (1.1) site conditions 0311. Three events recovered with sequelae (Group A, Death 1 (2.2) 1 0 (0.0) (O. 1 (1.1) 1 foot fracture Subject 504 and wound complication Subject Infections and infestations 0 (0.0) 1 (2.2) 1 (1.1) Cellulitis 0 (0.0) (O. 1 (2.2) 1 1 (1.1) 1 508; Group B, right foot cellulitis Subject 524). One sub Injury, poisoning and procedural 1 (2.2) 0 (0.0) 1 (1.1) ject in each treatment group reported death (Group A, complications unknown cause Subject 903; Group B, myocardial infarc Foot fracture 1 (2.2) 1 0 (0.0) (O. 1 (1.1) 1 tion Subject 827). Metabolism and nutrition disorders 0 (0.0) 1 (2.2) 1 (1.1) Diabetes mellitus inadequate control 0 (0.0) (O. 1 (2.2) 1 1 (1.1) 1 0312 Table 28 summarizes SAEs by MedDRA system organ class and preferred term. Five (5.4%) subjects (Group Note: Percentage was calculated by taking respective column headergroup countas denomina A: 2 subjects; Group B: 3 subjects) reported SAEs where foot tor. fracture (Subject 504) and death due to unknown cause (Sub PlMyocardial infarction led to death of the subject. ject 903) were reported by Group A subjects; and myocardial US 2013/0267471 A1 Oct. 10, 2013 29
0313. The 5 SAEs with the outcome are summarized in 0318. The secondary endpoints of the study were to assess Table 29 below: mean percent wound closure at Week 4. Subject assessment of pain, and time to complete wound closure. At Week 4 in the TABLE 29 PP population, Group A subjects showed statistically signifi cantly higher percent reduction of wound area from Baseline SAES by Severity, Outcome, and Action Taken compared to Group B subjects (p-value=0.0128; Wilcoxon Subject Relationship to Study Mann-Whitney U test). There was no statistically significant ID Severity Drug Study Treatment Action Taken Outcome difference in intensity of pain as assessed by the Subject from 903 Sewere Not related None Fatal Baseline to Week 12 between the 2 treatment groups 827 Sewere Not related None Fatal (p-value=0.8484; Wilcoxon Mann-Whitney U test), hence 2O1 Sewere Not related None Recovered GranexinTM Gel did not affect the intensity of pain experi SO4 Severe Not related Discontinued Recovered enced by the Subjects as recorded on a Visual Analogue Scale Study drug with of 1 to 10. sequelae 524 Moderate Not related Discontinued Recovered 0319 Complete wound closure was defined as 100% epi Study drug with thelialization of the wound and the absence of drainage from sequelae the wound. Group A subjects in the PP population took sig nificantly less time to achieve 100% wound closure than Group B subjects in the PP population (median duration: 0314 Safety Conclusions: Overall, 21 subjects reported Group A, 6 weeks; Group B, 14.64 weeks; p-value=0.0057; 26 AEs of which majority were mild in intensity. A total of 4 log-rank test). However, in the PP population the time taken moderate and 4 severe AEs were reported in this study. In for 50% wound closure was not significantly lower in Group Group A, death and foot fracture were the severe AEs and A as compared to Group B (median duration: Group A, 2 myocardial infarction and inadequate control of diabetes mel weeks; Group B, 4.07 weeks; p-value=0.2084; log-rank test). litus were severe AEs reported in Group B. All the severe AEs The categorical analysis of incidence of 100% and 50% were not related to study treatment. None of the AES reported wound closure at Week 12 was conducted and the subjects were related to GranexinTM Gelor study treatment. Of the 26 who had 100% wound closure were considered as responders. events, 18 (69.2%) recovered and 3 (11.5%) events recovered The number of responders in Group A in the PP population with sequelae. No action was taken against 22 AES. Study was significantly higher than in Group B (100% wound clo drug or treatment was discontinued in a 2 subjects each in sure. p-value=0.0104: 50% wound closure: p-value=0.0063: Group A and Group B. None of the subjects withdrew due to Chi-square analysis), indicating that the wounds treated with an AE. Five subjects reported 5 SAEs. None of the SAEs, GranexinTM Gel plus SoC have a higher frequency of 100% including 1 fatal event in Group A, were related to and 50% epithelialization than wounds treated with SoC GranexinTM Gel. alone. 0315 Discussion and Overall Conclusions: 0320 Cox Proportional Hazard Regression Model was 0316. This was a double blind, randomized, prospective, used to compare the event-time distribution function between parallel group, multi-center Phase II study conducted at 8 the treatment groups. This analysis was conducted to evaluate centers in India to evaluate the efficacy and safety of if one or more covariates were associated with the time to GranexinTM Gel in the treatment of DFU. The total duration of complete 100% or 50% wound closure. Cox Proportional the study was 12 months, including 6 months enrollment Hazard Regression Analysis for time to 100% wound closure period and 6 months for study procedures (3 months each for showed that in the PP population though treatment was not a efficacy and safety analyses). Hence, the study duration for an significant factor affecting wound closure. Group A Subjects individual subject was approximately 6 months, including 18 demonstrated 2.3 times higher wound closure than Group B visits. For interim analysis, data up to 12 weeks (Visit 15) was subjects. In addition, baseline wound depth and BMI were not considered. A total of 98 subjects with DFU were screened for significant factors but affected wound closure by 1.715 times this study and 6 were found to be screen failures. The remain and 1.050 times, respectively. All other parameters, including ing 92 subjects were randomized in the study to receive wound size, wound duration, and use of dressings did not GranexinTM Gel plus SoC or only SoC in the treatment of have a statistically significant difference in affecting wound DFU. Of these subjects, 76 (82.6%) completed the study closure for both the treatments. Cox Proportional Hazard treatment visits up to Visit 15 and are now in the follow-up Regression Analysis for time to 50% wound closure showed stage of the study. that none of the covariates (treatment group, wound size, 0317. The primary efficacy endpoint of the study was to wound duration, baseline wound depth, and BMI) were sig evaluate mean percent wound closure from Baseline to Week nificantly associated with time to 50% wound closure. How 12. The non-parametric Wilcoxon Mann-Whitney Utest was ever, Group A subjects had 1.186 times more wound closure used to analyze mean percent reduction of wound area from than Group B subjects. Baseline to Week 12 in both the treatment groups since the 0321. The safety variable was to evaluate the incidence of data did not follow a normal distribution, as confirmed by a treatment related AEs. Out of 92 subjects, 21 (22.8%) sub Shapiro-Wilk p-value <0.0001 and a non-linear Q-Q plot. In jects reported 26 AEs. There was no significant difference in the PP population, subjects treated with GranexinTM Gel plus the number of subjects with at least 1 AE between the 2 SoC (Group A) showed Statistically significantly higher per treatment groups (Group A, 11: Group B, 10; p-value=0. cent wound closure from Baseline to Week 12 compared to 8038). A total of 4 AEs were of severe intensity. None of the subjects treated with SoC alone (Group B) (p value=0.0069), AEs were related to the GranexinTM Gel. None of the subjects suggesting that at Week 12 wounds treated with GranexinTM withdrew due to an AE. A total of 5 (5.4%) subjects reported Gel plus SoC healed better than the wounds treated with SoC 5 SAEs of which 2 were Group A subjects and 3 were Group alone. B subjects, none of the SAEs was related to GranexinTM Gel US 2013/0267471 A1 Oct. 10, 2013 30 or study treatment. In Group A, 1 death was reported due to an 0335 Inclusion Criteria: unknown cause and 1 death in Group B occurred due to A subject was enrolled in this study if he/she met the follow myocardial infarction. ing criteria: 0336 2. Male or female aged 18 years and older 0322. In conclusion, GranexinTM Gel along with SoC has 0337 2a. Female subjects were post-menopausal or sur shown to accelerate wound healing in subjects with DFU, gically sterilized, or making it an efficacious, safe, and well tolerated therapeutic 0338 2b. Female of child-bearing potential must have had option for the treatment of DFU. a negative pregnancy test at Screening, and agreed to use hormonal contraceptive or intra-uterine device or dia Example 4 phragm with spermicide or condom with spermicide or abstinence throughout the study 0339. 3. Subjects with ulcers of venous origin, as clini Treatment of Venous Leg Ulcers cally determined by the Investigator by a positive venous reflux test (venous refilling <20 seconds) using Doppler 0323 A Phase II, Randomized, Prospective, Double Ultrasound for at least 4 weeks prior to screening day, Blind, Parallel Group, Multi-Center Study to Determine the which have not adequately responded to conventional ulcer Safety and Efficacy of GranexinTM Gel in the Treatment of therapy Venous Leg U1 0340 4. Ulcers that extend through the epidermis but not 0324. This was a double blind, multi-center, phase II study through muscle, tendon, or bone (Stage II or III ulcers as to evaluate the efficacy and safety of GranexinTM Gel in the defined by the International Association of Enterostomal treatment of Venous Leg Ulcer (VLU). Total duration of the Therapists) study was 11 months. The enrollment period of the study was (0341 5. Surface ulcer between 0.5 cm and 40 cm post 5 months and 6 months for study procedures. Study duration debridement for individual Subjects was 6 months and each subject was to 0342 6. Signed informed consent form attend a total of 18 visits during the study. Subjects were 0343 Exclusion Criteria: randomized to 1 of 2 treatment groups: Group A: GranexinTM A subject was not eligible for this study if he/she met any of Gel plus Standard Of Care (SoC) in treatment of VLU Group the following criteria: B: SoC in treatment of VLU Subjects in Group A received 0344) 1. Decrease or increase in the ulcer size by 30% or GranexinTM Gel (100 uMACT1 peptide) plus SoC compared more during 7 day Screening period to SoCalone in Group B. The total study duration for efficacy 0345 2. Cannot tolerate or comply with compression assessments was 12 weeks with additional 12 weeks follow therapy up for safety evaluations. The study procedures were divided 0346 3. An ulcer which showed signs of severe clinical into 3 phases: Screening Phase (Day -7 through Day 0), infection, defined as pus oozing from ulcer site 0347. 4. The ulcer to be treated required operative debri Treatment Phase (Day 0 through Week 12), and Follow-up dement Phase (Month 4 through Month 6). 0348 5. An ulcer positive for B-hemolytic Streptococcus 0325 Subjects enrolled in the study may have had mul upon culture tiple ulcers but only one ulcer was considered as the target 0349 6. The ulcer had more than 50% slough, significant ulcer. According to the randomization schedule, Subjects had necrotic tissue, osteomyelitis, bone, tendon, or capsule their ulcer site treated with either GranexinTM Gel and SoC or exposure or avascular ulcer beds SoC alone. Each subject in Group A was treated up to 14 0350 7. Highly exuding wounds (wounds that required a applications of GranexinTM Gel over the initial 12 weeks of daily dressing change) df the study period. Other than the application of GranexinTM 0351 8. Ankle Brachial Pressure Index (ABPI)<0.65 Gel for Group A Subjects, management of the wound sites was 0352 9. With active systemic infections similar in both groups. 0353 10. With clinically significant medical conditions as 0326 Each subject was assessed weekly for the first 12 determined by the Investigator which would impair wound weeks, followed by monthly assessments for the next 3 healing including renal, hepatic, hematologic, neurologic, months. Wound photographs were used to assess wound clo or immune disease. Examples included but were not lim sure in all subjects. Safety was determined by Treatment ited to Emergent Adverse Events (TEAEs). 0354) a. Renal insufficiency as an estimated glomerular filteration rate which was <30 mL/min/1.7 m 0327. The primary end point of the study is: 0355 b. Abnormal blood biochemistry defined as 3 0328. The mean percent wound closure from Baseline times that of the upper limit of the normal range to 12 weeks 0356 c. Hepatic insufficiency defined as total bilirubin 0329. The secondary end points are: >2 mg/dL or serum albumin <25 g/L 0357 d. HbA1c >9.0% 0330. Mean percent wound closure at 4 weeks 0358 e. Hemoglobin <10 g/dL 0331 Time to 50% wound closure 0359 f. Hematocrit<0.30 0332 Time to complete wound closure where complete 0360 g. Platelet count <100,000 wound closure will be defined as 100% epithelialization 0361) 11. Presence of an active systemic or local cancer or of the wound and the absence of drainage from the tumor of any kind (with the exception of non-melanoma wound skin cancer) 0362. 12. With active osteomyelitis of the study foot 0333 Subject self-assessment of intensity of pain 0363 13. With severe rheumatoid arthritis (with more than 0334 Incidence of treatment related adverse events 20 persistently inflamed joints, or below lower normal (AE) limit blood albumin level, or evidence of bone and cartilage US 2013/0267471 A1 Oct. 10, 2013 31
