EP4633733A1 - Oxidized carbon nanoparticles for treating oxidative skin disorders - Google Patents

Abstract

A transcutaneous composition for treating an oxidative skin disorder of a mammal in need is disclosed containing an effective oxidative skin disorder treating amount of covalently-substituted oxidi zed activated charcoal (OACs) nanoparticles dissolved or dispersed in an aqueous composition containing a thickening agent providing a viscosity of about 1000 to about 20,000 cps, and about 5 to about 20 wt % of a skin permeation enhancer. A substituent of the substituted OAC comprises an average of about 2 to about 5 polyethylene glycol ( PEG) chains covalently linked to each OAC, or an average of about 2 to about 5 metal ion chelating groups covalently linked to each OAC, or an average of about 2 to about 5 PEG chains and an average of about 2 to about 5 metal ion chelating groups linked to each OAC. A method of treating an oxidative skin disorder of a mammal is also disclosed.

Description

OXIDIZED CARBON NANOPARTICLES

FOR TREATING OXIDATIVE SKIN DISORDERS

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to US applications Serial No . 63/ 433 , 333 filed on December 16 , 2022 , whose disclosures are incorporated herein by reference .

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant number R01NS 094535 awarded by the National Institutes of Health . The government has certain rights in the invention . This invention was also made with the support of the Welch Foundation under grant number BE- 0048 .

FIELD OF THE INVENION

The present invention relates to the field of carbon nanomaterials and to their therapeutic uses in treating skin diseases .

BACKGROUND ART

Therapy to improve outcome from chronic venous stasis dermatitis ( CVSD) , a condition that af flicts up to 20 million in the US , remains elusive , in part because of the complexity of the inj ury and its consequences . Especially at risk are patients with obesity, diabetes , hypertension and inactivity .

The skin of the lower extremity is exposed to high venous pressures due to incompetent ( refluxing) valves , venous varicosities and/or obstruction . Venous capillaries become excessively permeable or even disrupted causing red blood cells to leak into the skin . The red blood cells undergo lysis in the tissue releasing hemoglobin that is then broken down into toxic materials including hemin and free iron .

Iron has been postulated to induce oxidative stress in conditions like chronic venous stasis dermatitis ( CVSD) ^1-5, and iron has been implicated in other conditions in the iron-mediated cell death program, " f erroptosis" leading to lipid peroxidation and cell death⁶. In addition to ferroptosis , our own research has found that hemin causes cellular " senescence" in neurons and endothelial cells⁷ , a condition in which cells can over time become a nidus for inflammation and ultimately cell dys function and recruit neighboring cells . ⁸

It is believed that these blood breakdown products contribute to the vicious cycle of swelling, hemorrhage , pain and ultimately ulceration and infection seen in venous stasis ulcers . Indeed, the amount of iron in cells af fected by CVSD has been associated with activating tissue breakdown enzymes² . There is no FDA-approved therapy for CVSD and current therapies are largely symptomatic and do not address the underlying disease mechanisms . ⁹

Some of the present inventors and their coworkers developed oxidi zed carbon nanoparticles ( OCNPs ) . OCNPs are biologically active materials derived from the acidic oxidation of carbon rich sources , such as single walled graphene nanotubes , coal or the preferred material , activated charcoal . These OCNPs are versatile and highly ef fective in a variety of pre-clinical models¹' ^10-12 . These nanoparticles are similar, but chemically and physically di f ferent from the previously described hydrophilic carbon clusters (HCCs ) ⁷ as is described hereinafter .

A new indication for recently described²⁷ new class of OCNPs is disclosed hereinafter . These materials are derived from medicinal grade activated charcoal and are reacted to form poly ( ethylene glycol ) - functionali zed oxidi zed activated charcoal ( PEG-OACs ) , that among their other properties catalytically dismutate superoxide and shuttle electrons between mitochondrial constituents²⁷ . The OACs-PEG particles can also be utili zed to form further substituted particles with the iron ion chelating drug deferoxamine⁷ ( DEF) to form DEF-OAC-PEG particles , or the oxidi zed activated charcoal can be reacted with both DEF and PEG at one time to form DEF-OAC-PEG particles .

SUMMARY

In accordance with the preset invention, oxidative skin disorders such as photosensitivity, aging, some malignancies and allergic disorders , as well as vascular disorders such as the common health care problem of chronic venous stasis dermatitis ( CVSD) , a painful condition characteri zed by edema, venous varicosities , and hemosiderin skin deposits¹³ are treated with a composition of substituted OACs discussed herein . These OACs possess remarkable attributes that make them particularly amenable to relieving pain, reducing inflammation, oxidative stress and edema, and in preventing ulceration .

A transcutaneous composition for treating an oxidative skin disorder of a mammal in need is contemplated that comprises an ef fective oxidative skin disorder treating amount of covalently- substituted oxidi zed activated charcoal nanoparticles ( OACs ) dissolved or dispersed in an aqueous composition . The composition ( formulation) contains an amount of thickening agent suf ficient to provide a viscosity of about 1000 to about 20 , 000 cps and about 5 to about 20 weight percent of a skin permeation enhancer . A substituent of the above substituted OAC has 1 ) an average of about 2 to about 5 polyethylene glycol ( PEG) chains covalently linked to the OAC, or 2 ) an average of about 2 to about 5 metal ion chelating groups covalently linked to the OAC, or 3 ) an average of about 2 to about 5 PEG chains and an average of about 2 to about 5 metal ion chelating groups linked to each OAC .

An ef fective amount of the transcutaneous composition is applied to the af fected area of the skin and gently massaged until it is appropriately sorbed ( absorbed and/or adsorbed) . The transcutaneous formulation utili zes one or more skin permeation enhancer agents to assist the composition' s sorption ( absorption and/or adsorption) into the skin of a mammalian subj ect in need . Alternatively, or in addition, the transcutaneous composition can be used together with elastic or inelastic compressive support . In some cases , the transcutaneous composition can be used together with antibiotics, emollients, or other cutaneous treatments such as the Unna boot.

Thus, the OCNP chemical structure utilized here can include further substituents such as the illustrative chelating drug deferoxamine⁷ (DEF) , to address the toxic effects of blood breakdown products hemin and iron that likely perpetuate the viscous cycle of venous insufficiency, bruising, skin breakdown and ulceration in CVSD⁷'¹⁴. A preferred transcutaneous formulation to treat CVSD contains an effective amount of DEF-substituted and PEG- substituted OACs (DEF-OCNP-PEG) dissolved or dispersed in a pharmaceutically acceptable diluent (carrier) . An effective amount of the transcutaneous composition is applied to the affected area of the mammalian subject's skin and gently rubbed (massaged) until it is appropriately sorbed (absorbed and/or adsorbed) .

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures forming a part of this disclosure,

Fig. 1 is a graph showing the permeation of DEF-PEG-OACs through pig ear skin using different enhancers. For these studies the OAC concentration (10%) , CARBOPOL® 934 polymer concentration (2%) , and skin permeation enhancer concentration (10%) were held constant, and the remainder of the gel was water. The integrity of pig ear skin was tested by measuring the trans-epidermal water loss and made sure it was constant. A Franz diffusion cell³⁰ was set up with water at 32 degrees Celsius and a phosphate buffer solution. The curves shown here compare the diffusion of the OAC when mixed with three different permeation enhancers [polyethylene glycol 400 (PEG 400) , propylene glycol (PG) , and diethylene glycol monomethyl ether ( Transcutol®, TC) and the control (no permeation enhancer; CNP Solution) ) .

Fig. 2 shows a schematic synthesis of PEG- OACs . Here, GMP activated charcoal (AC) is oxidized via fuming nitric acid at a temperature of 100 °C for 6 hours (h) followed by PEGylation to first form OACs and then PEG-OACs .

Fig. 3, in three panels as Figs. 3A, 3B and 3C, show representative HR-TEM images of particles oxidized at 100 °C for 2 h OACOAC, 4 h OAC, and 6 h OAC, respectively. Scale bar = 5 nm. The average carbon core diameter of the 2 h OAC, 4 h OAC, and 6 h OAC particles is 13 nm, 7 nm, and 2.5 nm, respectively .

Fig. 4, in two panels as Figs. 4A and 4B, are HR-TEM images of HCCs and PEG-HCCs, respectively, spanning empty domains on lacy carbon grids that illustrate both HCC preparations have aggregated during the sample preparation, which consisted of placing an aqueous dispersion of the respective HCCs on the lacy carbon grid and drying the sample at 70 °C for 16 h, and appear as Figures la and 2b, respectively, of Berlin et al.²⁹.

Fig. 5 is a graph similar to that of Fig. 1 showing comparative permeation studies of pig ear skin and human skin using an illustrative composition containing 10 wt% PEG-OAC-DEF (0.065 wt% or 0.65 mg/ml carbon nanoparticles) , 20 wt% of Transcutol® as a permeation enhancer and 80% aqueous Carbopol® 934 NF (2 wt% gel in water) over a 24 h time period as discussed in Study 2 hereinafter and using the data from Table 3.

Not otherwise defined abbreviations used: h = hour(s) ; min = minute (s) ; d = days; rt = room temperature; g - gram(s) ; kDa = kilodalton ( s ) ; mg = milligram ( s ) ; pg = microgram ( s ) ; ng = nanogram(s) ; Mol = mole(s) ; mmol = millimole ( s ) ; L = liter(s) ; mL = milliliter ( s ) ; cm - centimeter ( s ) ; wt% = weight percent; mm =millimeter ( s ) ; m = micrometer ( s ) ; nm = nanometer ( s ) ; MWCO = molecular weight cut off; UV = ultraviolet light; DMF = dimethylformamide; and ACN = acetonitrile .

