AU2018255294B2 - Parenteral non-systemic administration of buffering agents for inhibiting metastasis of solid tumors, hyperpigmentation and gout - Google Patents

Abstract

Parenteral dosing (other than direct systemic dosing) of pH raising and other anti-metastasis agents alone or in combination is effective and overcomes disadvantages of intravenous and oral administration in the treatment of solid tumors, melasma and gout.

Description

PARENTERAL NON-SYSTEMIC ADMINISTRATION OF BUFFERING AGENTS FOR INHIBITING METASTASIS OF SOLIDTUMORS, HYPERPIGMENTATION ANDGOUT

Cross-Reference to Related Applications

[0001] This application claims priority from provisional applications US Serial No. 62/602,227 filed 17 April 2017; US Serial No. 62/602,358 filed 20 April 2017; US Serial No. 62/602,359 filed 20 April2017; US Serial No. 62/559,947 filed 18 September2017; US Serial No. 62/559,360 filed 15 September 2017; US Serial No. 62/562,725 filed 25 September 2017; US Serial No. 62/609,982 filed 22 December 2017; and US Serial No. 62/639,904 filed 7 March 2018. The contents of these documents are incorporated herein by reference in their entirety.

Technical Field

[0002] The invention relates to the use of anti-metastatic agents, including buffering agents administered parenterally, non-systemically, in particular topically, to result in resistance of tumors to metastasis. Buffering agents that raise pH are also useful in treating hyperpigmentation and gout. Both topical and other parenteral administrations that are themselves non-systemic are contemplated as overcoming difficulties with oral administration and systemic parenteral administration of buffers designed to raise local pH.

Background Art

[0003] Progression to metastasis remains the highest mortality risk for cancer patients, despite significant efforts to therapeutically target metastatic lesions. Tumor invasion and metastasis associated with neoplastic progression are the major causes of cancer deaths and understanding the mechanisms determining metastatic spread of malignant cells via invasion to distant tissues is, perhaps, the central question in oncology.

[0004] It is known that microenvironmental acidosis in a primary tumor increases cellular motility and invasiveness, leading to increased metastasis and that solid tumors exist in a microenvironment of relatively low p, presumably because of the hypoxic nature of such tumors, increased glycolytic metabolism of glucose and poor perfusion. As early as 1979, Turner, GA, Experienia (1979) 35:1657-1658 reported that acid pH encouraged the release of tumor cells by collagenase, thus encouraging metastases. Curvier, C. etal., Cin. Exp Metastasis (1997) 15:19-25 reported enhanced invasive capacity of tumor cells due to glucose starvation, hypoxia and acidosis. Rofstad,E.K. metal ,Cancer Res(2006)66:6699 6706 reported that the acidic extracellular pH of human melanoma cells promotedmetastasis in mice.

[00051 The extracellular pH of malignant solid tumors is acidic, in the range of 6.5 to 6.9, whereas the p1 of normal tissues is significantly more alkaline, 7.2 to 7.5. These observations have led to the "acid-mediated invasion hypothesis," wherein tumor-derived acid facilitates tumor invasion by promoting normal cell death and extracellular matrix (ECM) degradation of the parenchyma surrounding growing tumors.

[00061 According to miscellaneous sources in the popular press, certain physicians have experimented with intravenous sodium bicarbonate as a method of inhibiting metastatic cancer.

[0007] A series of articles by Robey, I.F. et al., beginning with a publication in Cancer Res (2009) 69:2260-2267 demonstrated that oral bicarbonate reduces the formation of spontaneous metastases in mouse models of metastatic breast cancer. Additional publications such as Robey, IF. et al., BMC Cancer (2011) 11:235-245 and Robey, I[F., el al. BioMedRes International(2013) pages 1-10 and Robey, I.F. et al.,J. Integr.Uncol. (2015) 4:1-8 described oral administration of bicarbonate as an inhibitor of metastases first in mice, and then in human volunteers.

[00081 Ribeiro, M. de L., et al., J. NutrFood Sci (2013) 2:6-16 assigned various pH scores to foods (including wine) and also described oral administration of lysine buffer and bicarbonate in mice bearing prostate cancer. In addition, Silva, A.S. et al., Cancer Res (2009) 2677-2684 described the role of systemic buffers in reducing metastases. Sircus, M. published a book, Sodium Bicarbonate: Nature's Unique First Aid Remedy, Garden City Park, New York: Square One Publishers, 2014, advocating sodium bicarbonate as a remedy for various conditions.

100091 It has been found, however, that oral administration of buffers has side effects including diarrhea, nausea, vomiting and abdominal discomfort. Intravenous i e. systemic administration has also been discredited.

[00101 While treatment of cancer and prevention of metastasis is a significant application of the invention method, systemic regulation of pH is important in other contexts as well. These contexts include, for example, treatment of pain. Also, in recent unpublished results, it was demonstrated by ET Science of South Pasadena, California that topical application of sodium bicarbonate was effective in reducing post-exercise blood lactate as well as enhancing performance in cycling.