damage on X-ray, or inflammation in tissues other than 0378. This was a phase II, prospective, randomized, joints) and other collagen vascular diseases double blind, parallel group, and multi-center study con 0364. 14. With active connective tissue disease ducted in India in subjects with VLU. The total study duration 0365 15. Treatment with systemic corticosteroids (>15 was 11 months. The enrollment period was 5 months and 6 mg/day), or current immunosuppressive agents months for study procedures. Study duration for individual 0366 16. Previous or current radiation therapy or likeli subjects was 6 months and each subject attended a total of 18 hood to receive this therapy during study participation visits, including Screening (Visit 1), Baseline, (Visit 2), Treatment (Visit 3-15), and Follow-up (Visit 16-18) visits. 0367. 17. Pregnant or nursing subjects The study procedures were divided into 3 phases: Screening 0368 18. Known prior inability or unavailability to com Phase (Days -7 through Day 0), Treatment Phase (Day 0 plete required study visits during study participation through Week 12), and Follow-up Phase (Months 4 through 0369 19. A psychiatric condition (e.g. suicidal ideation) Month 6). Subjects were randomized to 1 of the 2 treatment or chronic alcohol or drug abuse problem, determined from groups: the subject’s medical history, which, in the opinion of the 0379 Group A: GranexinTM Gel plus SoC Investigator, may pose a threat to Subject compliance 0380 Group B: SoC only 0370 20. Use of any investigational drug ortherapy within 0381. Only one ulcer per subject was considered as the the 28 days prior to Screening target ulcer which was the largest ulcer. Each Subject in the 0371. 21. Any other factor which may have, in the opinion GranexinTM Gel treatment group was treated with up to 14 of the Investigator, compromised participation and follow applications of GranexinTM Gel over the initial 12 weeks of up in this study the study period. Other than the application of GranexinTM 0372 GranexinTM Gel (100 uM ACT1 peptide) was Gel, management of ulcer was similar in both the groups. applied topically on the wound on Day 0 (Baseline visit), Day 0382. The primary efficacy endpoint of the study was to 3, Week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The duration of evaluate mean percent wound closure from Baseline (Visit 2) the study was 11 months which included 5 months enrollment to Week 12 (Visit 15). The non-parametric Wilcoxon Mann period. Individual Subject participation was approximately Whitney Utest was used to analyze mean percent reduction of for the duration of 6 months. wound area from Baseline to Week 12 in both the treatment 0373 The reference therapy used in this study was SoC for groups since the data did not follow a normal distribution, as VLU. The ulcer was cleaned with sterile saline and bleeding confirmed by a Shapiro-Wilk p-value <0.0001 and a non was stopped before dressing. The ulcer was dressed accord linear Q-Q plot. Subjects treated with GranexinTM Gel plus ing to the following regimen: SoC showed Statistically significantly higher percent wound 0374. Applied a non-adherent dressing over the ulcer closure from Baseline to Week 12 compared to subjects which extended 0.5 inch beyond the ulcer perimeter and treated with SoC alone (ITT p-value=0.0238; mITT inflamed skin margins p-value=0.0105; PP p-value=0.0073), suggesting that at 0375. Applied a non-occlusive dressing such as fine Week 12, wounds treated with GranexinTM Gel plus SoC mesh gauze which was either folded or rolled as a bolster reduced in size when compared to wounds treated with SoC 0376 Applied a self adherent high compression four alone. layer elastic wrap (3M Elastic Adhesive Bandage) or a 0383 Mean percent wound closure at Week 4 time to compression crepe bandage from metatarsals to tibial complete (100% and 50%) wound closure, incidence of com plateau So that therapeutic compression is applied to the plete wound closure, and Subjectself assessment of pain were ulcer site also analyzed. At Week 4, a higher percentage reduction of 0377 This was a double blind, randomized, prospective, wound area was observed in GranexinTM Gel plus SoC group parallel group, multi-center Phase II study conducted at 10 than in the SoC group and this was statistically significant centers in India to evaluate the efficacy and safety of (ITT p-value=0.0006: mITT p-value=0.0001; PP p-value GranexinTM Gel in the treatment of VLU. The total duration of <0.0001 Wilcoxon Mann-Whitney U test). the study was 11 months, including 5 months enrollment (0384 Subjects treated with GranexinTM Gel plus SoC period and 6 months for study procedures. The study duration achieved 100% wound closure in median duration of 6 weeks for individual subjects was 6 months which included 18 vis in ITT, mITT, and PP population which was significantly its. A total of 101 subjects with VLU were screened for this faster compared to the SoC group in which the median dura study, 5 were found to be screen failures, and 4 subjects did tion was 12.14 weeks in the ITT population and not achieved not initiate participation in the study. A total of 92 subjects in both mITT and PP population during the 12 weeks of enrolled in the study had mean wound duration of 68.4 weeks efficacy assessments (ITT p-value=0.0006: mITT p-value=0. at Screening and mean wound area of 353.64 mm at Base 0001; PP p-value <0.0001: log-rank test). line. These subjects were randomized to receive GranexinTM (0385) Subjects treated with GranexinTM Gel plus SoC Gel plus SoC or SoCalone for the treatment of VLU. Of these, achieved 50% wound closure in 2.86 weeks in ITT, mITT and 70 completed the study and 22 subjects did not complete the PP population while subjects treated with SoC alone achieved study due to not meeting exclusion criterion number 1, con it in 6.86 weeks in ITT population and 8 weeks in both mITT sent withdrawal, loss to follow-up, non-compliance, or SAE. and PP population (ITT p-value=0.0002, mITT p-value Of 92 enrolled subjects, 15 subjects met exclusion criterion 1 <0.0001, PP p-value <0.0001). The categorical analysis of but were considered in the Safety and ITT population. These incidence of 100% and 50% wound closure at Week 12 was subjects were excluded from the mITT and PP populations. A conducted and the subjects who had 100% and 50% wound total of 83 (90.2%) subjects were males and 9 (9.8%) were closure were considered as responders. Overall in the ITT, females. The mean age of the study population was 49.8 mITT, and PP populations, 56.5%, 63.4%, and 74.3% of the years, mean weight was 70.47 kg, mean height was 168.35 subjects in GranexinTM Gel plus SoC group, respectively, cms, and mean BMI was 24.79 kg/m. responded to treatment with 100% wound closure as com US 2013/0267471 A1 Oct. 10, 2013 32 pared to 30.3% of the subjects in the SoC group. The number 0394 Visit 3-14: Treatment Visits (Day 3 through Week of responders were significantly higher in GranexinTM Gel 11) plus SoC group than in the SoC group (ITT. p-value=0.0061; The treatment visits were conducted at Day 3 and at Week 1, mITT p-value=0.0040; PP. p-value=0.0003; Chi-square 2,3,4,5,6,7,8,9, 10, and 11 after Baseline visit t1 day. The analysis). Similarly, in the PP population, 80.0% of the sub following procedures were performed at these treatment vis jects in GranexinTM Gel plus SoC group responded to treat its: ment and achieved 50% wound closure as compared to 51.5% 0395 Visit 15: End of Treatment Visit (Week 12) of the subjects in the SoC group. The number of responders 0396 Visit 16-18: Follow-up Visits including End of with 50% wound closure were significantly higher in Study Visit (Month 4 through 6): GranexinTM Gel plus SoC group than in the SoC group The Follow-up visits were conducted at Month 4-3 days, (p-value=0.0131; Chi-square analysis), indicating that the Month 5+3 days, and Month 6+3 days (End of Study EOS wounds treated with GranexinTM Gel plus SoC have higher visit). The following procedures were performed at this visit: incidences of 100% and 50% wound closure (epithelializa 0397 Standard of Care Treatment tion) than wounds treated with SoC alone. 0398. The SoC was followed in both treatment groups. 0386 Cox Proportional Hazard Regression Model analy The ulcer was cleaned with sterile saline and bleeding was sis was conducted to evaluate if one or more covariates Such stopped before dressing. The ulcer was dressed according to as treatment group, wound duration, baseline wound depth, the following regimen: and BMI etc. were associated with the time to complete 100% 0399. Applied a non-adherent dressing over the ulcer or 50% wound closure. It showed that treatment group was a which extended 0.5 inch beyond the ulcer perimeter and significant factor affecting wound closure and GranexinTM inflamed skin margins Gel plus SoC group subjects had ITT: 2.281; mITT: 2.776: 0400. Applied a non-occlusive dressing such as fine PP: 3.150 times more chances of 100% wound closure and mesh gauze which was either folded or rolled as a bolster ITT: 2.247; mITT: 2.501; PP: 2.61 times more chances of 04.01. Applied a self adherent high compression four 50% wound closure than the SoC group subjects. layer elastic wrap (3M Elastic Adhesive Bandage) or a 0387. At every visit, self assessment of intensity of pain compression crepe bandage from metatarsals to tibial score was completed by each Subject and the scores were plateau so that therapeutic compression is applied to the given in comparison to the last visit. Therefore significant ulcer site change in the pain scores was not observed and there was no (0402. In the GranexinTM Gel plus SoC group, the SoC as statistically significant difference in intensity of pain as mentioned above was followed along with the application of assessed by the subject at Week 12 between the 2 treatment Granexin TM Gel. The ulcer was cleaned with sterile saline and groups. bleeding was stopped before applying the GranexinTM Gel on 0388. The findings from the present study demonstrated a Days 0 and 3, and Weeks 1,2,3,4,5,6,7,8,9, 10, 11, and 12. good safety profile with GranexinTM Gel. Overall, 28 subjects The GranexinTM Gel was dispensed directly from its 5 ml reported 40AEs of which 24 AEs were reported by subjects in glass vial(s) to completely cover the wound bed. With a clean GranexinTM Gel plus SoC group; 16 AEs were reported in the sterile cotton Swab, Sterile tongue depressor, or a similar SOC group. The only 2 SAEs reported, were in the SoC application aid, the GranexinTM Gel was evenly spread over group. One SAE was infarction which resulted in death, and the wound surface. the other SAE was Deep Vein Thrombosis. Of the 40 AEs, 35 04.03 GranexinTM Gel is a topical gel with the active drug (87.5%) recovered. None of the AEs were related to substance Alpha-Connexin C-terminal peptide 1 (ACT1) GranexinTM Gel. None of the subjects were withdrawn from peptide developed at FirstString Research for the manage the study due to an AE. ment of wounds. ACT1 is a synthetic peptide (25 amino acid) 0389. No clinically significant abnormalities in the labo designed to mimic the Carboxy (C)-terminus of the ubiqui ratory parameters were observed in either of the treatment tous transmembrane gap junction protein Connexin 43 groups. The vital signs and ECG were normal and compa (CX43), with high binding specificity to Zona occludens, a rable between both the treatment groups. Anti-ACT1 peptide cytoplasmic tight junction protein. While the mechanism of antibodies were not detected in the serum samples collected action of ACT1 has not been completely elucidated, the pep at Screening or at Week 12. tide is soluble and engineered to directly translocate within 0390. In conclusion, GranexinTM Gelalong with SoC has cells. ACT1 peptide interacts with a known binding partner of shown to accelerate wound healing in subjects with VLU, Cx43—the Postsynaptic Density 95/Discs Large/Zonula making it an efficacious, safe, and well tolerated therapeutic Occludens (PDZ)-2 domain of Zonula Occludens-1 (ZO-1). option in the treatment of VLU. The binding of the peptide to specific CX43 C-terminus inter 0391) Description of Study Visits: The description of 18 action domains such as PDZ-2 on ZO-1 serves to competi tively inhibit its association with the Cx43 C-terminus. Con visits along with premature study discontinuation and nexins are gap junction proteins where as ZO-1 is a tight unscheduled visits is summarized below: junction associated protein. The binding of the 25 amino acid 0392 Visit 1: Screening Visit (Day -7): The Screening ACT1 peptide to the PDZ-2 domain of ZO-1 serves to stabi visit was held a maximum of 7 days prior to the Baseline visit. lize gap junctions as well as tight junctions 12. This junc 0393 Visit 2: Baseline Visit (Day 0) tional stability has been associated with faster healing and The Baseline visit was conducted within 7 days of the Screen reduced scarring. In addition, the low molecular weight of 25 ing visit. If wound size increased or decreased by 30% on the amino acid peptide is expected to reduce the risk of an immu day of randomization from Screening (exclusion criterion nogenic response. number 1), then the subject was considered ineligible for 0404 GranexinTM Gel is also designed to provide a local participation in the study. The following procedures were ized protective barrier against microbial colonization and a performed at the Baseline visit: moist environment to promote natural autolytic debridement US 2013/0267471 A1 Oct. 10, 2013
of necrotic tissue surrounding the wound. The lot number of TABLE 30-continued GranexinTM Gel used in this study was DP 1493. 04.05 Central block randomization (Block size of 2) list Disposition of Subjects was prepared by MNI independent Biostatistician using a validated computer program (Statistical Analysis Software Treatment Group SAS 9.1.3). Subjects were randomized 1:1 to either Group Granexin TM Gel A (GranexinTM Gel plus SoC) or Group B (SoC only). Ran plus SoC SoC Overall domization was further stratified by wound size (<10 cm and Category (N = 46) (N = 46) (N = 92) >10 cm) to avoid bias of undue number of subjects with Per-Protocol 35 (76.1%) 33 (71.7%) 68 (73.9%) Small or large wounds going into one arm of the study. Safety 46 (100.0%) 46 (100.0%) 92 (100.0%) 0406 Interactive web response system (IWRS) was used Total number of subjects 34 (73.9%) 36 (78.3%) 70 (76.1%) for treatment group assignment of the eligible Subjects. Being completed the study Total number of subjects 12 (26.1%) 10 (21.7%) 22 (23.9%) the double-blinded study, an unblinded cordinator designated not completed the study by the Investigator received the assigned treatment through Reason for discontinuation IWRS. 0407. Two studies, including a preclinical and clinical Consent Withdrawn 10 (83.3%) 4 (40.0%) 14 (63.6%) study tested the efficacy and safety of 100 uM concentration Death 0 (0.0%) 1 (10.0%) 1 (4.5%) of ACT1 formulated in GranexinTM Gel. In the preclinical Lost to follow up 2 (16.7%) 1 (10.0%) 3 (13.6%) study, the efficacy of GranexinTM Gel in wound healing was Protocol non-compliance 0 (0.0%) 4 (40.0%) 4 (18.2%) assessed on 5 mm diameter full thickness excisional wounds Note: in adult mice and pigs. Wounds treated with 100 uM of ACT1 Percentage was calculated taking respective column header group count as denominator, peptide closed faster, appeared less Swollen, inflamed and healed with a smoother and less discolored appearance as 0411. The following data sets were analyzed: compared to the wounds applied with the vehicle control. Subsequently, a Phase I, double blind, single-center, con ITT Population: trolled study was conducted to evaluate the safety and toler ability of 20, 50, 100, and 200 uM concentrations of ACT1 0412. The ITT population included all subjects random formulated in GranexinTM Gel versus placebo in 48 healthy ized to any treatment group, received at least one dose of subjects following punch biopsy. The results showed study medication or reference treatment, and performed GranexinTM Gel (ACT 1) at each concentration to be safe and study assessments within and/or outside the time window well tolerated with no evidence of localized or systemic AEs specified in the protocol. or Serious Adverse Events (SAEs). Based on preclinical stud mITT Population: ies and Phase I clinical trial, GranexinTM Gel with 100 uM 0413. The mITT population excluded all subjects that met concentration of the ACT1 peptide was chosen for Phase II exclusion criterion 1 or did not have adequate evaluable evaluation in the treatment of VLUs. wound photographic data post randomization 0408 For each subject, GranexinTM Gel (100 uMACT1 peptide) was applied at Days 0 and 3, and Weeks 1, 2, 3, 4, 5, PP Analysis Population: 6, 7, 8, 9, 10, 11, and 12. 04.09. A total of 101 subjects with VLU were screened. Of 0414. The PP analysis population was redefined to include these, 5 (4.9%) were screen failures and 4 (4.04%) subjects Subjects who did not have any major protocol violation. fulfilled the inclusion/exclusion criteria but did not come for the Baseline visit. The remaining 92 (91.1%) subjects were Safety Population: enrolled and randomized in the study. A total of 15 (16.3%) subjects met exclusion criteria number 1 or did not have 0415. The safety population included all subjects who adequate evaluable wound photographic data post random were randomized to either of the treatment groups in the ization and were excluded from the mITT and PP population. study. 0410. A total of 70 (76.1%) subjects completed the study 0416) The ITT population included 92 subjects; the mITT and the remaining 22 (23.9%) subjects did not complete the population included 77 subjects, PP population included 68 study of which 14 (63.6%) withdrew their consent, 4 (18.2%) Subjects; and the safety population included 92 Subjects. showed protocol non-compliance, 3 (13.6%) were lost to 0417 Modified Intent-to-Treat Population: The following follow up, and 1 (4.5%) subject (Subject 1115; SoC group) subjects were excluded from the mITT population: reported death due to Myocardial infarction (Table 30. GranexinTM Gel Plus SoC Group: TABLE 30 0418. Subject 304, Subject 501, Subject 503, Subject 505, Disposition of Subjects and Subject 906 Treatment Group SoC Group: Granexin TM Gel 0419 Subject 103, Subject 104, Subject 1004, Subject plus SoC SoC Overall 1006, Subject 1008, Subject 1017, Subject 1022, Subject Category (N = 46) (N = 46) (N = 92) 1026, Subject 1027, and Subject 1031 Population, n (%) 0420 Per Protocol Population: The following subjects were excluded from the PP population: Screened 101 Randomized 46 (100.0%) 46 (100.0%) 92 (100.0%) 0421 Meeting exclusion criterion Decrease or increase Intent-to-treat 46 (100.0%) 46 (100.0%) 92 (100.0%) in the ulcer size by 30% or more during 7 day screening Modified Intent-to-treat 41 (89.1%) 36 (78.3%) 77 (83.7%) period or did not have adequate evaluable wound photo graphic data post randomization (15 Subjects) US 2013/0267471 A1 Oct. 10, 2013 34