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The contemplated DEF-OACs have several remarkable features including acting as catalytic high-capacity mimics of superoxide dismutase, without exogenous regeneration, the important protective enzyme that is depleted in oxidative injury. They also catalyze the formation of polysulfides from hydrogen sulfide. They are rapidly taken up by cells and co-localize with mitochondria where they protect mitochondria from injury¹¹.

Moreover, their flexible chemistry permits attachment of a variety of molecules. Here, we have attached the chelating drug, deferoxamine (DEF) , facilitating its uptake into the cell and demonstrating excellent efficacy against the detrimental effects of iron and hemin in brain hemorrhage models.⁷ Oxidi zed carbon nanoparti cl es (OCNPs) :

OCNPs are biologically active materials derived from the acidic oxidation of carbon rich sources , such as single walled graphene nanotubes , coal or the preferred material , activated charcoal (AC ) . Medical grade ( GMP ) activated charcoal is a particularly preferred carbon-containing starting material because using a medicinal grade starting material removes one impediment to regulatory approval .

As used hereinafter, oxidi zed activated charcoal is abbreviated "OAC" . OACs are particularly preferred among OCNPs and the properties both chemical and physical between OACs and other OCNPs such as hydrophilic carbon clusters (HCCs ) and single walled graphene nanotubes ( S CNTS ) . Abbreviated plurals for these particles are formed by adding a lower-case " s" to the otherwise all upper-case letters of the abbreviation .

Reaction products are written as a three upper-case lettered abbreviation for the linked substituent linked by a hyphen to the three-letter abbreviation of the base particle . When two reactants are linked to a particle the abbreviation is written as one substituent-three-letter- abbr e via t ion-hyphen-par tide- abbreviation-hyphen- other substituent three-letter abbreviation . The position order of abbreviations for a substituent and the particle can be in either order ( substituent- particle or particle-substituent ) . When two substituents are linked to a particle , the particle abbreviation is preferably between the abbreviations for the two substituents . The OCNPs contemplated herein are derived from the harsh acid treatment of the carbon-rich activated charcoal source thereby generating nanometer si zed particles decorated with functional groups including carboxylates , permitting covalently bonding poly ( ethylene ) -glycol ( PEG) to improve biodistribution and cellular uptake , and quinones responsible for the enzyme-like catalytic reactions that benefit mitochondrial electron transport¹¹ .

The illustrative OCNPs used in the present studies are OACs that are typically PEGylated, and are also preferably reacted with the chelating drug, deferoxamine ( DEF) , as illustrated schematically in Fig . 2 and discussed hereinafter . The OACs , prepared as discussed herein, provide about 9 to about 25 percent carboxyl group carbon atoms , and preferably 20 to about 25 carboxyl group carbons by X-ray photoelectron spectroscopy (XPS ) . Those carboxyl groups can be reacted with an amine , preferably a primary amine , under amide- forming reaction conditions as with a water-soluble diimide compound to form carboxamide-linked substituents .

The oxidi zed particles also contain keto groups and adj acent keto groups as are found in quinones . Those keto groups can also be reacted with primary amines under reductive amination conditions to form amine-linked substituents .

In one aspect of the present invention, the OCNPs are reacted via covalent amido-con ugation of OCNPs and omega-monomethoxypolyethylene glycol amine . That reaction is referred to herein and in the art as "PEGylation" , and the product is referred to as PEGylated-OACs , or more simply as PEG-OACs . A preferred omega-methoxy poly ( ethylene glycol ) amine used can have a molecular weight of about 2000 to about 10 , 000 Da . Mixtures of these PEG derivatives having di f fering molecular weights can also be used . It is presently preferred to use an omega-methoxy poly ( ethylene glycol ) amine having an average molecular weight of about 5000 Da in the syntheses of a PEG-OAC preparation .

Amine-substituted poly ( ethyleneoxide ) - poly (propyleneoxide ) block copolymers [ also referred to as copolymers ] are a sub-group of PEG compound that can also be linked to OACs in a manner similar to that used herein and are hereby included in the terms "PEG" and "PEGylated" . Use of propylene oxidecontaining polymers can provide an increased lipophilicity to the resulting particles to improve fat solubility . Synthesis of amine-terminated PEG- PPG-containing polymers is discussed by Gyulai et al .³⁵ Similar molecular weight PEG-PPG polymers are used as with the PEG-only products . It is to be understood and is well known in the art that the commercially available PEG-containing products are mixtures of similar but di f fering molecular weights due to the di f fering number of ethylene glycol repeating units present . Thus , in a PEG product having a molecular weight of about 2000 Da, there are an average of about 60 repeating units . In a PEG product having a molecular weight of about 5000 Da, there are an average of about 112 repeating units , whereas in a PEG product having a molecular weight of about 10 , 000 Da, there are an average of about 225 repeating units . In some aspects of the invention, the carboxyl groups present can be PEGylated so that there would be an average of about 9 to about 25 percent carboxamidoPEG groups , and preferably 20 to about 25 percent carboxamidoPEG groups per particle by X-ray photoelectron spectroscopy (XPS ) . In other aspects , the particles are substituted with both PEG substituents and deferoxamine ( DEF) substituents . The reaction products are referred to herein as PEG- OAC-DEF or DEF-OAC-PEG particles .

The PEG-OAC, DEF-OAC and PEG-OAC-DEF particles can be used in treating oxidative skin conditions . The PEG-OAC-DEF particles are particularly preferred for treating oxidative skin conditions when hemorrhage and/or iron associated with the pathology are involved, such as where there is blood leakage beneath the skin, such as chronic venous stasis dermatitis ( CVSD) . These particles are also useful in treating a decubitus ulcer and nonhealing ulcers that occur in patients with venous disease , diabetes mellitus , and peripheral arterial disease .

PEG-OACs are synthesi zed using a general procedure previously described in WO 2021 /252382 . In brief , particles are preferably synthesi zed from coconut shell-derived activated charcoal ( coconut AC ) and oxidi zed with fuming nitric acid ( 90% HNOg ) followed by conj ugation with amino-poly ( ethylene glycol ) that produces carbon core particles having a mean diameter of about 3 nm .

The coconut AC is obtained from the manufacturer such as EnviroSupply & Service ( Irvine , CA) as a powder of varying average longest dimension . In one embodiment, the as-provided powder is passed through a sieve having a 20 m opening (U.S. Standard No. 635 openings) prior to the oxidation step of the through-put particles to provide oxidation product of greater uniformity of particle size. Unless stated otherwise al of the OACs made and used herein were prepared using coconut shell activated charcoal.

The oxidized particles are discoid in shape and form a solution or dispersion in water at a concentration of about 1 to about 5 mg/mL. The dispersion is stable to separation (non-separating) at ambient room temperature for at least seven days. The oxidized carbon nanoparticulate dispersion in water exhibits an UV absorbance maximum at about 220 nm. The particles contain about 5 to about 15 percent, and preferably about 9 to about 15 percent, carbon atoms as carbonyl groups by X-ray photoelectron spectroscopy (XPS) , and pass through a 0.22 m pore size polyethersulfone (PES) filter membrane (WO 2021/252382) .

Illustratively, starting with GMP grade activated charcoal (AC) . No in vivo toxicity has been observed.

Hydrophilic carbon clusters (HCCs) are high-capacity mimics of superoxide dismutase¹, the major early defense mechanism against oxidative stress and are prepared from single-walled carbon nanotubes. Their broad redox potential extends their action as a redox catalyst among mitochondrial constituents involved in electron transport. HCCs can be considered as a member of new class of material, a nanoparticle enzyme, or "nanozyme"¹¹. Notably, our HCCs are highly ef fective after IV inj ection in acute inj ury models¹' ¹² .

When tested against the ef fects of hemin in vascular endothelial and neuronal cell culture , PEG- HCCs dramatically reduced senescence⁷ . However, sensitivity to ferroptosis was increased⁷ , negating the benefit of reducing senescence , critical where iron levels are high such as in chronic venous stasis dermatitis ( CVSD) . Looking more speci fically at a model treatment for an oxidative skin condition, CVSD was selected for a second look using PEG-HCCs linked to an iron-chelating reagent as discussed below .

To address both mechanisms , the ability to covalently bond the chelating drug, deferoxamine ( DEF) , using the same chemistry for PEG bonds , was exploited creating a particle with both PEG and DEF ( PEG-HCCs-DEF) .

DEF is a clinically used iron chelating medication including in vi tro in skin derived cells^{21- 26} , although with maj or clinical shortcomings including inconsistent cellular uptake and therefore high doses increasing risk of toxicity, and alone is not as ef fective as treating in combination with the HCCs .

Tested against hemin, PEG-HCC-DEF prevented both senescence and ferroptosis , and with better cell survival than either the HCCs or DEF alone or together in their usual dose⁷ . Use of the PEG-HCC-DEF improved protection using a > 100-times lower DEF concentration than when DEF was used alone . PEG-HCC- DEF was also ef fective in vi vo in mice brains after experimental intracerebral hemorrhage⁷ . These favorable ef fects indicate this combination particle might be ef fective against key mechanisms by which CVSD causes its chronicity and complications . However, it is believed that the unexpectedly improved characteristics of present OACs provide an enhanced platform compared to the HCCs .