[0011] Not all cancers respond, however, to adjustment of microenvironmental pH by administration of buffers. Bailey KM, et al; Meoplasia, (2014) 16:354-364 showed that metastasis is not inhibited by buffers in some tumor models, regardless of the buffer used. In cellular phenotypes that utilize a pH-dependent mechanism for successful metastasis, a highly glycolytic phenotype is shown that acidifies the local tumor microenvironment resulting in morphologic changes, while buffer-resistant cell lines exhibit a pH-independent metastatic mechanism involving constitutive secretion of matrix-degrading proteases without elevated glycolysis.

100121 In addition to straightforward buffering to adjust pH, the unfavorable extracellular pH problemcan be addressed using the alternative target DNA/HW exchanger I (NE1) protein can be the target for anti-metastasis agents. Loo, S.Y. et al., Curr. Pharm. Des. (2012) 18:1372-1382, for example, describeNHE1 as a promising drug target as an anticancer strategy. Thus, therapeutic agents that target this protein are useful in the invention as well. Amith, S.R. and Fliegel, L. CancerRes. (2013) 73:1-6 lists a number of factors that also affect this protein and thus constitute suitable targets as well.

[0013] A number of other factors are also associated with tumor metastasis and in many cases, therapeutic agents that target them are in fact available. Among these are matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and certain cathepsins, in particular B, D and L. For instance, cathepsin B as a cancer target is described by Gondi, C.S. and Rao, J.S.

ExpertOpin. Other. Trgets (2013) 17:281-291 and cathepsins in general are described as such targets by Olson, O.C. and Joyce, J.A., Nat. Rev. Cancer (2015) 15:712-729. A general discussion of the role of MMPs in the progression of cancer is described in an article by Gialeli, C. et al., FEBSJ. (2011) 278:16-27. According to this article, MMPs play an extensive role in modeling the extracellular matrix, thus permitting cancer cells from solid tumors to escape.

[00141 Small molecule inhibitors of these proteins are known, for example, various N sulfonyl amino acid derivatives are described by Tamura, Y. et al., J. Med Chem. (1988) 41:640-649 and a number of additional agents have been developed since. In addition, there are naturally occurring endogenous cysteine protease inhibitors. Endogenous CPIs constitute a single protein superfamily, the cystatins, such as type I cystatins; stefins A (or cystatins A) with a molecular mass of 11,775 Da and stefinsB (or cystatinsB) with a molecular weight of 11,006 Da. Cysteine protease inhibitors (CPs) are very tight binding, pseudo-irreversible inhibitors.

[00151 Other anti-metastatic agents include inhibitors of the src homology region 2 containing protein tyrosinase phosphatase (Shp2). A multiplicity of inhibitors of this activity is known, including Fumosorine, PHPS (NSC-87877) and NSC-117199, phenylhydrazonopyrazolone sulfonate (PHPS1), DCA, cryptotanshinone, 11-B08 and #220 324.

[00161 Another anti-metastatic agent is based on the well-known role of epidermal growth factor receptor (EGFR) and/or integrins in driving tumor progression and it is well known that both of these classes of receptors stimulate NHE activity. Several anti-EGFR compounds (e.g. erlotinib) have been approved to inhibit metastasis and an anti-integrin drug (cilengitide) is in clinical trials. Cariporide, eniporide and/or amiloride have passed all clinical phases.

[00117] Finally, phytochemicals with antioxidative and anti-inflammatory properties are thought to play important roles in the prevention and/or treatment of cancer. Until now, numerous phytochemicals have been examined for their potential anticancer properties by using a diverse array of preclinical experimental models.

[00181 In particular, a large number of laboratory-based studies have demonstrated the anti-cancer effect of the medicinal plant, Withaniasomnifera. These anti-cancer properties are attributable to withanolides, a class of bioactive constituents that includes Withaferin A (WFA). Withanolides are a group of naturally occurring C28-steroidal lactones. They contain four cycloalkane ring structures, three cyclohexane rings and one cyclopentane ring. WFA is a steroidal lactone, derived from Acnistus arboescens,Withaniasonmifera and other members of Solanaceae family. It has been traditionally used in ayurvedic medicine. It is the first member of the withanolide class of ergostane type product to have been discovered.

[0019] Many of the foregoing agents can be used in combination as described below.

[0020] It has now been found that parenteral, non-systemic administration of biocompatible buffers and other metastatic agents, including topical administration of such buffers and agents has a positive effect on cancer, including inhibition of metastasis, inhibition of growth and in some cases, tumor shrinkage. Such administration of pH-raising buffers is also useful to treat gout and melasma (hyperpigmentation).

100211 Topical administration may employ penetrants that improve transdermal transit either locally or systemically. Suitable penetrants are described, for example, in PCT publications WO/2016/105499 and WO/2017/127834.

[00221 There is a variety of agents that have been suggested for the treatment of melasma including the tyrosinase inhibitors hydroquinone, kojic acid, azelaic acid, various phenols and arbutin. These appear not to be completely successful and have undesirable side effects. The causes of melasma are varied and the condition is characterized by a multiplicity of lesions of hyperpigmentation varying in size, shape and distribution. These lesions may suitably be treated by raising the local pH according to the methods of the invention described below.