GranexinTM Gel Plus SoC Group: TABLE 31-continued 0422. Subject 304, Subject 501, Subject 503, Subject 505, Summary of Subject Demographics at Screening and Subject 906 Treatment Group SoC Group: Granexin TM Gel 0423 Subject 103, Subject 104, Subject 1004, Subject Statistics plus SoC SoC Overall 1006, Subject 1008, Subject 1017, Subject 1022, Subject Category, n (%)"l (N = 46) (N = 46) (N = 92) 1026, Subject 1027, and Subject 1031 0424 Wound Photograph Data not Available (9 Subjects) Current 3 (6.5) 5 (10.9) 8 (8.7%) Past 1 (2.2) 1 (2.2) 2 (2.2%) Duration of GranexinTM Gel Plus SoC Group: Smoking (months) 0425. Subject 303, Subject 904, Subject 908, Subject 914, l 4 6 10 Subject 917, and subject 1102 Mean 3OO.O 345.7 327.4 SD 48.99 53.52 54.29 SoC Group: Median 3OO.O 33O.O 3OO.O Range (Min.:Max.) (240:360) (300:420) (240:420) 0426. Subject 502, Subject 1109, and Subject 1116 Missing O O O 0427 Demographics and Baseline Characteristics No. of Cigarettes 0428. Of 92 enrolled subjects, 83 (90.2%) were males and Smoked per day 9 (9.8%) were females. The meaniSD age was 49.8+12.71 years, weight was 70.47+14.89 kg, height 168.35+7.19 cm, l 3 5 8 and BMI was 24.79-4.55 kg/m, respectively. Ten (10.9%) Mean 8.8 8.8 8.8 subjects had a smoking habit with the meaniSD number of SD 9.67 6.87 7.33 cigarettes Smoked per day as 8.8+7.33. Median 3.5 6.0 6.O Range (Min.:Max.) (3:20) (2:20) (2:20) Missing 1 1 2 TABLE 31 Note: Summary of Subiect Demographics at Screening Percentages are calculated taking respective column header group count as denominator, Plcalculated age as: Age = (Informed consent signed Date (Screening) - Date of Birth + Treatment Group Plcalculated136525). Weight as: lbs 2.2 = kilograms. Granexin TM Gel Calculated Height as: (Height (cms) = Height in inches 2.538). Statistics plus SoC SoC Overall Plcalculated BMI as: {Weight (kg)/(Height (m)}. Category, n (%)"l (N = 46) (N = 46) (N = 92) Age (years) 0429 Ulcer Identification History l 46 46 92 0430. Thirty (32.6%) subjects reported wound location on Mean 48.2 51.5 49.8 SD 12.47 12.87 12.71 medial side of leg followed by 20 (21.7%) subjects each Median 48.0 53.0 S1.O reporting on anterior side of leg and lateral side of leg. The Range (Min.:Max.) (18:71) (22:79) (18:79) overall meant-SD duration of wound history was 68.4+149. Gender 65 weeks and mean wound area was 353.64+367,196 mm. Male 44 (95.7) 39 (84.8) 83 (90.2%) respectively (Table 32). Female 2 (4.3) 7 (15.2) 9 (9.8%) Weight (kg)l TABLE 32 l 46 46 92 Summary of Ulcer Identification History Mean 72.15 68.79 70.47 SD 15.O14 14.739 14.891 Treatment Group Median 69.40 67.60 69.00 Range (Min.:Max.) (48.0:118.0) (40.6:110.0) (40.6:118.0) Granexin TM Gel Height (cm) Statistics plus SoC SoC Overall Category, n (%) (n = 46) (n = 46) (N = 92) l 46 46 92 Mean 169:38 167.32 168.35 Wound Location SD S.918 8.223 7.199 Median 171.OO 168.00 169.85 Anteromedial 0 (0.0) 1 (2.2) 1 (1.1) Range (Min.:Max.) (156.0:18.0.0) (148.0:18.6.2) (148.0:18.6.2) Anterior side of leg 11 (23.9) 9 (19.6) 20 (21.7) Body Mass Lateral Malleolus 2 (4.3) 6 (13.0) 8 (8.7) Index (kg/m2)] Lateral Side of leg 12 (26.1) 8 (17.4) 20 (21.7) Medial Malleolus 3 (6.5) 5 (10.9) 8 (8.7) l 46 46 92 Medial side of leg 17 (37.0) 13 (28.3) 30 (32.6) Mean 25.114 24.478 24.796 Posterior side of leg 1 (2.2) 4 (8.7) 5 (5.4) SD 4.7553 4.3794 4.5572 Duration of Wound Median 23.930 23.807 23.807 History (weeks) Range (Min.:Max.) (16.04:38.97) (16.06:34.94) (16.04:38.97) Smoking Habits l 46 46 92 Mean 62.7 74.O 68.4 Yes 4 (8.7) 6 (13.0) 10 (10.9%) SD 119.46 175.92 149.65 No 42 (91.3) 40 (87.0%) 82 (89.1%) Median 16.0 16.0 16.0 If yes, Range (Min.:Max.) (4:511) (4:788) (4:788) US 2013/0267471 A1 Oct. 10, 2013
TABLE 32-continued TABLE 33-continued
Summary of Ulcer Identification History Mean Percent Reduction of Wound Area (mm) from Baseline to Week 12 Treatment Group (Parametric analysis) ITT Population (N = 92) Granexin TM Gel Treatment Group Statistics plus SoC SoC Overall Category, n (%) (n = 46) (n = 46) (N = 92) Granexin TM Gel plus Wound Area SoC SoC (mm) Statistics (N = 46) (N = 46) (Length * Width)
l 41 43 84 Difference Estimate 20.2O18 Mean 34747 359.51 353.64 SEl 11.2336 SD 369.266 369.484 367.196 95% CI (L:U.) (-1.99:42.39) Median 236.43 233.43 234.93 p-valuell-ll O.O741 Range (Min.:Max.) (19.0:1804.9) ( 0.0:1576.8) ( 0.0:1804.9) Model factorill Note: p-value Percentages are calculated taking respective column header group count as denominator, Treatment O.OO62 0431) Primary Efficacy Endpoint: Mean Percent Wound Visit TABLE 34 TABLE 35 Mean Percent Reduction of Wound Area (mm) from Baseline to Week 12 Mean Percent Reduction of Wound Area (mm) from Baseline to Week 12 Parametric analysis) InITT Population (N = 77 Parametric analysis) PP Population (N = 68 Treatment Group Treatment Group Granexin TM Gel plus Granexin TM Gel plus SoC SoC SoC SoC Statistics (N = 41) (N = 36) Statistics (N = 35) (N = 33) Total number of N 59) Total no. of subjects evaluated N = 58) Subjects evaluated Percent reduction of wound area from Baseline to Week 12 Percent reduction of wound area from Baseline to Week 12 (mm) l 33 25 l 33 26 Mean 78.957 28.63S Mean 78.957 31.380 SD 51.1148 189.1879 SD 51.1148 185.8931 Median 1OOOOO 85.225 Median 1OO.OOO 87.352 Range (Min.:Max.) (-68.85:100.00) (-849.50:100.00) Range (Min.:Max.) (-68.85:100.00) (-849.50:100.00) LS mean estimate 74.5294 53.5060 LS Mean Estimate 73.275 51.921 Difference estimate 21.0234 Difference Estimatell 21.3543 SEI3] 116048 SE3 11.456S 95% CI (L.:U.) (-1.92:43.97) 95% CI (L:U.) (-1.28:43.99) p-valuell-ll O.O722 p-value?-l-Isl O.O643 Model factorill Model factorill p-value p-value Treatment O.OO2S Treatment O.OO28 Visit 0439 Table 37 summarizes the non-parametric analysis Note: for mean percent reduction of wound area from Baseline to p-value was evaluated by using non-parametric WilcoxonMann-Whitney Utest between Week 12 in the mITT population. The WilcoxonMann-Whit treatment group, ney U test is a non-parametric test which determines whether 0441) Secondary Analysis: MeanPercentage Reduction of there is a difference in two samples of independent observa Wound Area from Baseline to Week 4 tions. In GranexinTM Gel plus SoC group, the actual sum of scores (1136.00) was found higher than the expected sum of 0442. The parametric evaluation of mean percent wound scores under the null hypothesis (990.00). In SoC group, the closure from Baseline to Week 4 in the ITT population is actual sum of scores (634.00) was found lower than the summarized in Table 39. Of 92 subjects in the ITT population, expected sum of the scores under the null hypothesis (780. non-missing observations for percent reduction of wound 00). The actual sum of scores was calculated by adding the area from Baseline to Week 4 were available for a total of 56 ranks of both groups after sorting data of wound size reduc subjects. The mean percent-SD reduction of wound area tion in ascending order and the expected Sum of scores was from Baseline to Week 4 was statistically significantly higher calculated as the sum of scores when there is no difference in GranexinTM Gel plus SoC group (76.99+29.74) than in SoC between the 2 groups. Subjects in GranexinTM Gel plus SoC group (39.080+42.80) (p-value=0.0040). The model factors group had statistically significantly higher percent reduction Such as treatment, visit, and ankle circumference were sig of wound area from Baseline to Week 12 than in SoC group nificantly related to the mean percent reduction of wound area (p-value=0.0105). from Baseline to Week 4 in the ITT population (treatment p-value=0.0062; Visit p-value <0.0001; treatment visit TABLE 37 p-value=0.0139; and Ankle Circumference p-value=0.0200). Mean Percent Reduction of Wound Area (mm) from Baseline to Week 12 TABLE 39 Non-parametric analysis) nITT Population (N = 77 Mean Percent Reduction of Wound Area (mm) from Baseline to Week 4 Treatment Group Parametric analysis) ITT Population (N = 92 Granexin TM Gel plus Treatment Group SoC SoC Statistics (N = 41) (N = 36) Granexin TM Gel plus SoC SoC Percentreduction of wound area from Baseline to Week 12 Statisctics (N = 46) (N = 46) Sum of score 1136.OO 634.OO Total no. of subjects evaluated N = 56) Expected under H. 990.OO 780.OO Percentreduction of wound area from Baseline to Week 4 p-valuel O.O1 OS l 30 26 Note: Mean 76.996 39.08O p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between SD 29.7470 42.8061 treatment group, General Note: Median 97.369 41.872 Mean percentage reduction from Baseline = ((Baseline - week X). Baseline) * 100 Range (Min.:Max.) (0.40:100.00) (-35.54:100.00) H: Null Hypothesis LS mean estimate 60.2593 27.0027 If wound was healed and all further visits were missing then 100% reduction had been Difference estimate 2 33.2566 carried forward to missing visits, SEB (3) 11.3883 US 2013/0267471 A1 Oct. 10, 2013 TABLE 39-continued TABLE 40-continued Mean Percent Reduction of Wound Area (mm) from Baseline to Week 4 Mean Percent Reduction of Wound Area (mm) from Baseline to Week 4 Parametric analysis) ITT Population (N = 92 Parametric analysis) InITT Population (N = 77 Treatment Group Treatment Group Granexin TM Gel plus Granexin TM Gel plus SoC SoC SoC SoC Statisctics (N = 46) (N = 46) Statistics (N = 41) (N = 36) 95% CI (L.:U.) (10.76:55.75) 95% CI (L.:U.) (13.21:58.73) p-value 4, 5 O.004O p-value 4, 5 O.0022 Model Factorill Model Factorill p-value p-value Treatment O.0062 Treatment O.OO28 Visit Note: Note: kTotal no. of subjects evaluated” represented subjects having non-missing observations kTotal no. of subjects evaluated” represented subjects having non-missing observations for dependent variable for dependent variable 2. Difference estimate for Granexin TM Gel plus SoC indicated (Granexin TM Gel plus 2. Difference estimate for Granexin TM Gel plus SoC indicated (Granexin TM Gel plus SoC)-SoC). SoC)-SoC). 3 SE of Granexin TM Gel plus SoC indicated Standard Error of Differences (Granexin TM 3 SE of Granexin TM Gel plus SoC indicated Standard Error of Differences (Granexin TM Gel plus SoC)-SoC). Gel plus SoC)-SoC). 4 p-value for Granexin TM Gel plus SoC indicated significance of treatment differences 4 p-value for Granexin TM Gel plus SoC indicated significance of treatment differences (Granexin TM Gel plus SoC)-(SoC). (Granexin TM Gel plus SoC)-(SoC). 5ANCOVA mixed model with repeated measures was used to compare average percentage 5ANCOVA mixed model with repeated measures was used to compare average percentage reduction in wound area with treatment, visit and treatment visit as factors and strata, reduction in wound area with treatment, visit and treatment visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software. PROC Mixed procedure of SAS software. 0443) The parametric evaluation of mean percent wound 0444 The parametric analysis of mean percentage reduc closure from Baseline to Week 4 in the mITT population is tion of wound area from Baseline to Week 4 in the PP popu summarized in Table 40. Of 77 subjects in the mITT popula lation is summarized in Table 41. Of 68 subjects in the PP tion, non-missing observations for percent reduction of population, non-missing observations for change from Base wound area from Baseline to Week 4 were available for a total line to Week 4 for percent reduction of wound area were of 54 subjects. The mean percent-SD reduction of wound available for a total of 53 subjects. The mean percent-SD area from Baseline to Week 4 was statistically significantly reduction of wound area (mm) from Baseline to Week 4 was higher in GranexinTM Gel plus SoC group (76.99+29.74) than statistically significantly higher in GranexinTM Gel plus SoC in SoC group (34.00+40.52) (p-value=0.0022). The model group subjects (76.99-29.74) than in SoC group subjects factors such as treatment, visit, and ankle circumference were (31.13+38.86) (p-value=0.0012). The model factors such as significantly related to the mean percent reduction of wound treatment, visit, treatment visit, and ankle circumference area from Baseline to Week 4 in the mITT population (treat were significantly related to the mean percent reduction of ment p-value=0.0028; Visit p-value <0.0001; treatment visit wound area from Baseline to Week 4 in the PP population p-value=0.0424; and Ankle Circumference p-value=0.0428). (treatment p-value=0.0025; Visit p-value <0.0001; treatment visit p-value=0.0228; and Ankle Circumference TABLE 40 p-value=0.0350). Mean Percent Reduction of Wound Area (mm) from Baseline to Week 4 TABLE 41 Parametric analysis) InITT Population (N = 77 Mean Percent Reduction of Wound Area (mm) from Baseline to Week 4 Treatment Group Parametric analysis) PP Population (N = 68 Granexin TM Gel plus Treatment Group SoC SoC Statistics (N = 41) (N = 36) Granexin TM Gel plus SoC SoC Total no. of subjects evaluated N = 54) Statistics (N = 35) (N = 33) Percentreduction of wound area from Baseline to Week 4 Total no. of subjects evaluated N = 53Ill l 30 24 Percentreduction of wound area from Baseline to Week 4 Mean 76.996 34.003 SD 29.7470 4.O.S227 l 30 23 Median 97.369 38.217 Mean 76.996 31.134 Range (Min.:Max.) (0.40:100.00) (-35.54:100.00) SD 29.7470 38.86OS LS mean estimate 60.394 24.421 Median 97.369 34.902 Difference estimate 2 35.9729 Range (Min.:Max.) (0.40:100.00) (-35.54:100.00) SE (3) 11.5.191 LS Mean Estimate 62.3396 23.83O4 US 2013/0267471 A1 Oct. 10, 2013 39 TABLE 41-continued than the expected Sum of scores under the null hypothesis (825.00). In SoC group, the actual sum of scores (439.00) was Mean Percent Reduction of Wound Area (mm) from Baseline to Week 4 found lower than the expected sum of the scores under the null Parametric analysis) PP Population (N = 68 hypothesis (660.00). GranexinTM Gel plus SoC group sub Treatment Group jects had statistically significantly higher percent reduction of wound area than SoC group subjects from Baseline to Week Granexin TM Gel plus 4 (p-value=0.0001). SoC SoC Statistics (N = 35) (N = 33) TABLE 43 Difference Estimate 38.5092 SEl 11.6027 Mean Percent Reduction of Wound Area (mm) from Baseline to Week 4 95% CI (L.:U.) (15.56:61.46) Non-parametric analysis) nITT Population (N = 77 p-value?