Approximately 20 million Americans are af flicted with CVSD, and CVSD is more prevalent in patients with diabetes , obesity, aging and a sedentary li festyle²⁶.

CVSD can progress to ulceration of the skin and hospitali zation . There is no definitive treatment⁸ and maj or complications include skin ulceration and infection requiring hospitali zation, intravenous antibiotics , and wound care^21-26.

OACs vs . HCCs

HCCs are prepared from single-walled carbon nanotubes by a harsh oxidation procedure employing oleum and nitric acid and have dimensions of about 1 nm wide and about 40 to about 60 nm long²⁹. More recent descriptions place those dimensions as about 3 nm wide and a length of about 30 to about 40 nm long¹' ⁷ .

Berlin et al .²⁹ report in regard to their Figure 5 that HCCs are insoluble in water and that their PEG-HCCs are dispersible in water, but precipitate on standing for 7 days . The present OACs are themselves stably soluble and/or dispersible in water or PBS for at least one week at 1 mg/mL at room temperature¹¹' ²⁷ . The PEG-OACs are even more soluble and/or dispersible . The OACs used herein are prepared by the oxidation of charcoal particles using refluxing fuming nitric acid (90 % nitric acid) . In preferred embodiments, a medical grade of coconut shell charcoal is used as the starting material. These particles are disc-shaped, with a longest dimension or diameter of about 2 to about 15 nm, depending upon the length of time of oxidative treatment between 2 and 6 hours. On the other hand, Berlin et al.² describe HCCs as being elongated structures approximately 40 nm in length.

Furthermore, as stated by Berlin et al.²⁹, HCCs are most likely somewhat protonated in water, therefore, there is the possibility that more interparticle hydrogen bonds form between HCC particles than between OACs. Hydrogen bonding is most likely the cause of the aggregation because the addition of NaOH to HCCs in water apparently eliminates aggregation in the composition Berlin et al ²⁹ Therefore, the physical as well as the chemical structure of the particles is an important factor in distinguishing these particles from previous particles .

As can be seen from the photomicrographs of Figs. 3 and 4, the OACs (Fig. 3) tend to appear as individual particles upon application and drying on a lacy carbon grid, whereas HCCs (Fig. 4) tend to agglomerate on the grid surface under similar conditions (Fig. 4A) . PEG-HOC particles also agglomerated (Fig. 4B) .

Derry et al.¹¹, Wu et al.²² and Wu et al.³ⁱ reported differences in the reduction and oxidation rate constants (Ki and K2) for the dismutation of superoxide by PEG-HCCs and PEG-OACs change their electronic properties. Thus, OACs are reduced by superoxide (OAC* + O2*“ -> OAC- + O2) faster than they are oxidized by superoxide (2H+ + 0AC“ + O2*- -> OAC* + H2O2) , whereas PEG-HCCs are oxidized faster than they are reduced.

It is thus seen that the HCCs and PEG-HCCs of Samuel et al.¹ and Dharmulingan et al.¹ differ chemically and physically from the OACs and PEG-OACs of Derry et al.¹¹, Wu et al.²¹ Berlin et al.²⁹ and Wu et al.³⁴ utilized herein.

A further type of carbon-containing nanoparticle are single-walled carbon nanotube that are often referred to as carbon nanotubes (CNTs) or SWCNTs. Oxidized forms of such materials are described in EP 1 428 793 Al, which after filing became US Patents No. 7,531,157 and No. 7,854,914.

As pointed out in that EP application, those materials are not soluble in water, and the problem said to be solved by those patents is the preparation of water-soluble particles. Therefore, there is a demonstrable difference in water solubility between the SWCNTs and the OACs used here.

Moreover, the method of solubilizing the SWCNTs described in EP 1 428 793 Al involves the formation of isocyanate groups from added urea which those inventors state in Paragraph [0063] EP 1 428 793 Al can react with several compound types such as alcohols to form urethanes, amines to form ureas, with urethanes to form allophanates , and so on. It is likely that the covalent polymerization or oligomerization of isocyanate derivatives on the SWCNTs increases their water solubility by enhancing the number of hydrogen bonds that can form with the SWCNTs .

A further distinction between the SWCNTs of EP 1 428 793 Al and the OACs used here is the percentage of oxidized carbon atoms. Paragraph [0070] of the EP application reports a total of about 1 percent oxidized carbon atoms. Table 1, hereinafter, teaches a percentage of oxidized carbon atoms in contemplated OACs of greater than 30 percent, which is due to the harsh oxidation process using fuming nitric acid imparts a high percentage of oxidized carbon atoms.

An important limitation to the use of carbon nanotubes in drug development is the necessity that they be small enough to interact with cells but not large enough to become toxic. As reviewed by Kobayashi et al., J. Occup Health 59:394-407 (2017) , carbon nanotubes with lengths in terms of microns are toxic through several routes and are known to bioaccumulate in the lungs. Therefore, a material which is chemically like a carbon nanotube is likely not suitable for use as a drug.

Claims 5 and 9 of the above-noted US Patent No. 7,857,194 recite the following uses for the carbon-nanotube product produced by the method disclosed in '194 patent: "a nanoelectronic device, a memory element, a field emission device, a sensor, an actuator, an electromechanical device, a composite material, a coating/paint/paste, a hydrogen storage device, a battery or fuel cell, a supercapacitor, a photoelectrochemical device, a photovoltaic device, an energy conversion device, a light emitting diode, a liquid crystal display, a probe scanning probe microscopy, a non-linear optical device or antenna, and a catalyst . " [US patent No . 7 , 854 , 914 ; claims 5 and 9 . ]

The ' 914 patent makes no mention of use of its water-soluble SWCNTs in a pharmaceutical product . There is mention in the background of interest in water-soluble CNTs for unstated biological applications .

It is thus seen that there are substantial di f ferences between the OACs used herein and either HCCs or SWCNTs .

Pa thophysi ol ogy of CVSD :

The skin of the lower extremity is exposed to high venous pressures in patients with CVSD, due to incompetent ( refluxing) valves , venous varicosity and or obstruction . Venous capillaries can become excessively permeable or even disrupted, causing red blood cells to leak into the skin . The red blood cells undergo lysis in the tissue releasing hemoglobin that is then broken-down releasing hemin and free iron, ultimately oxidi zed into hemosiderin . These breakdown products can then induce oxidative stress and the iron-mediated cell death program, ferroptosis leading to lipid peroxidation and death⁶' ⁷

In addition to f erroptosis-mediated cell death due to the high levels of free iron that are associated with venous disease , it was also found that hemin causes a dramatic phenotypic alteration termed " senescence"⁷ . The senescent phenotype is increasingly being recogni zed to be responsible for several pathological features of aging including generating an inflammatory state that can recruit neighboring cells into a perpetuating state of dys function⁸ .

Senescence can be stimulated by a variety of pathologies including an incomplete DNA damage response ( DDR) . In in-vi tro vascular endothelial and neuronal cell cultures , hemin-generated rapid and persistent DDR, remarkably similar to that caused by gamma radiation and etoposide , both well known to induce senescence⁷ are noted . Senescent cells persist in cell cycle arrest and become resistant to cell death pathways , including ferroptosis ; iron levels increase dramatically in senescence without cell death due to the many biochemical changes that accompany this phenotype¹⁵. Other oxida ti ve skin di sorders

The skin is very sensitive to oxidative damage , especially from UV light from the sun . A number of disorders are associated with oxidative stress that are targets of our therapy . These include photosensitivity, aging, some malignancies and allergic disorders . ^16-18 several are also influenced by vascular disorders sharing in common some mechanisms of CVSD . Our OACs are able to catalytically dismutate the superoxide radical , quench hydroxyl radical and catalyze the generation of polysul fides from hydrogen sul fide , which act as polysul fides and facilitate persul f idation of proteins as a post-translational modi fication . Disorders of hydrogen sul fide in the skin include allergic diseases such as psoriasis and dermatoses¹⁹' ²⁰ . Preparation of OACs

Powdered medical grade activated coconut shell charcoal ("c", 0.500 g, 41.7 mmol carbon) was dispersed in 50 mL fuming HNOg (90%, 1.2 mol) in a 250 mL round-bottom flask. The reaction mixture was placed in an oil bath pre-heated to 100 °C and stirred for 2 hours (h) , 4 h or 6 h, while under reflux. After heating, the reaction mixtures were removed from the heat source and permitted to return to room temperature (RT) . The RT reaction mixtures were separately quenched by carefully pouring the contents of the round-bottom flask over 70 mL of deionized (DI) ice in a 2 L beaker.

Each quenched solution was permitted to reach room temperature before pouring into a Spectra/Por® 7 (Repligen Co., Waltham, MA) dialysis membrane (regenerated cellulose, 1 kDa MWCO, 40 cm x 45 mm dimensions) . The OACs solutions were allowed to purify by bath dialysis, with continuous flow of DI water, for a total of 7 days. Each purified reaction mixture was removed from the dialysis bath and then filtered through a 0.22 pm poly (ethersulfone) [PES] membrane (Corning Inc., Corning, NY) .

X-ray photoelectron spectroscopy (XPS) measurements were acquired for the purified OACs using a PHI Quantera™ SXM scanning X-ray microprobe (Chigasaki, Japan) with a 100 pm X-ray beam under a base pressure of 5 x 10~⁹ Torr. Survey spectra were recorded using 0.5 eV step sizes with a pass energy of 140 eV. Elemental spectra were recorded using 0.1 eV step sizes with a pass energy of 26 eV. All of the XPS spectra were corrected using the C Is peaks (284.8 eV) as reference. Both C Is and 0 Is high resolution spectra, which reflect the characteristic binding states of carbon and oxygen atoms in the samples, were collected using solid, dried samples of the OACs .