-l-Isl O.OO12 Model Factorill Treatment Group p-value Granexin TM Gel plus Treatment O.OO2S SoC SoC Visit Sum of score 1 O60.00 S36.OO 0448 Time to 100% Wound Closure: Time taken for first Expected under H. 855.OO 741.00 complete (100%) wound closure for the ITT population is p-valuel O.OOO6 summarized in Table 45 and FIG. 13 Complete wound closure was defined as 100% epithelialization of the wound and Note: p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between absence of any drainage from the wound. Out of 92 subjects, treatment group, 28 (60.9% subjects in the GranexinTM Gel plus SoC group and 15 (32.6%) subjects in the SoC group had 100% wound 0446 Table 43 summarizes the non-parametric analysis closure by Week 12. The median duration of 100% wound for mean percent reduction of wound area from Baseline to closure was 6 weeks (90% CI: 4 to 8 weeks) in GranexinTM Week 4 in the mITT population. In GranexinTM Gel plus SoC Gel plus SoC group and 12.14 weeks (90% CI: 10.14 to not group, the actual sum of scores (1046.00) was found higher achieved) in SoC group. Subjects in Granexin TM Gel plus SoC US 2013/0267471 A1 Oct. 10, 2013 40 group took statistically significantly less time to achieve TABLE 46 100% wound closure than SoC group subjects (p-value=0. 0006). Five subjects (GranexinTM Gel plus SoC group: 4: SoC Summary of Time to First Complete (100%) Wound Closure by Week 12 group: 1) having missing value after Screening or having InITT Population (N = 77 missing visit date were not included in the analysis. The Treatment Group remaining 44 subjects (GranexinTM Gel plus SoC group: 14 Granexin TM Gel 30.4%; SoC group: 3065.2%) were censored due to rea plus SoC SoC Sons such as Subjects not having complete wound closure, Statistics) (N = 41) (N = 36) lack of photographic evaluation data, withdrawal of consent, Number (%) of subjects 28 (68.3%) 13 (36.1%) loss to follow-up, discontinued due to protocol non compli with complete (100%) ance, or death. wound closure Number (%) of censored 11 (26.8%) 23 (63.9%) Subjects TABLE 45 Median duration (weeks) of 6.00 (4.00; 7.00) NA (12.00; NA). complete wound closure (90% CI)(2) Summary of Time to First Complete (100%) Wound Closure by Week 12 p-value ITT Population (N = 92) Note: Percentage was calculated taking count of corresponding treatment groups as denomina Treatment Group tor. The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% CI was calculated for the median duration of 100% wound closure. Granexin TM Gel PlNA: Not achieved p-value was calculated using log-rank test for treatment groups using Proc Lifetest plus SoC SoC procedure of SAS software. Statistics) (N = 46) (N = 46) 0450 Time taken for 100% wound closure in the PP popu Number (%) of subjects with 28 (60.9%) 15 (32.6%) lation is summarized in Table 47 and FIG. 15. Out of 68 complete (100%) wound subjects, 40 (GranexinTM Gel plus SoC group: 2880.0%: closure SoC group: 12 36.4%) had 100% wound closure by Week Number (%) of censored 14 (30.4%) 30 (65.2%) 12. The median duration of 100% wound closure was 6 weeks Subjects (90% CI was 4 to 7 weeks) in GranexinTM Gel plus SoC group and median duation was not achieved (90% CI: 12.00 to NA) Median duration (weeks) of 6.00 (4.00; 8.00) 12.14 (10.14; NA) in 12 weeks of treatment evaluation in SoC group. Subjects in complete wound closure GranexinTM Gel plus SoC group took statistically signifi (90% CI)12 cantly less time to achieve 100% wound closure than SoC p-valuel O.OOO6 group subjects (p-value <0.0001). A total of 28 subjects (GranexinTM Gel plus SoC group: 720.0%; SoC: 21 (63. Note: 6%) were censored due to reasons such as Subjects not hav Percentage was calculated taking count of corresponding treatment groups as denomina tor. ing complete wound 100% closure, lack of photographic The median duration of complete wound closure was estimated by Kaplan-Meier method evaluation data, withdrawal of consent, loss to follow-up, and also 90% CI was calculated for the median duration of 100% wound closure. discontinued due to protocol non compliance, or death. Plp-value was calculated using log-rank test for treatment groups using Proc Lifetest procedure of SAS software. TABLE 47 0449 Time taken for first complete (100%) wound closure Summary of Time to First Complete (100%) Wound Closure by Week 12 for the mITT population is summarized in Table 46 and FIG. PP Population (N = 68 14 Error! Reference source not found. Complete wound clo Granexin TM Gel sure was defined as 100% epithelialization of the wound and plus SoC SoC absence of any drainage from the wound. Out of 77 subjects, Statistics (N = 35) (N = 33) 28 (68.3%) subjects in the GranexinTM Gel plus SoC group Number (%) of subjects with 28 (80.0%) 12 (36.4%) and 15 (36.1%) subjects in the SoC group had 100% wound complete (100%) wound closure by Week 12. The median duration of 100% wound closure Number (%) of censored 7 (20.0%) 21 (63.6%) closure was 6 weeks (90% CI: 4 to 7 weeks) in GranexinTM Subjects Gel plus SoC group and not achieved (90% CI: 12 to not Median duration (weeks) of 6.00 (4.00; 7.00) NA (12.00; NA) achieved) in SoC group. Subjects in Granexin TM Gel plus SoC complete wound closure group took statistically significantly less time to achieve (90% CI)12 100% wound closure than SoC group subjects (p-value p-valuel <0.0001). Five subjects (GranexinTM Gel plus SoC group: 4; Note: Percentage was calculated taking count of corresponding treatment groups as denomina SoC group: 1) having missing value after Screening or having tor. The median duration of complete wound closure was estimated by Kaplan-Meier method missing visit date were not included in the analysis. The and also 90% CI was calculated for the median duration of 100% wound closure. remaining 34 subjects (GranexinTM Gel plus SoC group: 11 Plp-value was calculated using Log-rank test for treatment groups using Proc Lifetest (26.8%); SoC group: 23 (63.9%) were censored due to rea procedure of SAS software. Sons such as Subjects not having complete wound closure, Time taken to achieve 50% wound closure in the ITT popu lack of photographic evaluation data, withdrawal of consent, lation is summarized in Table 48 and FIG. 16. Out of 92 loss to follow-up, discontinued due to protocol non compli subjects, 61 (GranexinTM Gel plus SoC group: 34 (73.9%: ance, or death. SoC group: 2554.3%) had 50% wound closure by Week 12. US 2013/0267471 A1 Oct. 10, 2013 The median duration of 50% wound closure was 2.86 weeks TABLE 49 (90% CI was 2.14 to 3 weeks) in GranexinTM Gel plus SoC group and 6.86 weeks (90% CI: 5.00 to 9.14) in SoC group. Summary of Time to First Complete 50% Wound Subjects in GranexinTM Gel plus SoC group took statistically Closure by Week 12 mLTT Population (N = 77 significantly less time to achieve 50% wound closure than Treatment Group SoC group subjects (p-value=0.0002). Five (5.5%) subjects Granexin TM having missing values after Screening or having missing visit Gel plus dates were not included in the analysis. A total of 28 subjects SoC SoC (GranexinTM Gel plus SoC group: 8 17.4%; SoC group: 20 Statistics (n = 41) (n = 36) 43.5%) were censored due to reasons such as subjects not Number (%) of subjects with 33 (80.5%) 23 (63.9%) having complete 50% wound closure, lack of photographic complete (50%) wound closure Number (%) of censored subjects 6 (14.6%) 13 (36.1%) evaluation data, withdrawal of consent, loss to follow-up, Median duration (weeks) of 2.86 (2.00; 3.00) 8.00 (5.14; 9.86) discontinued due to protocol non compliance, or death. complete wound closure (90% CI)(2) TABLE 48 p-valuel Note: Summary of Time to First Complete 50% Wound Closure by Week 12 Percentage was calculated taking count of corresponding treatment groups as denomina tor. ITT Population (N = 92) The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% confidence interwal was calculated for the median duration of 50% wound closure, p-value was calculated using Log-rank test for treatment groups using Proc Lifetest Treatment Group procedure of SAS software. Granexin TM Gel 0452. The time taken to achieve 50% wound closure in the plus SoC SoC PP population is summarized in Table 50 and Error! Refer ence source not found. 18 Out of 68 subjects, 54 (GranexinTM Statistics) (n = 46) (n = 46) Gel plus SoC group: 3291.4%; SoC group: 2266.7%) had 50% wound closure by Week 12. The median duration of 50% Number (%) of subjects with 34 (73.9%) 25 (54.3%) wound closure was 2.86 weeks (90% CI was 2 to 3 weeks) in complete (50%) wound GranexinTM Gel plus SoC group and 8 weeks (90%CI: 5.14 to closure 9.86) in SoC group. Subjects in GranexinTM Gel plus SoC Number (%) of censored 8 (17.4%) 20 (43.5%) group took statistically significantly less time to achieve 50% Subjects wound closure than SoC group subjects (p-value <0.0001). A total of 14 subjects (GranexinTM Gel plus SoC group: 3 Median duration (weeks) of 2.86 (2.14; 3.00) 6.86 (5.00; 9.14) 8.6%; SoC: 11 33.3%) were censored as they did not complete wound closure complete 50% wound closure during the study period, or due (90% CI)12 to lack of photographic evaluation data, withdrawal of con p-valuel O.OOO2 sent, loss to follow-up, discontinued due to protocol non compliance, or death. Note: Percentage was calculated taking count of corresponding treatment groups as denomina TABLE 50 tor. The median duration of complete wound closure was estimated by Kaplan-Meier method Summary of Time to First Complete (50%) Wound and also 90% confidence interwal was calculated for the median duration of 50% wound Closure by Week 12 PP Population (N = 68 closure, Plp-value was calculated using Log-rank test for treatment groups using Proc Lifetest procedure of SAS software Treatment Group Granexin TM 0451 Time taken to achieve 50% wound closure in the Gel plus mITT population is summarized in Table 49 and FIG. 17. Out SoC SoC of 77 subjects, 59 (GranexinTM Gel plus SoC group: 33 80. Statistics) (N = 35) (N = 33) 5%; SoC group: 23 63.9%) had 50% wound closure by Number (%) of subjects with 32 (91.4%) 22 (66.7%) Week 12. The median duration of 50% wound closure was complete (50%) wound closure Number (%) of censored subjects 3 (8.6%) 11 (33.3%) 2.86 weeks (90% CI was 2.14 to 3 weeks) in GranexinTM Gel Median duration (weeks) of 2.86 (2.00; 3.00) 8.00 (5.14; 9.86) plus SoC group and 8 weeks (90% CI: 5.14 to 9.86) in SoC complete wound closure group. Subjects in GranexinTM Gel plus SoC group took sta (90% CI)12 tistically significantly less time to achieve 50% wound clo sure than SoC group subjects (p-value <0.0001). Five (5.5%) Note: Percentage was calculated taking count of corresponding treatment groups as denomina Subjects having missing values after Screening or having tor. The median duration of complete wound closure was estimated by Kaplan-Meier method missing visit dates were not included in the analysis. A total of and also 90% CI was calculated for the median duration of 50% wound closure. 19 subjects (GranexinTM Gel plus SoC group: 614.6%; SoC Plp-value was calculated using Log-rank test for treatment groups using Proc Lifetest group: 13 36.1%) were censored due to reasons such as procedure of SAS software. subjects not having complete 50% wound closure, lack of 0453. Additional to the Kaplan-Meier Model, Cox Pro photographic evaluation data, withdrawal of consent, loss to portional Hazards Regression Model was also used to com follow-up, discontinued due to protocol non compliance, or pare the event-time distribution function between the two death. treatment groups. This analysis was conducted to evaluate if US 2013/0267471 A1 Oct. 10, 2013 42 one or more covariates were associated with the time to 100% treated with GranexinTM Gel plus SoC would have wound or 50% wound closure. Table 51 below summarizes the Cox closure 3.150 times more than the subjects treated with SoC Proportional Hazards Regression Analysis of time to com only. Wound size, wound duration, baseline wound depth, and plete wound closure in the ITT population. The effect of BMI were not associated with time to wound closure. covariates estimated by the proportional hazards model is reported as hazard ratios. As per the hazards ratio and p-value, TABLE 53 covariates Such as treatment group and wound size showed an association with time to completion of wound closure (treat ment group p-value=0.0406; wound size p-value=0.0265) Analysis of Time to First Complete (100%) Wound Closure (Cox's and the subjects treated with GranexinTM Gel plus SoC would Proportional Hazards Regression Analysis) PP Population (N = 68) have wound closure 2.281 times more than the subjects treated with SoC only. 95% CI for Hazard Ratio TABLE 51 Analysis of Time to First Complete (100%) Wound Closure (Cox's Statistics Hazard Ratiol Lower Upper p-value Proportional Hazards Regression Analysis) ITT Population (N = 92 95% CI for Hazard Treatment Group 3.150 1.354 7.326 O.OO77 Ratio Wound size O.999 O.997 1.OOO O.O714 Statistics Hazard Ratiol Lower Upper p-value Wound duration 1.OOO O.997 1.003 O.9942 Treatment group 2281 1.036 S.O21 O.04O6 Baseline wound depth 2.027 O.333 12.352 O.4435 Wound size O.998 O.997 1.OOO O.O265 BMI 1021 O.935 1.114 0.6449 Wound duration 1.OOO O.996 1.OO3 O.8431 Baseline wound depth 1.767 O.312 1O.OO6 0.5197 BMI 1.003 O.922 1092 O.9423 Note: lcox Proportional Hazard ratio with 95% CI and p-value was calculated using PROC Note: PHREG procedure. lcox Proportional Hazard ratio with 95% CI and p-value was calculated using PROC PHREG procedure. 0456 Time to 50% wound closure in the ITT population is 0454) Table 52 below summarizes the Cox Proportional Hazards Regression Analysis of time to complete wound Summarized in Table 54. As per the hazards ratio and p-value, closure in the mITT population. The effect of covariates esti covariates Such as treatment group and wound size, showed mated by the proportional hazards model is reported as haZ an association with time to completion of wound closure ard ratios. As per the hazards ratio and p-value, covariate Such (treatment group p-value=0.0143; wound size p-value=0. as treatment group showed an association with time to 0162) and the subjects treated with GranexinTM Gel plus SoC completion of wound closure (p-value=0.0139) and the sub would have wound closure 2.247 times more than the subjects jects treated with GranexinTM Gel plus SoC would have treated with SoC only. wound closure 2.776 times more than the subjects treated with SoC only. Wound size, wound duration, baseline wound TABLE 54 depth, and BMI were not associated with time to wound closure. Analysis of Time to First Complete (50%) Wound Closure (Cox's Proportional Hazards Regression Analysis) ITT Population (N = 92) TABLE 52 95% CI for Hazard Analysis of Time to First Complete (100%) Wound Closure (Cox's Ratio Proportional Hazards Regression Analysis) nITT Population (N = 77 Statistics Hazard Ratio Lower Upper p-value 95% CI for Hazard Ratio Treatment Group 2.247 1.176 4.294 O.O143 Statistics Hazard Ratio Lower Upper p-value Wound size O.999 O.997 1.OOO O.O162 Wound duration 1.OOO O.997 1.OO2 0.7702 Treatment group 2.776 1.230 6.26S O.O139 Baseline wound depth O.68O O.182 2.537 O.S.654 Wound size O.998 O.997 1.OOO OO629 BMI O.988 O.928 1.052 O.7046 Wound duration 1.OOO O.996 1.OO3 O.8838 Baseline wound depth 2.306 O409 13.004 O.3439 Note: BMI 1021 0.937 1113 O.6386 lcox Proportional Hazard ratio with 95% confidence interval and p-value was calculated Note: using PROCPHREG procedure. lcox Proportional Hazard ratio with 95% CI and p-value was calculated using PROC PHREG procedure. 0457. Time to 50% wound closure in the mITT population 0455 Cox Proportional Hazards Regression Analysis for is summarized in Table 55. As per the hazards ratio and time to 100% wound closure in the PP population is sum p-value, covariates Such as treatment group and wound size, marised in Table 53 below. The effect of covariates estimated showed an association with time to completion of wound by the proportional hazards model is reported as hazard closure (treatment group p-value=0.0067; wound size ratios. As per the hazards ratio and p-value, covariates such as p-value=0.0346) and the subjects treated with GranexinTM treatment group showed an association with time to comple Gel plus SoC would have wound closure 2.501 times more tion of wound closure (p-value=0.0077) and the subjects than the subjects treated with SoC only. US 2013/0267471 A1 Oct. 10, 2013 TABLE 55 TABLE 57-continued Analysis of Time to First Complete (50%) Wound Closure (Cox's Proportional Hazards Regression Analysis) InITT Population (N = 77 Wound Recurrence in the Overall Population 95% CI for Hazard Ratio Treatment Group Statistics Hazard Ratiol Lower Upper p-value Granexin TM Treatment Group 2.5O1 1.289 4.853 O.OO67 Wound size O.999 O.998 1.OOO O.O346 Gel plus SoC SoC Overall Wound duration 1.OOO O.997 1.OO2 O.8310 Recurrence, n (%)"l (N = 46) (N = 46) (N = 92) Baseline wound depth O.805 O.217 2.991 O.7461 BMI O.998 0.937 1.064 O.958S After Week 12 Note: lcox Proportional Hazard ratio with 95% confidence interval and p-value was calculated using PROCPHREG procedure. Yes 2 (4.3%) 2 (4.3%) 4 (4.3%) 0458 Time to 50% wound closure in the PP population is No 44 (95.7%) 44 (95.7%) 88 (95.7%) summarized in Table 56. As per the hazards ratio and p-value, p-value? 0.9999 covariates Such as treatment group and wound size, showed an association with time to completion of 50% wound closure (treatment group p-value=0.0059; wound size p-value=0. Note: 0442) and the subjects treated with GranexinTM Gel plus SoC Percentage was calculated by taking respective column headergroup countas denomina would have wound closure 2.611 times more than the subjects tor. Plp-value was calculated using the chi-square Test for comparison between two treatment treated with SoC only. groups. TABLE 56 0461 Table 58 summarizes the time points of wound Analysis of Time to First Complete (50%) Wound Closure (Cox's recurrence. Wound recurrence within 2 weeks was observed Proportional Hazards Regression Analysis) PP Population (N = 68 in 3 subjects (Subject 704, Subject 907, Subject 1002) in GranexinTM Gel plus SoC group and 2 subjects (Subject 208, 95% CI for Hazard Subject 902) in SoC group. Ratio Statistics Hazard Ratiol Lower Upper p-value TABLE 58 Treatment Group 2.611 1.319 S.169 O.OO59 Timepoints of Wound recurrence Wound size O.999 O.998 1.OOO O.0442 Wound duration 1.OOO O.997 1.OO2 O.8788 Visit Baseline wound depth O. 604 O.151 2.412 0.4755 Wound BMI O.998 O.935 1.064 O.9469 Subject Visit Wound Visit Wound Healed Number Treatment Group Healed recurred Again Note: lcox Proportional Hazard ratio with 95% confidence interval and p-value was calculated 206 Granexin TM Gel plus Visit 9 Visit 13 using PROCPHREG procedure. SOC 504 Granexin TM Gel plus Visit 8 Visit 18 0459 Wound Recurrence SOC 0460. Overall in ITT, mITT, and PP population, wound 704 Granexin TM Gel plus Visit 10 Visit 11 recurrence was reported in 10 subjects (GranexinTM Gel plus SOC SoC group: 5 subjects; SoC group: 5 subjects) for the entire 907 Granexin TM Gel plus Visit 15 Visit 16 Visit 17 duration of the study. Of these 10 subjects, wound recurrence SOC was observed in 2 subjects in GranexinTM Gel plus SoC group 1002 Granexin TM Gel plus Visit 7 Visit 8 Visit 12 SOC (Subject 504, Subject 907) and 2 subjects in SoC group (Sub 208 SOC Visit 10 Visit 11 Visit 16 ject 1003: Subject 1103) after completion of treatment at 12 902 SOC Visit 8 Visit 9 Visit 10 weeks. There was no statistically significant difference in 10O3 SOC Visit 9 Visit 16 wound recurrence between the 2 treatment groups (p-value 1103 SOC Visit 10 Visit 16 Visit 17 >0.9999). 1107 SOC Visit 11 Visit 13 TABLE 57 0462. The intensity of pain experienced by the subjects Wound Recurrence in the Overal Population was recorded on a Visual Analogue Scale of 1 to 10 where 1 Treatment Group indicated “no pain” and 10 indicated “extreme pain” at all Granexin TM visits. The Summary of subject self assessment of intensity of Gel plus SoC SoC Overall pain at all visits from Baseline to Week 12 for the ITT popu Recurrence, n (%) (N = 46) (N = 46) (N = 92) lation is presented in and Table 59. At any visit 0463 Table 59 summarizes the analysis of subject self assessment of intensity of pain at Week 12 for the ITT popu Yes 5 (10.9%) 5 (10.9%) 10 (10.9%) No 41 (89.1%) 41 (89.1%) 82 (89.1%) lation. The intensity of pain was not statistically significantly p-value? 0.9999 higher in subjects treated with GranexinTM Gel plus SoC as compared to those treated with SoC only (p-value=0.8393). US 2013/0267471 A1 Oct. 10, 2013 44 TABLE 59 TABLE 60-continued Analysis of Subject Self Assessment of Intensity Analysis of Subject Self Assessment of Intensity of Pain at Week 12 mITT Population (N = 77) of Pain at Week 12 ITT Population (N = 92) Treatment Group Treatment Group Granexin TM Gel plus SoC group SoC group Statistics (N = 41) (N = 36) Granexin TM Gel plus Missing O 1 SoC group SoC group p-value O. 1195 Statistics (N = 46) (N = 46) Note: p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between Intensity of pain at Week 12 treatment group, 0465 Wound Tracing were done for all subjects from l 39 38 Screening till Visit 15 or till the time the wound had healed. Mean O46 O.34 Qualitative Assessment of the wound site were done at the site SD 1.072 O.708 at every visit for the presence of granulation, necrotic tissue, Median O.OO O.OO exudate levels, wound odor, and escharas listed in the CRF. Range (MiniMax) (0.0:5.0) (0.0:3.0) 0466. The non-parametric Wilcoxon Mann-Whitney U test was used to analyze mean percent reduction of wound Missing O 1 area from Baseline to Week 12 in both the treatment groups p-valuel O.8393 since the data did not follow a normal distribution, as con firmed by a Shapiro-Wilk p-value of <0.0001 and a non-linear Note: Q-Q plot. The addition of ranks of both groups obtained after p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between sorting the data of wound size reduction in ascending order treatment group, showed that in GranexinTM Gel plus SoC subjects, the actual Sum of scores was higher than the expected Sum of Scores 0464) Table 60 Summarizes the analysis of subject self (ITT. actual: 1210.00, expected: 1071.00; mITT. actual: assessment of intensity of pain at Week 12 for the mITT 1136.00, expected: 990.00; PP: actual: 1123.00, expected: population. The intensity of pain was not statistically signifi 973.50). In SoC group, the actual sum of scores was lower cantly higher in subjects treated with GranexinTM Gel plus than the expected sum of the scores (ITT. actual: 743.00, SoC as compared to those treated with SoC only (p-value=0. expected: 882.00; mITT. actual: 634.00, expected: 780.00: 1663). PP: actual: 588.00; expected: 737.50). Therefore, subjects in GranexinTM Gel plus SoC group had a statistically signifi TABLE 60 cantly higher percent wound closure from Baseline to Week 12, as compared to subjects in SoC group (ITT p-value=0. Analysis of Subject Self Assessment of Intensity 0238; mITT p-value=0.0105; PP p-value=0.0073). of Pain at Week 12 mITT Population (N = 77) 0467. Non-parametric analysis for secondary endpoint Treatment Group showed that the mean percent reduction of wound area from Baseline to Week 4 in GranexinTM Gel plus SoC group was Granexin TM Gel plus SoC group SoC group statistically significantly higher, as compared to Subjects in Statistics (N = 41) (N = 36) SoC group (ITT p-value=0.0006: mITT p-value=0.0001; PP p-value <0.0001). Intensity of pain at Week 12 0468 Time to complete (100%) wound closure by Week l 34 29 12 was statistically significantly lesser in GranexinTM Gel Mean O.18 O.45 plus SoC group, as compared to SoC group (ITT p-value=0. SD O.387 O.783 0006: mITT p-value <0.0001; PP p-value <0.0001). In Median O.OO O.OO Range (MiniMax) (0.0:1.0) (0.0:3.0) GranexinTM Gel plus SoC group, 60.9% in the ITT popula Missing O 1 tion, 68.3% subjects in mITT population, and 80.0% subjects p-valuel O.1663 in the PP population had 100% wound closure by Week 12 as compared to 32.6% in the ITT population, 36.1% subjects in Treatment Group mITT population, and 36.4% in the PP population in the SoC Granexin TM Gel plus group. The median duration of 100% wound closure was 6 SoC group SoC group weeks in ITT, mITT, and PP population in the GranexinTM Gel Statistics (N = 46) (N = 46) plus SoC group. In the SoC group, the median duration of time to complete wound closure was about 12.14 weeks in the Intensity of pain at Week 12 ITT population and not achieved in the mITT and PP popu N 34 27 lation. Mean O.18 O.48 SD O.387 O.802 0469. The time taken to achieve 50% wound closure by Median O.OO O.OO Week 12 was statistically significantly lower in GranexinTM Range (MiniMax) (0.0:1.0) (0.0:3.0) Gel plus SoC group as compared to SoC group subjects (ITT p-value=0.0002, mITT p-value <0.0001, PP p-value <0.0001). In GranexinTM Gel plus SoC group, 73.9% subjects US 2013/0267471 A1 Oct. 10, 2013 in the ITT, 80.5% subjects in mITT population, and 91.4% 0472. The number of responders with 50% wound closure subjects in the PP population had 50% wound closure and in in GranexinTM Gel plus SoC group were 63% in ITT, 68.3% in SoC group, 54.3% subjects in ITT, 63.9% subjects in mITT mITT population as compared to 43.5% in ITT, 50% in mITT population in the SoC group which was not statistically sig population, and 66.7% in PP population had 50% wound nificant (ITT p-value=0.0600; mITT p-value=0.1025). The closure. In ITT, mITT, and PP population, the median dura number of responders with 50% wound closure in tion of 50% wound closure was 2.86 weeks in subjects treated GranexinTM Gel plus SoC group in the PP population were with GranexinTM Gel plus SoC. Subjects treated with SoC 80% as compared to 51.5% in the SoC group which was alone had median duration of 6.86 weeks in the ITT popula statistically significantly higher (p-value=0.0131). tion and 8 weeks in the both the mITT and PP population. 0473 Hence, in the PP population, the frequency of 100% and 50% wound closure (epithelialization) was significantly 0470. In Cox Proportional Hazard Regression Analysis for higher in wounds treated with Granexin TM Gel plus SoCs time to 100% wound closure covariates such as treatment than in SoC group Subjects. group showed a significant association with time to comple 0474 The intensity of pain experienced by the subjects tion of wound closure (ITT p-value=0.0406; mITT was recorded on a Visual Analogue Scale of 1 to 10 where 1 p-value=0.0139, PP p-value=0.0077) and the GranexinTM Gel indicated “no pain' and 10 indicated “extreme pain'. There plus SoC group subjects had ITT. 2.281; mITT p-value=2. was no statistically significant difference in Subject self 776; PP: 3.150 times more chances of 100% wound closure assessment of intensity of pain at the end of treatment at Week than the SoC group subjects. Wound duration, wound size, 12 between the 2 treatment groups (ITT p-value=0.8393; baseline wound depth, and BMI were not significant factors mITT p-value=0.1663, PP p-value=0.1195, Wilcoxon Mann affecting wound closure. Treatment group and wound size Whitney U test). showed a significant association with time to completion of 0475 Brief Summary of Adverse Events 50% wound closure (ITT. treatment group p-value=0.0143: 0476) Overall, 28 subjects reported 40 AEs of which 24 wound size p-value=0.0162; mITT. treatment group AEs were reported by subjects in GranexinTM Gel plus SoC p-value=0.0067: wound size p-value=0.0346: PP; treatment group; 16 AEs were reported in the SoC group. Out of 24 AEs, group p-value=0.0059; wound size p-value=0.0442) and the 21 (87.5%) in GranexinTM Gel plus SoC group and out of 16 GranexinTM Gel plus SoC group subjects had ITT: 2.247: AEs, 12 (75%) AEs in SoC group were mild. Three (12.5%) events in GranexinTM Gel plus SoC group and 3 (18.8%) mITT: 2.501 times; PP: 2.61 times more chances of wound events in SoC group were moderate; and 1 (6.3%) event in closure than the SoC group subjects. Wound duration, base SoC group was severe. None of the AES in either GranexinTM line wound depth, and BMI were not significant factors Gel plus SoC group or SoC group were related to the study affecting 50% wound closure. drug or study treatment. Of the 40 events, 35 (87.5%) recov 0471. The categorical analysis of incidence of 100% ered and 1 (2.5%) event recovered with sequelae, 3 (7.5%) wound closure at Week 12 was done using the Cochran AEs were ongoing when the subject completed the study. No Mantel-Haenszel analysis with Breslow-Day test and the action was taken against 39 (97.5%) AEs. None of the sub Chi-square test. The Chi-square test performed by combining jects withdrew due to an AE and 1 (1.1%) subject in SoC the data from all the study centers showed that the number of group discontinued the study treatment due to death. responders with 100% wound closure in GranexinTM Gel plus 0477. A total of 2 (2.2%) SAEs were reported in SoC SoC group (ITT: 56.5%: mITT: 63.4%; PP: 74.3%) was sta group one of which was death due to MI and the other was tistically significantly higher than in SoC group (ITT. 28.3%: Deep Vein Thrombosis. Both the SAEs were not related to the mITT. 30.6%; PP: 30.3%) (ITT. p-value=0.0061; mITT study treatment. No SAEs were reported in subjects in p-value=0.0040; PP: p-value=0.0003). GranexinTM Gel plus SoC group (Table 61). TABLE 61 Overview of Adverse Events-Safety Population (N = 92) Treatment Group Granexin TM Gel plus SoC SoC Overall Statistics (N = 46) (N = 46) (N = 92) Total number of AEs 24 16 40 reported Subjects reporting any 14 (30.4%) 14 (30.4%) 28 (30.4%) AEsil Subjects reporting 1 AE 8 (17.4%) 12 (26.1%) 20 (21.7%) Subjects reporting >1 AE 6 (13.0%) 2 (4.3%) 8 (8.7%) Subjects Reporting No 32 (69.6%) 32 (69.6%) 64 (69.6%) AEsil Number of AEs with severity of:Pl Mild 21 (87.5%) 12 (75.0%) 33 (82.5%) Moderate 3 (12.5%) 3 (18.8%) 6 (15.0%) Severe 0 (0.0%) 1 (6.3%) 1 (2.5%) US 2013/0267471 A1 Oct. 10, 2013 TABLE 