The spectra of relative intensity (in arbitrary units) vs. binding energy were deconvoluted into species of functional groups, C=C/C-C, C-OC/ C— 0, C=0, and 0-C=0, with varying atomic percentage of oxygen.

The thermograms of the PEG-OAC, and DEF- OAC-PEG, are all comparable. When heated to 800 °C, all nanoconjugates display a significant loss in weight from about 350 to about 450 °C. This weight loss is primarily attributed to the PEG because decomposition of the polymer begins about 350 °C.²⁹ The total weight loss for the PEG-OAC and DEF-OAC-PEG, is about 92%. After background subtracting the weight loss of the OAC carbon core, it is determined that the PEG-OAC is 81% PEG by weight of the nanoconjugate, the DEF-OAC-PEG is 81% DEF + PEG by weight of the nanoconjugate. Theoretical calculations based on these values indicate there are 3 PEG moieties per OAC molecule for the PEG-OAC, 4 DEF + 3 PEG moieties per OAC molecule for the DEF-OAC-PEG.

Transcutaneous Formulation

To obtain a transcutaneous formulation, an active agent such as OAC— PEG, OAC-DEF or a derivative such as PEG-OAC-DEF can be incorporated into an aqueous composition thickened with suitable gelling agents, including polymeric viscosity builders, penetration enhancers and stabilizers to optimize the formulation with respect to product efficacy, quality and required chemistry and manufacturing control elements .

Human skin is usually reported to have a pH value of about 4.5 to about 7.5. It is preferred that a contemplated formulation have a pH value on the acid side such as about 5.0 to about 6.5. The skin also tends to maintain its individual pH value after an aqueous liquid having a pH value outside that of the skin's individual pH value is applied.

The active agent is typically present at about 2 to about 20 weight percent (wt %) of the transcutaneous formulation. Preferably an amount of about 5 to about 15 wt % is present, and more preferably, an amount of about 8 to about 12 wt % is present dissolved or otherwise dispersed in the topical, transcutaneous formulation (composition) .

Skin penetration enhancers (or permeation enhancers) are used to help overcome barrier properties of stratum corneum. Preferred penetration enhancers include Cg-Cg polyols and their monomethyl ethers, and poly (Cg-Cg ) glycols and their monomethyl ethers having a molecular weight of about 1000 Da or less. Penetration enhancers can be screened through an in vitro permeability study using pig and human cadaver skin (skin diffusion study) : diethylene glycol monomethyl ether ( Transcutol®; DEGEE) , propylene glycol and polyethylene glycol 400 are illustrative PEG-type, Cg-Cg polyols and their monomethyl ether penetration enhancers. Commercially available products often contain one or two of these penetration enhancers and they are listed in the FDA's inactive ingredient database.

More specifically, the words "penetration enhancement" or "permeation enhancement" as used herein relate to an increase in the permeability of the skin or mucosal tissue to a selected pharmacologically active agent; i.e., so that the rate at which the drug such as the preferred PEG-OAC- DEF particles permeate through the skin or mucosal tissue is optimized. "Carriers" or "vehicles" as used herein refer to carrier materials suitable for transcutaneous or transmucosal drug administration, and include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is nontoxic and does not interact with other components of the composition in a deleterious manner.

In addition to the PEG-type penetration enhancement agents noted above, vasodilating agents are also useful penetration enhancement agents. Illustrative vasodilating agents include organonitrates such as molsidomine, linsidomine chlorhydrate and S-nitroso-N-acetyl-d, 1-penicillamine ("SNAP") . Broadly, organonitrate compounds are nitric oxide precursor vasodilators that can be coadministered (co-formulated) or administered separately in conjunction with a peroxide compound. Illustrative organonitrates or nitric oxide precursors include erythrityl tetranitrate (1, 2,3,4- butanetetrol tetranitrate) , isosorbide dinitrate, nitroglycerin, pentaerythritol tetranitrate, isosorbide mononitrate and nicorandil [N—[2— (nitroxy) ethyl ] -3-pyridinecarboxamide ] . An organonitrate compound is also used in a vasodilating effective amount.

Methods for measuring vasodilation using an organonitrate are well known as are vasodilating amounts for internal use by oral or buccal administration, as such compounds are commercially available and approved for such uses by governmental bodies of many countries including the US FDA. Determination of a topically applied vasodilating effective amount can therefore be readily determined by a skilled worker.

Further penetration enhancement agents include long and short acting a-blockers such as phenoxybenzamine, dibenamine, doxazosin, terazosin, phentolamine, tolazoline, prazosin, trimazosin, alfuzosin, tamsulosin and indoramin; ergot alkaloids such as ergotamine and ergotamine analogs, e.g., acetergamine, brazergoline, bromerguride, cianergoline, delorgotrile, disulergine, ergonovine maleate, ergotamine tartrate, etisulergine, lergotrile, lysergide, mesulergine, metergoline, metergotamine, nicergoline, pergolide, propisergide, proterguride and terguride; antihypertensive agents such as diazoxide, hydralazine and minoxidil; nimodepine, pinacidil, cyclandelate, dipyridamole and isoxsuprine; chlorpromazine; haloperidol; yohimbine; trazodone, vasoactive intestinal peptides and mixtures thereof. Prostaglandin E_lf an organonitrate and phentolamine are particularly preferred vasoactive agents for use in conjunction with the present method.

A further class of penetration enhancer compounds are described to a greater extent in US Patents No. 6,046,244 and No. 6,118,020 to Buyuktimkin et al. One contemplated penetration enhancer is an alkyl-2- (N, N-disubstituted amino ) alkanoate . Another is a (N, N-disubstituted amino ) alkanol alkanoate. A mixture of both can also be used. For convenient reference, alkyl-2- (N, N- disubstituted amino ) alkanoates and (N, N-disubstituted amino ) alkanol alkanoates can be grouped together and referred to as alkyl (N, N-disubstituted amino) esters .

An alkyl-2- (N, N-disubstituted amino ) alkanoate suitable for the present invention can be represented as follows: wherein n is an integer having a value of about 4 to about 18; R is a member of the group consisting of hydrogen, C7-C7 alkyl, benzyl and phenyl; Rl and R2 are the same or different and are hydrogen or C1-C7 alkyl; and R^ and R^ are the same or different and are each hydrogen, methyl or ethyl.

Preferred alkyl (N, N-disubstituted amino ) alkanoates are C4-C18 alkyl (N, N-disubstituted amino ) acetates and C4-C78 alkyl (N, N-disubstituted amino ) propionates . Exemplary specific alkyl-2- (N, N- disubstituted amino ) -alkanoates include dodecyl 2- (N,N dimethylamino ) propionate (DDAIP) ; and dodecyl 2- (N, N-dimethylamino ) acetate (DDAA) . Alkyl-2- (N, N-disubstituted amino) alkanoates are known compounds. In addition, alkyl-2- (N, N- disubstituted amino ) alkanoates can be synthesized from more readily available compounds as described in US Patent No. 4,980,378 to Wong et al.

Suitable (N, N-disubstituted amino ) alkanol alkanoates for use in a contemplated composition can be represented by the formula: wherein n is an integer having a value of about 5 to about 18; y is an integer having a value of zero to about 5; and Rl, R2 , he same or different and are hydrogen, Cj_-Cg alkyl, or Cg-Cg aryl; and R^ is a hydrogen, hydroxyl, Cj_-Cg alkyl or Cg-Cg aryl.

Preferred (N, N-disubstituted amino ) alkanol alkanoates are Cg-C^g carboxylic acid esters. Exemplary specific (N, N-disubstituted amino ) alkanol alkanoates include 1- (N, N-dimethylamino ) -2-propanol dodecanoate (DAIPD) ; 1- (N, N-dimethylamino ) -2-propanol myristate (DAIPM) and 1- (N, N-dimethylamino ) -2- propanol oleate (DAIPO) . These materials are also known compounds and are readily synthesized.

Generally, the amount of skin penetration enhancer is about 5 percent to about 20 percent, and preferably at about 8 to about 15 percent based on the total weight of the composition. Preferably, the penetration enhancer is present at about 10 wt % of the composition.

Dosage forms include various pharmaceutically acceptable topical vehicles also referred to in the art as carriers such as gels, creams, ointments. These compositions contain substantial amounts of water; i.e., about 50 to about 90 percent by weight, and more preferably about 60 to about 80 weight percent, or can be substantially free of water, as in the case of an ointment.

Aqueous creams and thixotropic gels are preferred. A cream is an emulsion that preferably has water as the external phase (an oil-in-water emulsion) and has a semi-solid viscosity that resembles the viscosity of mayonnaise. A thixotropic gel is also a semi-solid, that is somewhat thicker or more viscous than mayonnaise. Such a composition exhibits thixotropic, non-Newtonian flow characteristics in which the flow is characterized by 1) a yield point, 2) pseudoplastic behavior, 3) a reduction in viscosity on continued shearing, visible over a finite time, and 4) a tendency to rebuild viscosity and/or yield point on standing.

A more precise measure of viscosity is by dynamic viscosity that reports in units of centipoise (cps) as measured at a specified temperature. A contemplated transcutaneous composition typically exhibits a viscosity of about 1000 to about 20,000 cps at ambient room temperature. More preferably, that viscosity is about 1500 to about 10,000 cps. The table below provides viscosities of several common materials as set out in a published table³¹.