61-continued Overview of Adverse Events-Safety Population (N = 92 Treatment Group Granexin TM Gel plus SoC SoC Overall Statistics (N = 46) (N = 46) (N = 92) Number of AEs with relationship of: Not Related 24 (100.0%) 16 (100.0%) 40 (100.0%) Unlikely 0 (0.0%) 0 (0.0%) 0 (0.0%) Possible 0 (0.0%) 0 (0.0%) 0 (0.0%) Probable 0 (0.0%) 0 (0.0%) 0 (0.0%) Definite 0 (0.0%) 0 (0.0%) 0 (0.0%) Number of AES by outcome: Recovered 22 (91.7%) 13 (81.3%) 35 (87.5%) Recovered with sequelae 0 (0.0%) 1 (6.3%) 1 (2.5%) On-going when subject 2 (8.3%) 1 (6.3%) 3 (7.5%) completed the study Death 0 (0.0%) 1 (6.3%) 1 (2.5%) Unknown 0 (0.0%) 0 (0.0%) 0 (0.0%) Number of AES by action aken:l None 24 (100.0%) 15 (93.8%) 39 (97.5%) Discontinued study drug 0 (0.0%) 1 (6.3%) 1 (2.5%) Subjects reporting AES 0 (0.0%) 0 (0.0%) 0 (0.0%) eading to withdrawal Subjects reporting SAEs 0 (0.0%) 2 (4.3%) 2 (2.2%) Subjects reporting death 0 (0.0%) 1 (2.2%) 1 (1.1%) Note: Percentage was calculated by taking respective column headergroup count as denominator, 'Percentage was calculated by taking count of Total Number of AEs Reported in corresponding treatment group as denominator, 0478 Display of Adverse Events 0479. The AEs by MedDRA system organ class and pre ferred term are summarized in 0480. There was no statistically significant difference in the number of subjects with at least 1 AE between the treat ment groups (p-value 0.9999). More than 5% safety popu lation reported venous ulcer pain (10 10.9%) and wound complication (12 13.0% subjects). TABLE 62 Summary of Adverse Events by MedDRA System Organ Class and Preferred Tern Safety Population (N = 92 Treatment Group n (% l Granexin TM Gel plus System Organ Class/Preferred SoC SoC Overall Term (n = 46) (n = 46) (N = 92) p-value? Total number of subjects with at 14 (30.4%) 14 (30.4%) 28 (30.4%) 0.9999 least one AE Total number of AES reported 24 16 40 Cardiac disorders 0 (0.0%) 1 (2.2%) 1 (1.1%) Myocardial infarction 0 (0.0%)O 1 (2.2%)1 1 (1.1%)1 General disorders and 0 (0.0%) 2 (4.3%) 2 (2.2%) administration site conditions Pyrexia 0 (0.0%)O 2 (4.3%)2 2 (2.2%)2 Infections and infestations 2 (4.3%) 1 (2.2%) 3 (3.3%) Wound infection 2 (4.3%)2 1 (2.2%)2 3 (3.3%)4 Injury, poisoning and procedural 2 (4.3%) 4 (8.7%) 6 (6.5%) complications Blister 1 (2.2%)1 0 (0.0%)O 1 (1.1%)1 Wound complication 7 (15.2%)11 5 (10.9%)5] 12 (13.0%) 16 US 2013/0267471 A1 Oct. 10, 2013 47 TABLE 62-continued Summary of Adverse Events by MedDRA System Organ Class and Preferred Term Safety Population (N = 92 Treatment Group n (% l Granexin TM Gel plus System Organ Class/Preferred SoC SoC Overall Term (n = 46) (n = 46) (N = 92) p-value Respiratory, thoracic and 2 (4.3%) 0 (0.0%) 2 (2.2%) mediastinal disorders Cough 1 (2.2%)1 0 (0.0%)O 1 (1.1%)1 Pneumonitis 1 (2.2%)1 0 (0.0%)O 1 (1.1%)1 Skin and Subcutaneous tissue 7 (15.2%) 4 (8.7%) 11 (12.0%) disorders Venous ulcer pain 6 (13.0%)6] 4 (8.7%)4 10 (10.9%)10] Dermatitis allergic 1 (2.2%)1 0 (0.0%)O 1 (1.1%)1 Pruritus 1 (2.2%)1 0 (0.0%)O 1 (1.1%)1 Vascular disorders 0 (0.0%) 1 (2.2%) 1 (1.1%) Bleeding varicose vein 0 (0.0%)O 1 (2.2%)1 1 (1.1%)1 Deep vein thrombosis 0 (0.0%)O 1 (2.2%)1 1 (1.1%)1 Note: Percentage was calculated by taking respective column headergroup count as denominator, 2 p-value was calculated by comparing two treatment group using Chi-square test, Myocardial infarction led to death of the subject. 0481. Of the 40 AEs reported in this study, one severe 0484 Immunogenicity Testing: Using universal precau event of MI was reported by a subject in the SoC group None tions recommended for biological samples, the blood of the AEs (GranexinTM Gel plus SoC group: 24 100%; SoC samples were collected in yellow top gel Vaccutainers. They group: 16 100.0% had any definite, probable, or possible were properly labeled with date and time of collection, site relationship to GranexinTM Gel or the study treatment. One number, visit number, Screening and randomization number, subject (Subject 1115) in SoC group was discontinued from and the sample type. Using acceptable Venipuncture tech the study treatment due to death. No other subjects were nique 7 mL of whole blood was collected and allowed to stand withdrawn or discontinued from the study due to an AE. Out for 15 min for the clot to form. Serum was separated by of 40 AEs, 35 (87.5%) recovered, 1 (2.5%) AE (venous ulcer centrifuging the blood samples at 3500 rpm. Approximately 2 pain) in SoC group (Subject 102) recovered with sequelae, 3 mL of the separated serum was pipetted out into two aliquots (3.3%) AEs of wound complication (GranexinTM Gel plus and refrigerated at 2-6° C. till shipped. These tubes were SoC group: 2 subjects Subject 702, 916: SoC group: 1 labeled with date and time of collection, site number, visit subject Subject 1113) were on-going when the subjects number, Screening and randomization number, and the completed the study, and 1 subject (Subject 1115, SoC group) sample type. These samples were shipped to a central storage died due to MI. One subject (Subject 1115, SoC group) died facility (Metropolis Laboratory, Mumbai) under appropriate due to MI. temperature conditions with dry ice or frozen cold packs. 0482. At Screening visit, of the 9 female subjects in the 0485 The samples were stored at -20° C. at Metropolis study, 2 were Surgically sterilized and 4 were post meno and shipped in batches to Wuxi Laboratories at Philadelphia, pausal. Pregnancy test was done for 3 (3.3%) subjects and Pa., under appropriate temperature conditions with serum doppler waveform analysis for all 92 subjects. The mean sample shipment forms. Doppler waveform reading for GranexinTM Gel plus SoC 0486 Anti-ACT1 antibodies were not detected in the group subjects was 11.60 cm/s and 9.57 cm/s for SoC group serum in any of the subjects at Screening and Week 12. subjects. X-ray was taken for 86 (93.5%) subjects where 0487. The systolic and diastolic blood pressure, heart rate, X-ray finding was normal for 81 (88.0%) and abnormal for 5 respiratory rate, and body temperature were comparable (5.4%) subjects, which was not clinically significant. None of between the treatment groups at Screening visit and at Week the target wounds were infected with Bhemolytic Streptococ 12. cus culture. The eGFR for males in GranexinTM Gel plus SoC 0488. In the overall safety population, the ECG was nor group was 95.18 mL/min and 88.50 mL/min in SoC group mal in 81 (88.0%) subjects. Changes in ECG impression subjects. However, the eGFR for female and ABPI were com reported in the remaining Subjects were not clinically signifi parable between the treatment groups. cant at the Screening visit (End-of-Text Table 14.3.6) 0483 None of the subjects in the study had clinically 0489. Overall, 28 (30.4%) subjects reported 40 AEs of significant hematological parameters at Screening visit or which 24 AEs were reported by subjects in GranexinTM Gel Week 12. Two subjects in GranexinTM Gel plus SoC group plus SoC group; 16 AEs were reported in the SoC group. Out had clinically significant HbA1c at Screening visit but none of 24 AEs, 21 (87.5%) in GranexinTM Gel plus SoC group and of the subjects had clinically significant HbA1c at 12 weeks. out of 16 AEs, 12 (75%) AEs in SoC group were mild. Three None of the Subjects had any clinically significant creatinine, (12.5%) events in GranexinTM Gel plus SoC group and 3 uric acid, BUN, potassium, sodium, chloride, bicarbonate, (18.8%) events in SoC group were moderate; and 1 (6.3%) albumin, AST, ALT, and total cholesterol at Screening visitor event in SoC group was severe. None of the AEs in either at Week 12. GranexinTM Gel plus SoC group or SoC group were related to US 2013/0267471 A1 Oct. 10, 2013 48 the study drug or study treatment. Of the 40 events, 35 (87. also analyzed. At Week 4, the wound area percent of reduction 5%) recovered and 1 (2.5%) event recovered with sequelae. in GranexinTM Gel plus SoC group was statistically signifi None of the subjects withdrew due to an AE and 1 (1.1%) cantly higher than in SoC group (ITT p-value=0.0006; mITT Subject in SoC group discontinued the study treatment due to p-value=0.0001; PP p-value <0.0001 Wilcoxon Mann-Whit death. ney U test). 0490. A total of 2 (2.2%) SAEs were reported in SoC group one of which was death due to Myocardial Infarction 0496 Subjects treated with GranexinTM Gel plus SoC and the other was Deep Vein Thrombosis. Both the SAEs achieved 100% wound closure in median duration of 6 weeks were not related to the study treatment. No SAEs were in ITT, mITT, and PP population which was significantly reported by subjects in GranexinTM Gel plus SoC group Over faster compared to the SoC group in which the median dura all, there was no significant difference in the number of sub tion was 12.14 weeks in the ITT population and not achieved jects reporting AES between the 2 treatment groups in both mITT and PP population during the 12 weeks of (Granexin TM Gel plus SoC group, 14: SoC group, 14; p-value efficacy assessments (ITT p-value=0.0006: mITT p-value=0. >0.9999). 0001; PP p-value <0.0001: log-rank test). 0491. At Screening visit, no clinically significant abnor 0497 Subjects treated with GranexinTM Gel plus SoC malities were observed in any of the treatment groups. The achieved 50% wound closure in 2.86 weeks in both ITT and vital signs and ECG were normal and comparable between PP population while subjects treated with SoC alone achieved both the treatment groups. Anti-ACT1 peptide antibodies it in 6.86 weeks in ITT population and 8 weeks in both mITT were not detected in any of the subjects in both Screening and and PP population, respectively (ITT p-value=0.0002, mITT EOS serum samples. p-value <0.0001, PP p-value <0.0001). The categorical analy 0492. This was a double blind, randomized, prospective, sis of incidence of 100% and 50% wound closure at Week 12 parallel group, multi-center Phase II study conducted at 10 was conducted and the subjects who had 100% wound closure centers in India to evaluate the efficacy and safety of were considered as responders. Overall in the ITT, mITT, and GranexinTM Gel in the treatment of VLU. The total duration of PP populations, 56.5%, 63.4%, and 74.3% of the subjects in the study was 11 months, including 5 months enrollment GranexinTM Gel plus SoC group, respectively, responded to period and 6 months for study procedures. The study duration treatment with 100% wound closure as compared to 30.3% of for individual subjects was 6 months which included 18 vis the subjects in the SoC group. The number of responders were its. A total of 101 subjects with VLU were screened for this significantly higher in GranexinTM Gel plus SoC group than in study, 5 were found to be screen failures, and 4 subjects did the SoC group (ITT. p-value=0.0061; mITT p-value=0.0040: not initiate participation in the study. A total of 92 subjects PP: p-value=0.0003; Chi-square analysis). Similarly, in the enrolled in the study had mean wound duration of 68.4 weeks PP population, 80.0% of the subjects in GranexinTM Gel plus at Screening and mean wound area of 353.64 mm at Base SoC group responded to treatment and achieved 50% wound line. These subjects were randomized to receive GranexinTM closure as compared to 51.5% of the subjects in the SoC Gel plus SoC or SoCalone for the treatment of VLU. Of these, group. The number of responders with 50% wound closure 70 completed the study and 22 subjects did not complete the were significantly higher in GranexinTM Gel plus SoC group study due to consent withdrawal, loss to follow-up, non than in the SoC group (p-value=0.0131; Chi-square analysis), compliance, or SAE. indicating that the wounds treated with GranexinTM Gel plus 0493) Of 92 enrolled subjects, 15 subjects met exclusion SoC have higher incidences of 100% and 50% wound closure criterion number 1 or did not have adequate evaluable wound (epithelialization) than wounds treated with SoC alone. photographic data post randomization but were considered in 0498 Cox Proportional Hazard Regression Model analy the Safety and ITT population. These subjects were excluded sis was conducted to evaluate if one or more covariates Such from the mITT and PP populations. A total of 83 (90.2%) as treatment group, wound duration, baseline wound depth, subjects were males and 9 (9.8%) were females. The mean and BMI etc. were associated with the time to complete 100% age of the study population was 49.8 years, mean weight was or 50% wound closure. It showed that treatment group was a 70.47 kg, mean height was 168.35 cms, and mean BMI was significant factor affecting wound closure and GranexinTM 24.79 kg/m. Gel plus SoC group subjects had ITT: 2.281; mITT: 2.776: 0494 The primary efficacy endpoint of the study was to PP: 3.150 times more chances of 100% wound closure and evaluate mean percent wound closure from Baseline (Visit 2) ITT: 2.247; mITT: 2.501; PP: 2.61 times more chances of to Week 12 (Visit 15). The non-parametric Wilcoxon Mann 50% wound closure than the SoC group subjects. Whitney Utest was used to analyze mean percent reduction of wound area from Baseline to Week 12 in both the treatment 0499. At every visit, self assessment of intensity of pain groups since the data did not follow a normal distribution, as score was completed by each subject and the scores were confirmed by a Shapiro-Wilk p-value <0.0001 and a non given in comparison to the last visit. Therefore significant linear Q-Q plot. Subjects treated with GranexinTM Gel plus change in the pain scores was not observed and there was no SoC showed Statistically significantly higher percent wound statistically significant difference in intensity of pain as closure from Baseline to Week 12 compared to subjects assessed by the subject at Week 12 between the 2 treatment treated with SoC alone (ITT p-value=0.0238; mITT groups. p-value=0.0105; PP p-value=0.0073), suggesting that at 0500. The findings from the present study demonstrated a Week 12, wounds treated with GranexinTM Gel plus SoC had good safety profile with GranexinTM Gel. Overall, 28 subjects a significant reduction in size as compared to wounds treated reported 40AEs of which 24 AEs were reported by subjects in with SoC alone. GranexinTM Gel plus SoC group; 16 AEs were reported in the 0495 Mean percent wound closure at Week 4 time to SOC group. The only 2 SAEs reported, were in the SoC complete (100% and 50%) wound closure, incidence of com group. One SAE was Myocardial infarction which resulted in plete wound closure, and Subjectself assessment of pain were death, and the other SAE was Deep vein thrombosis. Of the US 2013/0267471 A1 Oct. 10, 2013 49 40AEs, 35 (87.5%) recovered. None of the AEs were related and materials are described herein. All publications cited to GranexinTM Gel. None of the subjects were withdrawn herein are incorporated herein by reference for the purpose of from the study due to an AE. disclosing and describing specific aspects of the invention for 0501. No clinically significant abnormalities in the labo which the publication is cited. ratory parameters were observed in either of the treatment 0504 The publications discussed herein are provided groups. The vital signs and ECG were normal and compa solely for their disclosure prior to the filing date of the present rable between both the treatment groups. Anti-ACT1 peptide application. Nothing herein is to be construed as an admission antibodies were not detected in the serum samples collected that the present invention is not entitled to antedate such at Screening or at Week 12. publication by virtue of prior invention 0502. In conclusion, GranexinTM Gel along with SoC has 0505 While the invention has been described in connec shown to accelerate wound healing in subjects with VLU, tion with specific embodiments thereof, it will be understood making it an efficacious, safe, and well tolerated therapeutic that it is capable of further modifications and this application option in the treatment of VLU. is intended to cover any variations, uses, or adaptations of the 0503. Unless defined otherwise, all technical and scien invention following, in general, the principles of the invention tific terms herein have the same meaning as commonly under and including Such departures from the present disclosure as stood by one of ordinary skill in the art to which this invention come within known or customary practice within the art to belongs. Although any methods and materials, similar or which the invention pertains and as may be applied to the equivalent to those described herein, can be used in the prac essential features hereinbefore set forth and as follows in the tice or testing of the present invention, the preferred methods Scope of the appended claims. TABLE 9 Wound Healing Gel Qualitative and Quantitative Composition Concentration Ingredients Grade Function (% w/w) Grams/kg Batch Peptide 328967 (ACT1) Active 0.0072; 0.018 0.072; 0.18 0.036; 0.072 0.36; 0.72 Methylparaben NF Preservative O.17 1.7 Propylparaben NF Preservative O.O2 O.2 Glycerin USP Solvent 5 50 Sodium Phosphate Monobasic USP Buffer Agent O.263 2.63 Sodium Phosphate Dibasic USP Buffer Agent O.O44 0.44 Propylene Glycol USP Solvent 3 30 Edetate Disodium (EDTA) USP Chelating Agent O.OS O.S D-Mannitol USP Stabilizer O.OS O.S Hydroxyethylcellulose, 25OHHX NF Gelling Agent 1.25 12.5 Purified Water, qsad USP Solvent 1 1 kg SEQUENCE LISTING <16 Os NUMBER OF SEO ID NOS: 92 <21 Os SEQ ID NO 1 &211s LENGTH: 2O 212s. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1 Pro Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp 1. 5 1O 15 Asp Lieu. Glu Ile 2O <21 Os SEQ ID NO 2 &211s LENGTH: 9 212s. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 2 Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 1. 5 US 2013/0267471 A1 Oct. 10, 2013 50 - Continued <210s, SEQ ID NO 3 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated ACT 3 peptide sequence <4 OOs, SEQUENCE: 3 Arg Pro Arg Pro Asp Asp Lieu. Glu Val 1. 5 <210s, SEQ ID NO 4 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated ACT 4 peptide sequence <4 OOs, SEQUENCE: 4 Arg Pro Arg Pro Asp Asp Val Pro Val 1. 5 <210s, SEQ ID NO 5 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated ACT 5 peptide sequence <4 OOs, SEQUENCE: 5 Lys Ala Arg Ser Asp Asp Lieu. Ser Val 1. 5 <210s, SEQ ID NO 6 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 6 agacct cqgc Ctgatgacct ggagatt 27 <210s, SEQ ID NO 7 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Drosophila melanogaster <4 OO > SEQUENCE: 7 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Llys 1. 5 1O 15 <210s, SEQ ID NO 8 &211s LENGTH: 36 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 2 fusion peptide sequence <4 OOs, SEQUENCE: 8 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Llys 1. 5 1O 15 Pro Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp 2O 25 3O Asp Lieu. Glu Ile 35 US 2013/0267471 A1 Oct. 10, 2013 51 - Continued <210s, SEQ ID NO 9 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 9 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Llys 1. 5 1O 15 Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 <210s, SEQ ID NO 10 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 3 fusion peptide sequence <4 OOs, SEQUENCE: 10 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Llys 1. 5 1O 15 Arg Pro Arg Pro Asp Asp Lieu. Glu Val 2O 25 <210s, SEQ ID NO 11 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 4 fusion peptide sequence <4 OOs, SEQUENCE: 11 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Llys 1. 5 1O 15 Arg Pro Arg Pro Asp Asp Val Pro Val 2O 25 <210s, SEQ ID NO 12 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 5 fusion peptide sequence <4 OOs, SEQUENCE: 12 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Llys 1. 5 1O 15 Lys Ala Arg Ser Asp Asp Lieu. Ser Val 2O 25 <210s, SEQ ID NO 13 &211s LENGTH: 74 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 1 fusion nucleotide sequence <4 OOs, SEQUENCE: 13 cggcagcc.ca agatctggitt CCC calaccgg C9gaa.gc.cct ggaagaa.gcg gcc.cggc.ccg 6 O US 2013/0267471 A1 Oct. 10, 2013 52 - Continued acgacctgga gatc 74 <210s, SEQ ID NO 14 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Human immunodeficiency virus <4 OOs, SEQUENCE: 14 Gly Arg Llys Lys Arg Arg Glin Arg Pro Pro Glin 1. 5 1O <210s, SEQ ID NO 15 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Drosophila melanogaster <4 OOs, SEQUENCE: 15 Arg Glin Ile Lys Ile Trp Phe Glin Asn Arg Arg Met Lys Trp Llys Llys 1. 5 1O 15 <210s, SEQ ID NO 16 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Drosophila melanogaster <4 OOs, SEQUENCE: 16 Arg Glin Ile Ala Ile Trp Phe Glin Asn Arg Arg Met Lys Trp Ala Ala 1. 5 1O 15 <210s, SEQ ID NO 17 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Human immunodeficiency virus <4 OOs, SEQUENCE: 17 Arg Llys Lys Arg Arg Glin Arg Arg Arg 1. 5 <210s, SEQ ID NO 18 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated Buforin II peptide sequence <4 OOs, SEQUENCE: 18 Thir Arg Ser Ser Arg Ala Gly Lieu. Glin Phe Pro Val Gly Arg Val His 1. 5 1O 15 Arg Lieu. Lieu. Arg Llys 2O <210s, SEQ ID NO 19 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated Transportan peptide sequence <4 OOs, SEQUENCE: 19 Gly Trp Thir Lieu. Asn. Ser Ala Gly Tyr Lieu. Lieu. Gly Lys Ile Asn Lys 1. 5 1O 15 Ala Lieu Ala Ala Lieu Ala Lys Lys Ile Lieu 2O 25 US 2013/0267471 A1 Oct. 10, 2013 53 - Continued <210s, SEQ ID NO 2 O &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: model amphipathic peptide <4 OOs, SEQUENCE: 2O Llys Lieu Ala Lieu Lys Lieu Ala Lieu Lys Ala Lieu Lys Ala Ala Lieu Lys 1. 5 1O 15 Lieu Ala <210s, SEQ ID NO 21 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated K-FGF peptide sequence <4 OOs, SEQUENCE: 21 Ala Ala Wall Ala Lieu Lleu Pro Ala Wall Lieu. Lieu Ala Lieu. Lieu Ala Pro 1. 5 1O 15 <210s, SEQ ID NO 22 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated Ku70 peptide sequence <4 OOs, SEQUENCE: 22 Val Pro Met Leu Lys Pro Met Leu Lys Glu 1. 5 1O <210s, SEQ ID NO 23 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated Prion peptide sequence <4 OOs, SEQUENCE: 23 Met Ala Asn Lieu. Gly Tyr Trp Lieu. Leu Ala Leu Phe Val Thr Met Trp 1. 5 1O 15 Thir Asp Val Gly Lieu. Cys Llys Lys Arg Pro Llys Pro 2O 25 <210s, SEQ ID NO 24 &211s LENGTH: 18 212. TYPE: PRT <213s ORGANISM: Mus musculus <4 OOs, SEQUENCE: 24 Lieu. Lieu. Ile Ile Lieu. Arg Arg Arg Ile Arg Lys Glin Ala His Ala His 1. 5 1O 15 Ser Lys <210s, SEQ ID NO 25 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2013/0267471 A1 Oct. 10, 2013 54 - Continued <223> OTHER INFORMATION: mutated Pep-1 peptide sequence <4 OOs, SEQUENCE: 25 Lys Glu Thir Trp Trp Glu Thir Trp Trp Thr Glu Trp Ser Glin Pro Llys 1. 5 1O 15 Llys Lys Arg Llys Val 2O <210s, SEQ ID NO 26 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated SynB1 peptide sequence <4 OOs, SEQUENCE: 26 Arg Gly Gly Arg Lieu. Ser Tyr Ser Arg Arg Arg Phe Ser Thir Ser Thr 1. 5 1O 15 Gly Arg <210s, SEQ ID NO 27 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated Pep-7 peptide sequence <4 OOs, SEQUENCE: 27 Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Glin Tyr 1. 5 1O 15 <210s, SEQ ID NO 28 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated HN-1 peptide sequence <4 OOs, SEQUENCE: 28 Thir Ser Pro Lieu. Asn. Ile His Asn Gly Glin Llys Lieu. 1. 5 1O <210s, SEQ ID NO 29 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Gallus gallus <4 OOs, SEQUENCE: 29 Pro Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp 1. 5 1O 15 Lieu. Glu Ile <210s, SEQ ID NO 3 O &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 30 Gly Ser Asn Llys Ser Thr Ala Ser Ser Lys Ser Pro Asp Pro Lys Asn 1. 5 1O 15 Ser Val Trp Ile 2O US 2013/0267471 A1 Oct. 10, 2013 55 - Continued <210s, SEQ ID NO 31 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Gallus gallus <4 OOs, SEQUENCE: 31 Gly Ser Asn Llys Ser Ser Ala Ser Ser Lys Ser Gly Asp Gly Lys Asn 1. 5 1O 15 Ser Val Trp Ile 2O <210s, SEQ ID NO 32 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 32 Gly Arg Ala Ser Lys Ala Ser Arg Ala Ser Ser Gly Arg Ala Arg Pro 1. 5 1O 15 Glu Asp Lieu Ala Ile 2O <210s, SEQ ID NO 33 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 33 Gly Ser Ala Ser Ser Arg Asp Gly Llys Thr Val Trp Ile 1. 5 1O <210s, SEQ ID NO 34 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Pan troglodytes <4 OOs, SEQUENCE: 34 Pro Arg Val Ser Val Pro Asn Phe Gly Arg Thr Glin Ser Ser Asp Ser 1. 5 1O 15 Ala Tyr Val <210s, SEQ ID NO 35 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Gallus gallus <4 OOs, SEQUENCE: 35 Pro Arg Met Ser Met Pro Asn Phe Gly Arg Thr Glin Ser Ser Asp Ser 1. 5 1O 15 Ala Tyr Val <210s, SEQ ID NO 36 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 36 Pro Arg Ala Gly Ser Glu Lys Gly Ser Ala Ser Ser Arg Asp Gly Lys 1. 5 1O 15 US 2013/0267471 A1 Oct. 10, 2013 56 - Continued Thir Thr Val Trp Ile 2O <210s, SEQ ID NO 37 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 37 Gly Tyr His Ser Asp Lys Arg Arg Lieu. Ser Lys Ala Ser Ser Lys Ala 1. 5 1O 15 Arg Ser Asp Asp Lieu. Ser Val 2O <210s, SEQ ID NO 38 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 38 Pro Lieu. Ser Arg Lieu. Ser Lys Ala Ser Ser Arg Ala Arg Ser Asp Asp 1. 5 1O 15 Lieu. Thir Wall <210s, SEQ ID NO 39 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 39 Pro Asn His Val Val Ser Lieu. Thir Asn. Asn Lieu. Ile Gly Arg Arg Val 1. 5 1O 15 Pro Thr Asp Leu Glin Ile 2O <210s, SEQ ID NO 4 O &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Rattus norvegicus <4 OOs, SEQUENCE: 4 O Pro Ser Cys Val Ser Ser Ser Ala Val Lieu. Thir Thr Ile Cys Ser Ser 1. 5 1O 15 Asp Glin Val Val Pro Val Gly Lieu Ser Ser Phe Tyr Met 2O 25 <210s, SEQ ID NO 41 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Ovis airies <4 OOs, SEQUENCE: 41 Gly Arg Ser Ser Lys Ala Ser Lys Ser Ser Gly Gly Arg Ala Arg Ala 1. 5 1O 15 Ala Asp Lieu Ala Ile 2O <210s, SEQ ID NO 42 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2013/0267471 A1 Oct. 10, 2013 57 - Continued <4 OOs, SEQUENCE: 42 Lieu. Cys Tyr Lieu. Lieu. Ile Arg Tyr Cys Ser Gly Llys Ser Lys Llys Pro 1. 5 1O 15 Wall <210s, SEQ ID NO 43 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 43 Gly Glin Llys Pro Pro Ser Arg Pro Ser Ser Ser Ala Ser Lys Lys Glin 1. 5 1O 15 Tyr Val <210s, SEQ ID NO 44 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 44 Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp 1. 5 1O 15 Lieu. Glu Wall <210s, SEQ ID NO 45 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 45 Arg Pro Llys Pro Asp Asp Lieu. Glu Ile 1. 5 <210s, SEQ ID NO 46 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 46 Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Llys Pro Asp Asp 1. 5 1O 15 Lieu. Glu Ile <210s, SEQ ID NO 47 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 47 Arg Pro Llys Pro Asp Asp Lieu. Asp Ile 1. 5 US 2013/0267471 A1 Oct. 10, 2013 58 - Continued <210s, SEQ ID NO 48 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 48 Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp 1. 5 1O 15 Lieu. Asp Ile <210s, SEQ ID NO 49 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 49 Ser Ser Arg Ala Ser Thr Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp 1. 5 1O 15 Lieu. Glu Ile <210s, SEQ ID NO 50 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 50 Arg Pro Arg Pro Glu Asp Lieu. Glu Ile 1. 5 <210s, SEQ ID NO 51 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative Cx43 variant <4 OOs, SEQUENCE: 51 Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Glu Asp 1. 5 1O 15 Lieu. Glu Ile <210s, SEQ ID NO 52 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative CX45 variant <4 OOs, SEQUENCE: 52 Gly Asp Gly Lys Asn. Ser Val Trp Val 1. 5 <210s, SEQ ID NO 53 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2013/0267471 A1 Oct. 10, 2013 59 - Continued <223> OTHER INFORMATION: conservative CX45 variant <4 OOs, SEQUENCE: 53 Ser Lys Ala Gly Ser Asn Llys Ser Thr Ala Ser Ser Llys Ser Gly Asp 1. 5 1O 15 Gly Lys Asn. Ser Val Trp Val 2O <210s, SEQ ID NO 54 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conservative CX37 variant <4 OOs, SEQUENCE: 54 Gly Glin Llys Pro Pro Ser Arg Pro Ser Ser Ser Ala Ser Lys Llys Lieu. 1. 5 1O 15 Tyr Val <210s, SEQ ID NO 55 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: non-active control peptide <4 OO > SEQUENCE: 55 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Ile 1. 5 1O 15 Glu Lieu. Asp Asp Pro Arg Pro Arg 2O <210s, SEQ ID NO 56 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: HIV-Tat/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 56 Gly Arg Llys Lys Arg Arg Glin Arg Pro Pro Glin Arg Pro Arg Pro Asp 1. 5 1O 15 Asp Lieu. Glu Ile 2O <210s, SEQ ID NO 57 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Penetratin/ACT 1 fusion peptide sequence <4 OO > SEQUENCE: 57 Arg Glin Ile Lys Ile Trp Phe Glin Asn Arg Arg Met Lys Trp Llys Llys 1. 5 1O 15 Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 <210s, SEQ ID NO 58 &211s LENGTH: 25 212. TYPE: PRT US 2013/0267471 A1 Oct. 10, 2013 60 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antip-3A/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 58 Arg Glin Ile Ala Ile Trp Phe Glin Asn Arg Arg Met Lys Trp Ala Ala 1. 5 1O 15 Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 <210s, SEQ ID NO 59 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Tat/ACT 1 fusion peptide sequence <4 OO > SEQUENCE: 59 Arg Llys Lys Arg Arg Glin Arg Arg Arg Arg Pro Arg Pro Asp Asp Lieu 1. 5 1O 15 Glu Ile <210s, SEQ ID NO 60 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Buforin II/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 60 Thir Arg Ser Ser Arg Ala Gly Lieu. Glin Phe Pro Val Gly Arg Val His 1. 5 1O 15 Arg Lieu. Lieu. Arg Lys Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 3O <210s, SEQ ID NO 61 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Transportan/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 61 Gly Trp Thir Lieu. Asn. Ser Ala Gly Tyr Lieu. Lieu. Gly Lys Ile Asn Lys 1. 5 1O 15 Ala Lieu Ala Ala Lieu Ala Lys Lys Ile Lieu. Arg Pro Arg Pro Asp Asp 2O 25 3O Lieu. Glu Ile 35 <210s, SEQ ID NO 62 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAP/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 62 Llys Lieu Ala Lieu Lys Lieu Ala Lieu Lys Ala Lieu Lys Ala Ala Lieu Lys 1. 5 1O 15 Lieu Ala Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 US 2013/0267471 A1 Oct. 10, 2013 61 - Continued <210s, SEQ ID NO 63 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: K-FGF/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 63 Ala Ala Wall Ala Lieu Lleu Pro Ala Wall Lieu. Lieu Ala Lieu. Lieu Ala Pro 1. 