*Data reported at about ambient room temperature.³¹

Other dosage forms for topical and local applications can include but are not limited to, sprays, aerosols, aerosol foams, powders, solutions, suspensions, and emulsions. A formulation can contain all necessary and appropriate pharmaceutical ingredients such as acceptable solvents including alcohols, polyols, esters, and ethers and the like, as well as synthetic, semi-synthetic and natural polymers that act as thickening agents.

A polymeric thickener can be present in a thickening effective amount as noted previously. Such amounts differ with the polymeric agent and composition, but are readily determined by a formulator of ordinary skill. Illustrative polymeric thickeners that are particularly useful in an aqueous composition include semi-synthetic polymers such as the starch derivatives Zeina B862 hydroxypropyl starch phosphate (Grain Processing Corporation, Muscatine, IA) , Zeina B860 hydroxypropyl starch, a synthetic polymer such as the lightly cross-linked polyacrylic acids discussed hereinafter and the natural polymers such as the polysaccharide gums that are discussed below. A polysaccharide gum is another useful thickener that can be present in a contemplated composition . Suitable representative gums are those in the galactomannan gum category .

A galactomannan gum is a carbohydrate polymer containing D-galactose and D-mannose units , or other derivatives of such a polymer . There are a relatively large number of galactomannans , which vary in composition depending on their origin . The galactomannan gum is characteri zed by a linear structure of P-D-mannopyranosyl units linked ( 1^-6 ) . Single membered a-D-manopyranosyl units , linked ( 1^-6 ) with the main chain, are present as side branches . Galactomannan gums include guar gum, which is the pulveri zed endosperm of the seed of either of two leguminous plants ( Cyamposi s tetragonal obus and psoral oids) and locust bean gum, which is found in the endosperm of the seeds of the carob tree ( Cera tonia siliqua ) . Locust bean gum is preferred for the present invention .

Other suitable representative gums include agar gum, carrageenan gum, ghatti gum, karaya gum, rhamsan gum and xanthan gum . A composition of the present invention can contain a mixture of various gums , or mixture of gums and acidic polymers .

Gums , and galactomannan gums in particular, are well-known materials ³²' ³³ . See for instance , most gums are commercially available in various forms , commonly a powder, and ready for use in foods and topical compositions . For example , locust bean gum in powdered form is available from Tic Gums Inc . (Belcam, Md . ) . A polysaccharide gum, when used, is present at about 0.5 percent to about 5 percent, based on the total weight of the composition, with the preferred amount being about 0.5 percent to about 2 percent.

An alternative or addition to the polysaccharide gum is a polyacrylic acid polymer. A common variety of polyacrylic acid polymer is known generically as "carbomer". Carbomer polymers are polyacrylic acid polymers lightly cross-linked with polyalkenyl polyether such as polyallyl sucrose or polyallyl pentaerythritol containing an average of at least three allyl groups per molecule, and present at about 0.75 to about 3 wt % , and preferably about 1 to about 2 wt % . These materials are commercially available from the Lubrizol Corp., Wickliffe, Ohio under the designation "CARBOPOL®". A particularly preferred variety of carbomer are those designated as "CARBOPOL 940" and "CARBOPOL 934". (See, Fig. 1.)

Other lightly cross-linked polyacrylic acid polymers suitable for use in practicing this invention are those commercially available under the designations "Pemulen®" and "POLYCARBOPHIL®" , both of which are now also available from Lubrizol Corp. The Pemulen® polymers are copolymers of C]_Q to Cgg alkyl acrylates and one or more monomers of acrylic acid, methacrylic acid or one of their simple esters that are cross-linked with an allyl ether of sucrose or an allyl ether of pentaerythritol. POLYCARBOPHIL® is a polyacrylic acid cross-linked with a about 0.05 to about 1.5 weight percent divinyl glycol such as 3,4- dihydroxy-1 , 5-hexadiene, or 2 , 5-dimethyl-l , 5- hexadiene. POLYCARBOPHIL® is substantially free from polyalkenyl polyethers such as polyallyl sucrose or polyallyl pentaerythritol containing an average of at least three allyl groups per molecule. POLYCARBOPHIL® is used in the vaginal moisturizer disclosed in US Patents No. 4, 615, 697, and No.

5, 474,768.

A non-aqueous topical vehicle is also contemplated, as noted before. Here, the vehicle and the resulting composition contain small amounts of water such as less than about 5 percent by weight and preferable less than about 2 percent by weight, and most preferably less than about 1 weight percent.

Illustrative vehicles are comprised of hydrophobic materials such as petrolatum or CQ-C-22 fatty acid esters of C]_-Cg alcohols. Illustrative materials include methyl palmitate, hexyl laurate, butyl stearate, isopropyl eicosanoate (arachidate) , isopropyl behenate and the like. These materials are often used as emollients in the cosmetics industry and are well known in that art. Polyethylene glycols, polypropylene glycols and polyethylenepolypropylene block copolymers, known in the art as poloxamers, that are solid and semi-solid at room temperature are also useful in preparing non-aqueous topical vehicles.

Methods Illustrative Synthesis of PEG-OAC-DEF Synthesis of OAC The synthesis of OACs by nitric acid is similar to that previously reported by our group. Powdered medical grade coconut activated charcoal (0.506 g, 42.2 mmol carbon) was added to a 250 mL round-bottom flask, followed by the addition of fuming (90%) HNOg (50 mL, 1.2 mol) . The reaction mixture was placed in an oil bath pre-heated to 100 °C and was stirred under reflux for the 6 h. After heating, the reaction mixture was removed from the heat source and cooled to room temperature. The reaction was quenched by pouring the contents of the round-bottom flask over 70 mL of deionized (DI) ice in a 2 L beaker.

The quenched solution was permitted to reach room temperature before pouring into one Spectra/Por® 7 dialysis membrane 237 (regenerated cellulose, 1 kDa MWCO, 40 cm x 45 mm dimensions) . The OAC solution was purified via bath dialysis for 7 d. The purified reaction mixture was then removed from the dialysis bath and filtered through a 0.22 pm poly (ethersulfone) (PES) membrane.

Prior to functionalizing the OAC with PEG, or both DEF and PEG, the aqueous material was dried by lyophilization (Labconco Freeze Dry System/ Freezone 4.5) .

Synthesis of PEG-OACs

The lyophilized OACs (35.8 mg, 2.98 mmol) was suspended in DMF (30 mL) and cup horn sonicated for 60 min at 50% amplitude (Cole-Parmer® Ultrasonic Processor CP 750) . Then 5 kDa methoxy-PEG-amine (0.3588 g, 0.07176 mmol) was added to the reaction vessel. The mixture was bath-sonicated for 20 min (Cole-Parmer® 08849-00 ultrasonic cleaner) .

Next, the reaction vessel was removed from the sonicator and N, N'-diisopropylcarbodiimide (DIG; 0.358 mL, 2.32 mmol) was added. The solution was stirred for 48 h at rt . Prior to purification by bath dialysis, excess DMF was removed from the reaction mixture by centrifugation (NuAire NU-C200R, parameters: 4500 x g, 30 min, rt) using Amicon® Ultra-15 Centrifugal Filters (regenerated cellulose, 10 kDa MWCO) . The resulting retentate was diluted in Milli-Q® water and then transferred to one Float-A-Lyzer® G2 dialysis device (Repligen Corporation, Waltham, MA; regenerated cellulose, MWCO 50 kDa, 10 mL volume capacity) . To dialyze the material, the filled device was placed in a 2 L beaker containing Milli-Q water. During a period of 48 h, the water bath was continuously stirred by a magnetic stir bar/stir plate. The water was exchanged 6-8 times over the course of this time. After dialysis, the PEG-OAC particles were sterile-f iltered by passage through a 0.22 pm PES membrane.

Synthesis of DEF-OAC-PEG

The lyophilized OACs (17.0 mg, 1.42 mmol) were suspended in DMF (15 mL) and cup horn sonicated for 60 min at an amplitude of 50% (Cole-Parmer® Ultrasonic Processor CP 750) . Then deferoxamine mesylate salt (20.6 mg, 0.0314 mmol) and N,N- diisopropylethylamine (0.027 mL, 0.16 mmol) were added to the reaction vessel, followed by 5 kDa methoxy-PEG-amine (0.1705 g, 0.03410 mmol) . The mixture was bath-sonicated for 20 min (Cole-Parmer® 08849-00 ultrasonic cleaner) .

Next, the reaction vessel was removed from the sonicator and DIG (0.204 mL, 1.32 mmol) was added. The solution was stirred for 48 h at room temperature. Prior to purification by bath dialysis, excess DMF was removed from the reaction mixture by centrifugation (NuAire NU-C200R, parameters: 4500 x g, 30 min, room temperature) using Amicon® Ultra-15 Centrifugal Filters (regenerated cellulose, 10 kDa MWCO) .

The resulting retentate was diluted in Milli-Q® water and then transferred to one Float-A- Lyzer® G2 dialysis device (regenerated cellulose, MWCO 50 kDa, 10 mL volume capacity) . To dialyze the material, the filled device was placed in a 2 L beaker containing Milli-Q® water. During a period of 48 h, the water bath was continuously stirred by a magnetic stir bar/stir plate. The water was exchanged 6-8 times over the course of this time. After dialysis, the DEF-OAC-PEG particles were sterile-f iltered by passing through a 0.22 pm PES membrane .

High-resolution transmission electron microscopy (HR- TEM)

High-resolution transmission electron microscopy (HR-TEM) imaging was performed on a JEOL 2100F field emission electron microscope [Jeol. Akishima, Tokyo, Japan] . Samples were prepared by adding OACs in water and sonicating the mixture until well dispersed. The solution was drop-cast onto a 300-mesh holey lacey carbon TEM grid supported on copper (Ted Pella, Inc., Redding, CA) . Prior to imaging, the drop-cast OACs were dried in a vacuum desiccator. The hydrodynamic diameter of each nanomaterial was characterized through dynamic light scattering [Brookhaven, ZetaPALS with BI-9000AT digital autocorrelator, 1 = 656 nm) ; Spectra research, Mississauga, Ontario, Canada] . X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS) measurements were acquired for the purified OACs using a PHI Quantera SXM scanning X-ray microprobe (Chigasaki, Japan) with a 100 pm X-ray beam under a base pressure of 5xl0^_ Torr. Survey spectra were recorded using 0.5 eV step sizes with a pass energy of 140 eV. Elemental spectra were recorded using 0.1 eV step sizes with a pass energy of 26 eV. All of the XPS spectra were corrected using the C Is peaks (284.8 eV) as reference.

Both C Is and 0 Is high resolution spectra, which reflect the characteristic binding states of carbon and oxygen atoms in the samples, were collected using powderized, dried samples of the OACs. The spectra of relative intensity (in arbitrary unit) vs. binding energy were deconvoluted into species of functional groups, C=C/C-C, C-OC/C-O, C=O, and O-C=O, with varying atomic percentage of oxygen .

Attenuated total reflectance Fourler-transform infrared spectroscopy , (ATR-FTIR)

ATR-FTIR spectra were acquired on a Thermo Scientific Nicolet 6700 attenuated total reflectance Fourier-transform infrared spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) . OAC, PEG-OAC, and DEF-OAC-PEG, samples were dried and powderized for data collection. Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was performed on a Mettler Toledo TGA/DSC 3+system. Alumina crucibles were used for the sample and reference pans. Prior to data collection, dried and powderized cOCNP was pre-treated by ramping to 120 °C at a rate of 10 °C/min under nitrogen. The temperature was held at 120 °C for 10 min to remove any excess moisture in the sample. Thermograms were acquired from 120 °C to 800 °C at a ramp rate of 10 °C/min under nitrogen. OAC, PEG-OAC, and DEF-OAC-PEG samples were dried and powderized for data collection .

Resul ts

Structural and chemical characterization of the OAC indicates the nanoparticles are an amenable size for biological applications, while also being comprised of sufficient sp2 content and oxygencontaining functionalities. HR-TEM images of the OAC show the nanoparticles are disc-like in shape with average diameters of 2.5 nm for the 6 h OAC, compared to 7 nm and 13 nm for the 4 h OAC and 2 h OAC, respectively, (Fig. 3) . As the oxidation duration is lengthened, the diameter of the OAC carbon core decreases, whereas the homogeneity of the size distribution increases. Longer oxidation durations permit greater free edge etching of the liberated cAC sp2 domains, resulting in smaller particles.

The carbon core of the nanomaterial displays a high degree of sp2 hybridization, as is noted by the presence of C=C content in the deconvoluted high-resolution C Is XPS spectrum and the C=C band (about 1610 cm^_l) in the ATR-FTIR spectrum. Furthermore, Raman spectroscopy suggests the OAC core is hybridized as there are apparent G (1600 cm-l) and D peaks (1390 cm-l) in the spectrum, which are characteristic of graphene. The honeycomb lattice structure of the OAC core is important for the SOD-mimetic behavior of the nanozyme. The hybridization of highly conjugated systems lowers the activation energy for acquisition of electrons; therefore, the sp2 lattice of the OAC facilitates the transfer of electrons between the nano-antioxidant and SO¹.

The Table 1 below illustrates the elemental composition and oxygen-functionality distributions of cOCNP particles after 2, 4 and 6 h of oxidation in fuming nitric acid as discussed above, approximated from XPS survey scans and high-resolution C Is peak spectra .

Table 1

Although the OACs such as those in the above Table 1 are primarily composed of sp2 carbon, the oxidation with nitric acid still affords a product with a high degree of oxygen-containing functionalities. The C:0 content ratio of the nanomaterial is 2.6, with the oxygen moieties that comprise the OAC being C-O-C/C-O ( ether/hydroxyl ) , C=0 (carbonyl) and 0-C=0 (carboxylic acid) in nature. Additionally, ATR-FTIR further suggests the presence of C=0 and 0-C=0 functionalities showing an evident C=0 stretch (about 1710 cm-l) and a broad O-H stretch (about 3700-2000 cm-l) , representative of the COO-H, in the spectrum.

Considerable weight loss is also observed when the sample is heated up to 800 °C, with the initial reduction in weight occurring at about 190 °C. This finding further corroborates that the OAC is highly oxidized because about 190-210 °C is the temperature range at which oxygen functionalities begin to decompose²⁸.

The Table 2 below illustrates the elemental composition and oxygen-functionality distributions of OAC and its functionalized derivatives, approximated from XPS survey scans and high-resolution C Is peak spectra. Data for PEG-OAC and PEG-OAC-DEF were determined from separate preparations.

Table 2

Successful functionalization of the OAC with PEG + DEF was analyzed by ATR-FTIR, XPS, and TGA. The characterization for the DEF-OAC-PEG was compared to PEG-OAC as a baseline. ATR-FTIR spectra of the various functionalized OAC nanoconjugates is similar to that of PEG with strong aliphatic C-H stretches representative of the PEG ethyleneoxy repeat unit. Because the primary component of the nanocon ugates is PEG, identifying signals for the DEF are oversaturated by the polymer. It should be noted that the OAC carbon core C=O stretch (about 1710 cm-l) and C=C stretch (about 1610 cm-l) are still observed in the spectra, albeit at lower intensities. XPS analysis, however, reveals the presence of the appended moieties (e.g., PEG and DEF) .

Regarding the PEG-OACs, as a control, it is apparent that the OACs have been functionalized with the amine-terminated polymer. Unmodified OAC does not contain detectable amounts of nitrogen (N) , by XPS, whereas the PEG-OAC does contain N. The increase in nitrogen content suggests potential amide bond formation between the nanoparticle and the PEG, because any unreacted PEG would have been removed during the purification process.

Furthermore, when DEF is added to the particles, the nitrogen content of the nanoconjugates shows a greater increase from that of the PEG-OAC. The methoxy-PEG-amine only has one nitrogen atom per molecule, whereas DEF has six nitrogen atoms per molecule. From the deconvoluted C Is high-resolution XPS spectra, a significant increase in the C-O-C/C-O content and a decrease in the O-C=O content of the functionalized OAC nanoparticles is also observed.

The higher percentage of C-O-C/C-O arises due to the oxyethylene groups of the PEG, whereas the O-C=O content is reduced as a result of the OAC carboxyl-groups being converted to amides upon reaction with the methoxy-PEG-amine and DEF. The DEF-OAC-PEG nanocon ugates have lower C-O-C/C-O and O-C=O content than the PEG-OAC. This further corroborates the metal-chelating constructs are functionali zed with DEF because these addenda do not possess ether groups , like PEG .

Additionally, because twice the number of addenda ( substituents ) were added for the synthesis of the nanoconj ugates , it is expected that the free carboxylic acid content for the DEF-OAC-PEG would be less than that of the PEG-OAC, as is observed . Lastly, the C=O content of the DEF-OAC-PEG is greater than the OAC parent particle . This increase is attributed to the hydroxamic acid and amide functional groups of the DEF moieties .

The particles made using about equal molarities of each of the PEG-amine and DEF-amine reactants resulted in product DEF-OAC-PEG particles having about equimolar amounts of each substituent linked to each particle . Those about equimolar amounts come out to be an average of about two to about five of each of the two substituents per particle , and more typically an average of about three to about four of each substituent per particle .

Topical Transcutaneous and Transdermal Formulation

Study 1

Four illustrative transcutaneous formulations containing PEG-OAC-DEF with an average number of about three or four reacted PEG and DEF groups per particle were prepared and assayed . Three formulations contained 10% PEG-OAC-DEF, 10% of one or the other of three permeation enhancers and 80% aqueous Carbopol® 934 NF ( 2 % gel in water ) . The fourth formulation contained added water instead of the permeation enhancer . The permeation enhancers were polyethylene glycol 400 ( PEG 400 ) , propylene glycol (PG) , and diethylene glycol monomethyl ether

( Transcutol®, TC) . All percentages for the formulation are weight percentages of the total composition .

The formulations were evaluated for permeation through pig ear skin (thickness, 1 mm) using Franz cell³⁰ with a diffusion area of 1.767 cm² and volume capacity of 12 ml of the receptor medium ( 0.2M phosphate buffer pH 7.4) .

The pig ear skin was carefully placed between the donor and receptor compartments followed by placement of 1 gram of the formulation over donor chamber. The receptor medium was stirred constantly at 350 rpm by magnetic bar during the studies. The system was maintained at a constant temperature of 3210.5 °C through thermostatic bath circulation. Aliquots of 0.3 mL were collected at time 0.5, 1, 2, 3, 4, 5, 6, and 24 hours (h) . A withdrawal sample was replaced with buffer maintained at 32 ± 0.5 °C. The samples were analyzed by SEC-HPLC and quantified using regression equation. The cumulative amount of PEG-OAC-DEF permeated through the membrane was calculated in ,g/cm².