5 1O 15 Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 <210s, SEQ ID NO 64 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Ku70/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 64 Val Pro Met Lieu Lys Pro Met Lieu Lys Glu Arg Pro Arg Pro Asp Asp 1. 5 1O 15 Lieu. Glu Ile <210s, SEQ ID NO 65 &211s LENGTH: 37 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Prion/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 65 Met Ala Asn Lieu. Gly Tyr Trp Lieu. Leu Ala Leu Phe Val Thr Met Trp 1. 5 1O 15 Thir Asp Val Gly Lieu. Cys Llys Lys Arg Pro Llys Pro Arg Pro Arg Pro 2O 25 3O Asp Asp Lieu. Glu Ile 35 <210s, SEQ ID NO 66 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: pWEC/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 66 Lieu. Lieu. Ile Ile Lieu. Arg Arg Arg Ile Arg Lys Glin Ala His Ala His 1. 5 1O 15 Ser Lys Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 <210s, SEQ ID NO 67 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Pep-1/ACT 1 fusion peptide sequence <4 OO > SEQUENCE: 67 US 2013/0267471 A1 Oct. 10, 2013 62 - Continued Lys Glu Thir Trp Trp Glu Thir Trp Trp Thr Glu Trp Ser Glin Pro Llys 1. 5 1O 15 Llys Lys Arg Llys Val Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 3O <210s, SEQ ID NO 68 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: SynB1/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 68 Arg Gly Gly Arg Lieu. Ser Tyr Ser Arg Arg Arg Phe Ser Thir Ser Thr 1. 5 1O 15 Gly Arg Arg Pro Arg Pro Asp Asp Lieu. Glu Ile 2O 25 <210s, SEQ ID NO 69 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Pep-7/ACT 1 fusion peptide sequence <4 OOs, SEQUENCE: 69 Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Glin Tyr Arg 1. 5 1O 15 Pro Arg Pro Asp Asp Lieu. Glu Ile 2O <210s, SEQ ID NO 70 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: HN-1/ACT 1 fusion peptide sequence <4 OO > SEQUENCE: 7 O Thir Ser Pro Lieu. Asn. Ile His Asn Gly Glin Llys Lieu. Arg Pro Arg Pro 1. 5 1O 15 Asp Asp Lieu. Glu Ile 2O <210s, SEQ ID NO 71 &211s LENGTH: 122 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 71 Lys Gly Lys Ser Asp Pro Tyr His Ala Thir Ser Gly Ala Lieu. Ser Pro 1. 5 1O 15 Ala Lys Asp Cys Gly Ser Glin Llys Tyr Ala Tyr Phe Asin Gly Cys Ser 2O 25 3O Ser Pro Thr Ala Pro Leu Ser Pro Met Ser Pro Pro Gly Tyr Lys Lieu. 35 4 O 45 Val Thr Gly Asp Arg Asn. Asn. Ser Ser Cys Arg Asn Tyr Asn Lys Glin SO 55 6 O Ala Ser Glu Glin Asn Trp Ala Asn Tyr Ser Ala Glu Glin Asn Arg Met 65 70 7s 8O US 2013/0267471 A1 Oct. 10, 2013 63 - Continued Gly Glin Ala Gly Ser Thr Ile Ser Asn Ser His Ala Gln Pro Phe Asp 85 90 95 Phe Pro Asp Asp Asn Glin Asn. Ser Lys Llys Lieu Ala Ala Gly His Glu 1OO 105 11 O Lieu. Glin Pro Lieu Ala Ile Val Asp Glin Arg 115 12 O <210s, SEQ ID NO 72 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Gallus gallus <4 OOs, SEQUENCE: 72 Lys Thr Asp Pro Tyr Ser His Ser Gly Thr Met Ser Pro Ser Lys Asp 1. 5 1O 15 Cys Gly Ser Pro Llys Tyr Ala Tyr Tyr Asn Gly Cys Ser Ser Pro Thr 2O 25 3O Ala Pro Leu Ser Pro Met Ser Pro Pro Gly Tyr Lys Lieu Val Thr Gly 35 4 O 45 Asp Arg Asn. Asn. Ser Ser Cys Arg Asn Tyr Asn Lys Glin Ala Ser Glu SO 55 6 O Glin Asn Trp Ala Asn Tyr Ser Ala Glu Glin Asn Arg Met Gly Glin Ala 65 70 7s 8O Gly Ser Thr Ile Ser Asn Ser His Ala Glin Pro Phe Asp Phe Ala Asp 85 90 95 Glu. His Glin Asn. Thir Lys Llys Lieu Ala Ser Gly His Glu Lieu. Glin Pro 1OO 105 11 O Lieu. Thir Ile Val Asp Glin Arg Pro 115 12 O <210s, SEQ ID NO 73 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 73 Lieu. Gly Phe Gly. Thir Ile Arg Asp Ser Lieu. Asn. Ser Lys Arg Arg Glu 1. 5 1O 15 Lieu. Glu Asp Pro Gly Ala Tyr Asn Tyr Pro Phe Thr Trp Asn Thr Pro 2O 25 3O Ser Ala Pro Pro Gly Tyr Asn Ile Ala Val Llys Pro Asp Glin Ile Glin 35 4 O 45 Tyr Thr Glu Lieu. Ser Asn Ala Lys Ile Ala Tyr Lys Glin Asn Lys Ala SO 55 6 O Asn Thr Ala Glin Glu Gln Glin Tyr Gly Ser His Glu Glu Asn Lieu Pro 65 70 7s 8O Ala Asp Lieu. Glu Ala Lieu. Glin Arg Glu Ile Arg Met Ala Glin Glu Arg 85 90 95 Lieu. Asp Lieu Ala Val Glin Ala Tyr Ser His Glin Asn. Asn Pro His Gly 1OO 105 11 O Pro Arg Glu Lys Lys Ala Lys Val 115 12 O <210s, SEQ ID NO 74 &211s LENGTH: 120 212. TYPE: PRT US 2013/0267471 A1 Oct. 10, 2013 64 - Continued <213> ORGANISM: Gallus gallus <4 OOs, SEQUENCE: 74 Gly Phe Gly. Thir Ile Arg Asp Thir Lieu. Asn. Asn Lys Arg Lys Glu Lieu. 1. 5 1O 15 Glu Asp Ser Gly Thr Tyr Asn Tyr Pro Phe Thir Trp Asn Thr Pro Ser 2O 25 3O Ala Pro Pro Gly Tyr Asn Ile Ala Val Llys Pro Asp Gln Met Glin Tyr 35 4 O 45 Thr Glu Lieu. Ser Asn Ala Lys Met Ala Tyr Lys Glin Asn Lys Ala Asn SO 55 6 O Ile Ala Glin Glu Glin Glin Tyr Gly Ser Asn. Glu Glu Asn. Ile Pro Ala 65 70 7s 8O Asp Lieu. Glu Asn Lieu. Glin Arg Glu Ile Llys Val Ala Glin Glu Arg Lieu 85 90 95 Asp Met Ala Ile Glin Ala Tyr Asn. Asn Glin Asn. Asn Pro Gly Ser Ser 1OO 105 11 O Ser Arg Glu Lys Llys Ser Lys Ala 115 12 O <210s, SEQ ID NO 75 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 75 Pro Tyr Lieu Val Asp Cys Phe Val Ser Arg Pro Thr Glu Lys Thir Ile 1. 5 1O 15 Phe Ile Ile Phe Met Leu Val Val Gly Lieu. Ile Ser Lieu Val Lieu. Asn 2O 25 3O Lieu. Lieu. Glu Lieu Val His Lieu. Lieu. Cys Arg Cys Lieu. Ser Arg Gly Met 35 4 O 45 Arg Ala Arg Glin Gly Glin Asp Ala Pro Pro Thr Glin Gly Thr Ser Ser SO 55 6 O Asp Pro Tyr Thr Asp Glin Val Phe Phe Tyr Lieu Pro Val Gly Glin Gly 65 70 7s 8O Pro Ser Ser Pro Pro Cys Pro Thr Tyr Asn Gly Lieu Ser Ser Ser Glu 85 90 95 Glin Asn Trp Ala Asn Lieu. Thir Thr Glu Glu Arg Lieu Ala Ser Ser Arg 1OO 105 11 O Pro Pro Leu Phe Lieu. Asp Pro Pro 115 12 O <210s, SEQ ID NO 76 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Rattus norvegicus <4 OO > SEQUENCE: 76 Cys Gly Ser Lys Glu. His Gly Asn Arg Llys Met Arg Gly Arg Lieu. Lieu. 1. 5 1O 15 Lieu. Thir Tyr Met Ala Ser Ile Phe Phe Llys Ser Val Phe Glu Val Ala 2O 25 3O Phe Leu Lieu. Ile Gln Trp Tyr Lieu. Tyr Gly Phe Thr Lieu Ser Ala Val 35 4 O 45 US 2013/0267471 A1 Oct. 10, 2013 65 - Continued Tyr Ile Cys Glu Glin Ser Pro Cys Pro His Arg Val Asp Cys Phe Leu SO 55 6 O Ser Arg Pro Thr Glu Lys Thr Ile Phe Ile Leu Phe Met Leu Val Val 65 70 7s 8O Ser Met Val Ser Phe Val Lieu. Asn Val Ile Glu Lieu Phe Tyr Val Lieu. 85 90 95 Phe Lys Ala Ile Lys Asn His Lieu. Gly Asn. Glu Lys Glu Glu Val Tyr 1OO 105 11 O Cys Asn Pro Val Glu Lieu Gln Lys 115 12 O <210s, SEQ ID NO 77 &211s LENGTH: 239 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: enhanced green fluorescent protein <4 OO > SEQUENCE: 77 Met Val Ser Lys Gly Glu Glu Lieu Phe Thr Gly Val Val Pro Ile Leu 1. 5 1O 15 Val Glu Lieu. Asp Gly Asp Val Asn Gly His Llys Phe Ser Val Ser Gly 2O 25 3O Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Lieu. Thir Lieu Lys Phe Ile 35 4 O 45 Cys Thir Thr Gly Lys Lieu Pro Val Pro Trp Pro Thr Lieu Val Thir Thr SO 55 6 O Lieu. Thir Tyr Gly Val Glin Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 7s 8O Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Glin Glu 85 90 95 Arg Thir Ile Phe Phe Lys Asp Asp Gly Asn Tyr Llys Thr Arg Ala Glu 1OO 105 11 O Val Llys Phe Glu Gly Asp Thir Lieu Val Asn Arg Ile Glu Lieu Lys Gly 115 12 O 125 Ile Asp Phe Lys Glu Asp Gly Asn. Ile Lieu. Gly His Llys Lieu. Glu Tyr 13 O 135 14 O Asn Tyr Asn. Ser His Asn Val Tyr Ile Met Ala Asp Llys Glin Lys Asn 145 150 155 160 Gly Ile Llys Val Asn. Phe Lys Ile Arg His Asn. Ile Glu Asp Gly Ser 1.65 17O 17s Val Glin Lieu Ala Asp His Tyr Glin Glin Asn Thr Pro Ile Gly Asp Gly 18O 185 19 O Pro Val Lieu. Lieu Pro Asp Asn His Tyr Lieu. Ser Thr Glin Ser Ala Lieu. 195 2OO 2O5 Ser Lys Asp Pro Asn. Glu Lys Arg Asp His Met Val Lieu. Lieu. Glu Phe 21 O 215 22O Val Thir Ala Ala Gly Ile Thr Lieu. Gly Met Asp Glu Lieu. Tyr Lys 225 23 O 235 <210s, SEQ ID NO 78 &211s LENGTH: 60 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 78 US 2013/0267471 A1 Oct. 10, 2013 66 - Continued C cct cotc.cc gggcct cotc ccgggcct Co. tcc.cggCCCC ggc.ccgacga Cctggagat C 6 O <210s, SEQ ID NO 79 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 79 cggcc.ccggc ccgacgacct ggagatc 27 <210s, SEQ ID NO 8O &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated ACT 3 nucleotide sequence <4 OOs, SEQUENCE: 80 cggcc.ccggc ccgacgacct ggaggtg 27 <210s, SEQ ID NO 81 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated ACT 4 nucleotide sequence <4 OOs, SEQUENCE: 81 cggcc.ccggc ccgacgacgt gcc.cgtg 27 <210s, SEQ ID NO 82 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: mutated ACT 5 nucleotide sequence <4 OOs, SEQUENCE: 82 aaggc.ccggit ccgacgacct gtc.cgtg 27 <210s, SEQ ID NO 83 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Drosophila melanogaster <4 OOs, SEQUENCE: 83 cggcagcc.ca agatctggitt CCC calaccgg C9gaa.gc.cct ggaagaag 48 <210s, SEQ ID NO 84 &211s LENGTH: 108 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 2 fusion nucleotide sequence <4 OOs, SEQUENCE: 84 cggcagcc.ca agatctggitt CCC calaccgg C9gaa.gc.cct ggaagaa.gcc ctic ct cocgg 6 O gcct cotcCC gggcct cotic ccggcc.ccgg ccc.gacgacc tigagat C 108 <210s, SEQ ID NO 85 &211s LENGTH: 75 &212s. TYPE: DNA US 2013/0267471 A1 Oct. 10, 2013 67 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 1 fusion nucleotide sequence <4 OOs, SEQUENCE: 85 cggcagcc.ca agatctggitt CCC calaccgg C9gaa.gc.cct ggaagaa.gcg gcc.ccggcc C 6 O gacgacctgg agatc <210s, SEQ ID NO 86 &211s LENGTH: 75 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 3 fusion nucleotide sequence <4 OOs, SEQUENCE: 86 cggcagcc.ca agatctggitt CCC calaccgg C9gaa.gc.cct ggaagaa.gcg gcc.ccggcc C 6 O gacgacCt99 aggtg <210s, SEQ ID NO 87 &211s LENGTH: 75 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 4 fusion nucleotide sequence <4 OO > SEQUENCE: 87 cggcagcc.ca agatctggitt CCC calaccgg C9gaa.gc.cct ggaagaa.gcg gcc.ccggcc C 6 O gacgacgtgc ccgtg <210s, SEQ ID NO 88 &211s LENGTH: 75 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Antp/ACT 5 fusion nucleotide sequence <4 OOs, SEQUENCE: 88 cggcagcc.ca agatctggitt CCC calaccgg C9gaa.gc.cct ggaagaagaa ggc.ccggtc.c 6 O gacgacctgt CC9tg <210s, SEQ ID NO 89 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Danilo rerio <4 OOs, SEQUENCE: 89 Pro Cys Ser Arg Ala Ser Ser Arg Met Ser Ser Arg Ala Arg Pro Asp 1. 5 1O 15 Asp Lieu. Asp Val 2O <210s, SEQ ID NO 90 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Gallus gallus <4 OOs, SEQUENCE: 90 Pro Arg Val Ser Val Pro Asn Phe Gly Arg Thr Glin Ser Ser Asp Ser 1. 5 1O 15 US 2013/0267471 A1 Oct. 10, 2013 - Continued Ala Tyr Val <210s, SEQ ID NO 91 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Danilo rerio <4 OOs, SEQUENCE: 91 Pro Arg Met Ser Met Pro Asn Phe Gly Arg Thr Glin Ser Ser Asp Ser 1. 5 1O 15 Ala Tyr Val <210s, SEQ ID NO 92 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Cx43 isoleucine deletion <4 OOs, SEQUENCE: 92 Arg Glin Pro Lys Ile Trp Phe Pro Asn Arg Arg Llys Pro Trp Llys Llys 1. 5 1O 15 Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp Lieu. Glu 2O 25 1. A method of treating a chronic wound in a subject, 13. The method of claim 1, wherein the chronic wound is comprising administering to the Subject a topical formulation selected from the group consisting of venous leg ulcers, dia comprising at least one alpha connexin polypeptide, wherein betic foot ulcers, and pressure ulcers. the formulation is administered in a dosing regimen effective 14. The method of claim 1, wherein the method reduces the for the treatment of the chronic wound. symptoms of the chronic wound. 2. The method of claim 1, wherein the formulation is 15. The method of claim 1, wherein the formulation does administered daily or weekly. not induce excessive levels of side effects. 3. The method of claim 1, wherein the formulation is 16. The method of claim 1, wherein the method reduces the administered in a dosing regimen at day 0, day 3, week 1, time to 100% wound closure as compared to standard of care week 2, week 3, week 4, week 5, week 6, week 7, week 8, treatment for wound healing. week 9, week 10, week 11, and week 12. 17. The method of claim 1, wherein the method reduces the 4. The method of claim 1, wherein the formulation further time to 50% wound closure at compared to standard of care comprises hydroxycellulose at a concentration of about treatment for wound healing. 1.25% (w/w). 18. The method of claim 1, wherein the method reduces the 5. The method of claim 4, wherein the hydroxycellulose gel time to 50% wound closure as compared to standard of care is present at a concentration of about 1.25% (w/w). treatment for wound healing. 6. The method of claim 1, wherein the at least one isolated 19. The method of claim 1, wherein the method increases polypeptide comprises the carboxy terminal-most 4 to 30 the average percent of wound closure at 12 weeks, as com contiguous amino acids of an alpha Connexin, or a conserva pared to standard of care treatment for wound healing. tive variant thereof. 20. The method of claim 1, wherein the method reduces wound area as compared to the wound area reduced by stan 7. The method of claim 1, wherein the alpha connexin dard of care treatment. polypeptide is connexin 37, connexin 40, connexin 43, or 21. The method of claim 1, wherein the chronic wound is connexin 45. treated in the absence of clinically significant abnormalities. 8. The method of claim 1, wherein the alpha connexin 22. The method of claim 1, wherein the formulation does polypeptide is linked at its amino terminus to a cellular inter not induce production of anti-alpha connexin polypeptide nalization transporter. antibodies in the subject. 9. The method of claim 8, wherein the cellular internaliza 23. The method of claim 1, wherein the method increases tion transporter is an antennapedia sequence. the incidence or frequency of 100% complete wound closure 10. The method of claim 1, wherein the formulation com compared to standard of care treatments for wound healing. prises a polypeptide comprising the amino acid sequence of 24. The method of claim 1, wherein the method is used to SEQID NO:9. treat an ulcer lacking Sufficient wound size reduction within 11. The method of claim 1, wherein the chronic wound is one, four, 12, or 24 weeks of Standard of care. an ulcer. 25. The method of claim 1, wherein the method is used to 12. The method of claim 1, wherein the chronic wound is a treat an ulcer with less than 50% wound closure within one lower extremity ulcer. four, 12 or 24 weeks of standard of care. US 2013/0267471 A1 Oct. 10, 2013 69 26. The method of claim 1, wherein the method is used to treat an ulcer lacking Sufficient wound size reduction within one, four, 12, or 24 weeks of Standard of care. 27. The method of claim 1, wherein the method is used to treat an ulcer lacking Sufficient wound size reduction within one, four, 12, or 24 weeks of Standard of care. 28. The method of claim 1, wherein the method is used to treat a patient with an ulcer possessing wound area and dura tion characteristics of a chronic wound. 29. The method of claim 1, wherein the method is used to treat a patient with an ulcer possessing a wound with a non healing wound trajectory. 30. The method of claim 1, wherein the method is used to treat an ulcer possessing wound area and time duration char acteristics of a chronic wound. 32. A method of treating a chronic wound in a Subject, comprising administering to the Subject a topical formulation comprising at least one alpha connexin polypeptide in addi tion to standard of care compression therapy, wherein the chronic wound is healed at a faster rate and/or increased frequency than achieved with standard of care compression therapy alone.