The results of this study are shown in Fig. 1. As can be seen from Fig. 1, although three of the four formulations produced similar results, the diethylene glycol monomethyl ether (darkened circles; Transcutol®) provided the fastest penetration and greatest concentration of PEG-OAC-DEF at substantially all of the measured time points. Surprisingly, each of the three permeation enhancercontaining compositions provided greatly enhanced rates and amounts of penetration as well as greatly enhanced amounts of the delivered PEG-OAC-DEF particles. The diffusion study of the prepared hydrogel formulation showed 20% diffused within 1 h, 50% diffused within 6 h and 100% PEG-OAC-DEF diffused in 24 h through the pig ear skin with a diffusion flux of 8.41 pg/cm²h.

Study 2

A second, similar permeation study was carried out using a formulation containing 10 wt% PEG-OAC-DEF (0.065 wt% or 0.65 mg/ml carbon nanoparticles) , 20 wt% of Transcutol® as a permeation enhancer and 80% aqueous Carbopol® 934 NF (2 wt% gel in water) . Testing of permeation of DEF-OAC-PEG through pig ear skin and human skin samples was carried out using the DEF-OAC-PEG prepared above using equal molar amounts of DEF and amino-methyl PEG 5000 covalently linked to the OAC particles. Human skin was obtained from excess removed at surgery.

Skin samples for study were held in a commercially available vertical Franz diffusion cell. Circular excision (8 mm diameter) of skin explants (0.5 cm2) _were made by biopsy punches (#NC9677689;

Acuderm, Inc., Ft. Lauderdale, FL) . Topical application of 30 pL PEG-OAC-DEF (PEGylated carbon nanoparticle, 16 pg/mL formulation) was applied to one group, while the other group received hydrogel control .

Skin explants were cultured in keratinocyte growth medium (#PCS-200-040; ATCC, Manassas, VA) at 37°C for 24 h, then harvested for histological analysis, including H&E staining and immunofluorescence staining using anti-PEG antibody (#ab51257; Abeam, Cambridge, UK) and red fluorescing- labeled anti-CK14 antibody (#ab7800; Abeam) that binds to the basal layer of stratifying squamous and non-squamous epithelia. The blue-fluorescent DNA stain 4 ’ , 6-diamidino-2-phenylindole (DAPI) stains cell nuclei.

The nanoparticles, def eroxamine-linked PEG- OAC were quantified using HPLC with size exclusion. The conditions were:

Column: Shodex Ohpack SB-806 SEC at 40°C; Mobile phase: acetonitrile: water (60:40) ; Flow rate: 0.5 mL/min;

Total run time: 30 min;

Injection volume: 50 pl; and UV detection: 240 nm.

Results of the permeation study were as follows. Table 3, below, includes the percentage of nanomaterial that accumulated across the respective skin material and the graph of Fig. 5 illustrates the data used to calculate the amount that permeated in amount/cm2 of skin material.

The results indicate that the formulation employed at 24 hours, 75.3% of pig skin material permeated and 23.2% permeated human skin. The results also indicate successful permeation of skin from both species, with optimization still in progress for human skin.

Table 3 Cumulative Amount Permeated/cm2

There was minimal fluorescence in the skin samples not treated with nanoparticles. In the samples treated with labeled-nanoparticles, there was fluorescence indicating the presence of labeled- nanoparticles both on the surface of the skin and in deeper dermal layers shown in overview and higher power sections. These results are consistent with effective permeation through fresh human skin and indicates the successful development of a transcutaneous nanoparticle-containing formulation.

Pig Ear Diffusion Assay

A size-exclusion high pressure liquid chromatography (SEC-HPLC) method was developed and validated to quantify the PEG-OAC-DEF . The HPLC system used was 1260 Infinity Series HPLC (Agilent, Santa Clara, CA) . SEC separation was achieved using a Shodex OH pak® SB-806 column (300 mm x 8 mm, 13p; Showa Denka America, Inc., New York, NY) fitted with a Shodex OH pak® SB-G guard column (6 x 50 mm) . The flow rate was 0.5 mL/min. The mobile phase was ACN : water (60:40 v/v) . The column temperature was maintained at 40°C, and the injection volume was 50 pL . The UV detection wavelength was 230 nm. Data were collected using Agilent Open Lab CDS software. Analytical method suitability was determined by injecting 6 replicate injections of the system suitability standard (2.5 pg/mL) before validating the method. The method was validated for specificity, limit of detection (LOD) and quantification (LOQ) , linearity, accuracy, and precision for three days.

The analytical method met the preset criteria of system suitability parameters for relative standard deviation (RSD) : retention time (% RSD <2) , peak area (% RSD <2) , capacity factor (>3) , and USP tailing factor (<2) for CNP peak. The method also met ICH and USP requirements of validation parameters .

The method was specific, as the peak of PEG-OAC-DEF was not interfered with by mobile phase, solvent peak, and formulation components. The method was linear over 0.25 to 10 pg/mL concentration range. The correlation coefficient and slope were consistent in inter-day validation. The correlation coefficient was >0.999. Accuracy and precision were established across the analytical range of 0.25 to 10 pg/mL and calculated from the quality control (QC) samples of PEG-OAC-DEF.

Accuracy of PEG-OAC-DEF solutions at 3 concentrations 0.25, 5, and 10 pg/mL (LOQ, middle and high levels of linearity range) ranged from 100.79% to 101.79%, 99.64% to 100.99%, and 100.36% to 102.82% of the nominal values in 3 days' validation studies, respectively. The RSD of repeatability measurements of the samples of 0.25, 5, and 10 pg/mL QC samples were 1.58%, 1.62%, and 1.81%, respectively. Similarly, intermediate precision of 0.25, 5, and 10 pg/mL control samples was 1.52%-1.64%, 1.11%-1.95% and 1.391-2.15%, respectively.

Analytical method met The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and U.S. Food and Drug Administration (FDA) validation requirements. The developed method is simple, sensitive and specific to PEG-OAC-DEF and can quantify 0.08 pg/ml in the prepared topical formulation without interference with the topical formulation components.

An exemplary topical formulation of PEG- OAC-DEF was prepared as described above containing 10wt% Transcutol®. That formulation contained 10 wt% PEG-OAC-DEF, and 80 wt% aqueous Carbopol® 934 NF (2 wt% gel in water) . The formulation was evaluated for permeation through pig ear skin (thickness, 1mm) using Franz cell with a diffusion area of 1.767 cm² and volume capacity of 12 ml of the receptor medium ( 0.2M phosphate buffer pH 7.4) .

The pig ear skin was carefully placed between the donor and receptor compartments followed by placement of 1 gram of the formulation over donor chamber. The receptor medium was stirred constantly at 350 rpm by magnetic bar during the studies. The system was maintained at a constant temperature of 3210.5 °C through thermostatic bath circulation. Aliquots of 0.3 mL were collected at time 0.5, 1, 2, 3, 4, 5, 6, and 24 hours (h) . A withdrawal sample was replaced with buffer maintained at 32 ± 0.5 °C. The samples were analyzed by SEC-HPLC and quantified using regression equation. The cumulative amount of PEG-OAC-DEF permeated through the membrane was calculated in pg/cm².

Safety and Permeation Study

A safety study was performed to determine whether application of the nanomaterial

(def eroxamine-linked PEG-OAC) would interfere with wound healing in a mouse wound model. Briefly, the following procedure was performed: Skin wounds were generated bilaterally in normal mice (n=5 per group) . Three different doses of the above same OACs were administered (2 pg/ml, 8 pg/ml and 16 pg/ml) either every other day or every other week for 2 weeks. On one side, hydrogel only was applied, and, a nanoparticle formulation as noted above was applied to the other side. The hydrogel formulation was the same as the nanoparticle formula with water making up the difference for the nanoparticles in the formulations. No treatment controls and no surgery control groups were also designated.

Put differently, the 8 groups were treated for 2 weeks and then blood obtained after the last dose :

Group 1 16 pg/mL OAC (every other day)

Group 2 16 pg/mL OAC (every other week)

Group 3 8 pg/mL OAC (every other day)

Group 4 8 pg/mL OAC (every other week)

Group 5 2 pg/mL OAC (every other day)

Group 6 2 pg/mL OAC (every other week)

Group 7 no treatment control

Group 8 no surgery control Blood Chemistry tests:

Aspartate Aminotransferase, Globulin, Total bilirubin, albumin, creatinine, potassium (slight increase in Group 5 only) , phosphorous, sodium (slight increase in group 8 compared to group 7) , Lactate dehydrogenase, Alanine aminotransferase, Alkaline phosphatase, blood urea nitrogen, creatinine, calcium, chloride, glucose (lower glucose in 7 vs Group 8, no different from any other treatment group) , and total protein.

No differences were found in any of these parameters that were measured:

Red Blood Cells, Mean Corpuscular Volume, Hemoglobin, Hematocrit, Mean corpuscular hemoglobin concentration, white blood cells, segmented neutrophils (except for lower group 6 vs group 7, no other significant differences) , lymphocytes, basophils, eosinophils, monocytes, lymphocytes, basophils, platelets, red cell distribution width, Mean platelet volume, and reticulocyte count.

At every 3-day intervals, the wounds were photographed and the extent of wound analyzed by an image analysis program. Results indicated no significant difference in wound healing rates compared to hydrogel alone. Four of the five mice in the 8 ,g/ml every other day dose due to some presently unknown cage issue. Safety, defined as significant alteration in blood chemistry and hematology due to topical application of the nanoparticle formulation. There was no mortality in either the highest (16 mg every other day) or lowest dose (2 mg every other week) , with some mortality randomly distributed among other doses except for one cage in which 4/5 mice died, as noted above.

To assess location of permeation of the above formulation of nanoparticles, a fresh human skin sample was treated with the nanomaterial or hydrogel (to control for non-specific immunofluorescence) using fluorescence histological methods. The nanoparticles were detected by the fluorescence related to an antibody to the PEG covalently bonded to the particle 24 hours after application .

A human skin sample was procured by Dr. Aldona Spielgel, MD, Houston Methodist, Weill Cornell Medical College. Circular excision (8 mm diameter) of skin explants (0.5 cm2) _were made by biopsy punches (#NC9677689, Acuderm, Ft. Lauderdale, FL) . Topical application of 30 pL PEG-CNP-DEF (PEGylated carbon nanoparticle, 16 pg/mL formulation) was applied to one group, while the other group received hydrogel control.

Skin explants were cultured in keratinocyte growth medium (#PCS-200-040, ATCC) at 37°C for 24 h, then harvested for histological analysis, including H&E staining and immunofluorescence staining using anti-PEG antibody (#ab51257, Abeam, Inc., Cambridge, UK) and anti-CK14 antibody (#ab7800, Abeam) . DAPI stains cell nuclei. There is minimal fluorescence in the skin samples not treated with labeled-nanoparticles . In the samples treated with nanoparticles , there is fluorescence indicating the presence of labeled- nanoparticles both on the surface of the skin and in deeper dermal layers shown in overview and higher power sections . These results are consistent with ef fective permeation through fresh human skin and indicates the success ful development of a potential transcutaneous formulation .

Study 3

There is a great advantage in terms of use as a medicinal agent for the OACs compared to the HCCs i f only because the starting material for the OACs is already a readily available medicinal agent whereas the previously used HCC starting material is a SWCNT from a single source that is no longer available . In addition, prior to acid treatment , SWCNTs are a potentially hazardous material as stated earlier . Moreover, we were unable to generate as therapeutically ef fective nanoparticles from many commercially available SWCNTs compared to the original SWCNT starting material used for the HCCS .

A steady source of starting material is a crucial factor in the use of OACs as a therapeutic . Although it must go through the full approval process because its chemical characteristics are altered by the acid oxidation, a good manufacturing processgenerated starting material already used in many countries as a therapeutic or supplement is a highly beneficial step in the ultimate pharmaceutical product approval process . We therefore have restudied the ef fects of the present OACs on neurons cultured in the presence of hemin that causes toxic ef fects . That study replicated much of the beneficial ef fects against toxic ef fects of hemin of DEF-HCC-PEG using the DEF- OAC-PEG particle⁷ . Analogous to the unstably- dispersible PEG-HCC, the water-soluble PEG-OAC alone reduced senescence but increased susceptibility to ferroptosis , whereas the DEF-OAC-PEG reduced both senescence and ferroptosis .

Lipid peroxidation assay using fluorescence detection of reactive aldehydes in lipids as a reflection of ferroptosis in terminally di f ferentiated neurons shows that SOD-mimetic oxidi zed activated charcoal increases lipid peroxidation after addition of hemin, as reflected in increased fluorescence , while decreasing senescence as detected by colorimetric detection of senescence associated beta galactosidase . The combined chelator def eroxamine-oxidi zed activated charcoal ( DEF-OAC- PEG) reduced both lipid peroxidation and senescence .³⁶

Each of the patents , patent applications and articles cited herein is incorporated by reference . The articles "a" and "an" are used herein to refer to one or to more than one ( i . e . , to at least one ) of the grammatical obj ect of the article .

The foregoing description and the examples are intended as illustrative and are not to be taken as limiting . Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art . Citations

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Claims

WHAT IS CLAIMED

1 . A transcutaneous composition for treating an oxidative skin disorder of a mammal in need that comprises an ef fective oxidative skin disorder treating amount of covalently-substituted oxidi zed activated charcoal nanoparticles ( OACs ) dissolved or dispersed in an aqueous composition that contains an amount of thickening agent suf ficient to provide a viscosity of about 1000 to about 20 , 000 cps and about 5 to about 20 weight percent of a skin permeation enhancer, wherein a substituent of said substituted OAC comprises an average of about 2 to about 5 polyethylene glycol ( PEG) chains covalently linked to said OAC, or an average of about 2 to about 5 metal ion chelating groups covalently linked to said OAC, or an average of about 2 to about 5 PEG chains and an average of about 2 to about 5 metal ion chelating groups linked to each OAC .

2 . The transcutaneous composition according to claim 1 , wherein the average molecular weight of said substituent PEG chains is about 2000 to about 10 , 000 Da .

3 . The transcutaneous composition according to claim 1 , wherein water comprises about 50 to about 90 percent by weight of said transcutaneous composition .

4 . The transcutaneous composition according to claim 1 , wherein the transcutaneous composition exhibits a viscosity of about 1500 to about 10 , 000 cps .

5 . The transcutaneous composition according to claim 1 , wherein said thickener is a polymeric material selected from the group consisting of one or more of a polysaccharide gum, hydroxypropyl starch, hydroxypropyl starch phosphate , a water- soluble and/or water-dispersible cross-linked polyacrylic acid, polymethacrylic acid, mixed polyacrylic-polymethacrylic acid copolymer, and a copolymer of C]_ o to Cg g alkyl acrylates and one or more monomers of acrylic acid or methacrylic acid wherein said cross-linking agents are an allyl ether of sucrose or an allyl ether of pentaerythritol ,

6 . The transcutaneous composition according to claim 1 , wherein said skin permeation enhancer is selected from one or more of the group consisting of Cg-Cg polyols and their monomethyl ethers , poly ( Cg-Cg ) glycols and their monomethyl ethers having a molecular weight of about 1000 Da or less , vasodilating agents , a-blockers and an alkyl (N, N-disubstituted amino ) ester .

7 . The transcutaneous composition according to claim 6 , wherein said skin permeation enhancer is present in an amount of about 8 to about 15 percent by weight .

8 . The transcutaneous composition according to claim 1 , wherein said mammal is a human .

9 . The transcutaneous composition according to claim 1 , wherein said composition has a pH value of about 5 . 0 to about 6 . 5 .

10 . A method of treating an oxidative skin disorder that comprises the steps of administering a transcutaneous composition to the area of the skin of a mammal in need believed to be af fected by an oxidative skin disorder, said transcutaneous composition comprising an ef fective oxidative skin disorder treating amount of covalently-substituted oxidi zed activated charcoal nanoparticles ( OACs ) dissolved or dispersed in an aqueous composition that contains an amount of thickening agent suf ficient to provide a viscosity of about 1000 to about 20 , 000 cps and about 5 to about 20 weight percent of a skin permeation enhancer, wherein a substituent of said substituted OAC comprises an average of about 2 to about 5 polyethylene glycol ( PEG) chains covalently linked to said OAC, or an average of about 2 to about 5 metal ion chelating groups covalently linked to said OAC, or an average of about 2 to about 5 PEG chains and an average of about 2 to about 5 metal ion chelating groups linked to each OAC .

11 . The method according to claim 10 , wherein the average molecular weight of said substituent PEG chains is about 2000 to about 10 , 000

Da .

12 . The method according to claim 10 , wherein water comprises about 50 to about 90 percent by weight of said transcutaneous composition .

13 . The method according to claim 10 , wherein the transcutaneous composition exhibits a viscosity of about 1500 to about 10 , 000 cps .

14 . The method according to claim 10 , wherein said thickener is a polymeric material selected from the group consisting of one or more of a polysaccharide gum, hydroxypropyl starch, hydroxypropyl starch phosphate , a water-soluble and/or water-dispersible cross-linked polyacrylic acid, polymethacrylic acid, mixed polyacrylic- polymethacrylic acid copolymer, and a copolymer of CI O to C30 alkyl acrylates and one or more monomers of acrylic acid or methacrylic acid wherein said cross-linking agents are an allyl ether of sucrose or an allyl ether of pentaerythritol ,

15 . The method according to claim 10 , wherein said skin permeation enhancer is selected from one or more of the group consisting of Cg-Cg polyols and their monomethyl ethers , poly ( Cg-Cg ) glycols and their monomethyl ethers having a molecular weight of about 1000 Da or less , vasodilating agents , a-blockers and an alkyl (N, N-disubstituted amino ) ester .

16 . The method according to claim 15 , wherein said skin permeation enhancer is present in an amount of about 8 to about 15 percent by weight .

17. The method according to claim 10, wherein said transcutaneous composition is administered to the area of the skin that includes the oxidative skin disorder and the composition is rubbed into the skin until it is sorbed by the skin.

18. The method according to claim 10, wherein said administration is repeated.

19. The method according to claim 10, wherein said mammal is a human.

20. The method according to claim 10, wherein said composition has a pH value of about 5.0 to about 6.5.

21. The method according to claim 10, wherein the oxidative skin disorder is venous stasis dermatitis .

22. The method according to claim 10, wherein the oxidative skin disorder is a non-healing ulcer in a patient with venous disease, diabetes mellitus, or peripheral arterial disease.

23. The method according to claim 10, wherein the oxidative skin disorder is a decubitus ulcer .