[0023] The contents of all documents set forth above are incorporated herein by reference, as are the contents of any cited documents throughout the specification.

Disclosure of the Invention

[0024] Applicants have found that the drawbacks of intravenous and oral administration of buffers and other anti-metastatic agents can be overcome by administering these agents non-systemically parenterally, such as by intraperitoneal, subcutaneous or intramuscular injection and in particular by topical administration. Topical administration is most conveniently transdermal, but further includes transmembrane administration for example by suppository or intranasal application.

[0025] Thus, in one aspect, the invention is directed to a method to inhibit metastasis and/or growth of a solid tumor contained in asubject which method comprises administering by a non-systemicparenteral route to a subject in need of such inhibition an effective amount of an anti-metastasis agent. In some embodiments, the anti-metastasis agent may be buffer sufficient to increase the p1 of the microenvironment of the solid tumor, or a protease inhibitor, an inhibitor of Na-/H' exchanger activity, an inhibitor of epidermal growth factor receptor (EGFR), an inhibitor of src homology region 2-containing protein tyrosinase phosphatase (Shp2), withaferin A or combinations thereof Theadministration is by non systemic parenteral route includes topical administration, especially transdermal.

[0026] For transdermal topical administration in particular for agents other than buffer, a suitable formulation typically involves a penetrant that enhances penetration of the skin and is, in some embodiments, composed of chemical permeation enhancers (CPEs). In some cases it can also include peptides designed to penetrate cells i.e. cell penetrating peptides

(CPPs) also known as skin penetrating peptides (SPPs). The formulation may be applied as such, or by means of occlusive devices such as patches.

[00271 If the active agent is a buffer, the choice of buffer system is based on the criteria of capability of buffering at a suitable pH typically between 7 and 8, as well as biocompatibility of the buffer system itself and the compatibility of the buffer system with the remaining components of the formulation. Conversely, the formulation is chosen to be compatible with the buffer selected; amounts of penetrants are generally less than those advantageous for therapeutic agents in general.

[00281 When the pH is adjusted for the purpose of inhibiting metastasis, treatment is followed by assessment of effectiveness. More importantly, as noted above, it has been found that some tumors are resistant to buffer treatment- i.e., raising the pH does not have a metastasis inhibiting effect. See, for example, Bailey, KM, et al.,Neoplsia (2014) 16:354 364 (supra). It is therefore one aspect of the invention to evaluate the tumors of potential subjects for treatment with buffer, e.g., by culturing biopsies for sensitivity/resistance to p-I adjustment.

100291 Raising the pH in the vicinity of melasmas and gout is also an effective treatment. The methods of the invention can usefully be applied to treatment of these conditions as well. Thus, another aspect of the invention such methods of treatment directed to melasma and gout

Brief Description of the Drawings

[0030] Figure 1 shows the time course of urine pH immediately following topical administration of sodium bicarbonate in various formulations and dosage regimes.

[0031] Figure 2 shows the mean daily urine pH of experimental subjects as a function of time after administration of a formulation of sodium bicarbonate in the formulations and dosages of Figure 1.

[0032] Figure 3 shows the time course of urine pH over a 3-day period using alternative topical formulations.

Modes of Carrving Out the Invention

[0033] As noted above, one aspect of the invention is a method to inhibit cancer growth and metastasis, including diminution of cancer mass by non-systemic parenteral, including topical administration of antimetastatic agents, including those agents that result in buffering the immediate environment of tumor cells, including solid tumors and melanomas. For non systemic parenteral administration, such as intramuscular, intraperitoneal or subcutaneous administration standard formulations are sufficient. These formulations include standard excipients and other ancillary ingredients such as antioxidants, suitable salt concentrations and the like. Such formulations can be found, for example, in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA-a standard reference for various types of administration.

[0034] As the subjects of the invention method are, in addition to humans, veterinary subjects, formulations suitable for these subjects are also appropriate. Such subjects include livestock and pets as well as sports animals such as horses and greyhounds.

[0035] One important aspect of the invention is based on the above-noted recognition that some tumors do not respond to buffer treatment as their microenvironment is not acidic and at least some of these tumors achieve metastasis by elevation of certain proteolytic enzymes that break down the extracellular matrix (ECM). If buffer treatment is contemplated, tumor cells from the biopsy of a solid tumor in a subject are therefore preferably cultured and tested in advance of treatment to insure responsiveness to buffer. Such evaluation can be carried out by any suitable means, including measurement of pH, assessment of the levels of relevant proteases, and invasion assays as impacted by buffer treatment as described in Bailey, K.M. et al (2014) spra.One important such assay is a glycolytic stress assay as described therein. Cell cultures of biopsied tumors that appear not to respond to buffer treatment as shown by such assays may benefit from administration of other antimetastatic agents and inclusion of such agents in the compositions of the invention that include buffers would also be beneficial. Thus, treatment with buffer-containing compositions alone may be contraindicated and the subject is not administered buffer as the sole active agent, but diverted to alternative treatment. This does not mean, of course, that buffer is necessarily omitted from formulations used to administer alternative active agents.

'7

100361 For topical administration, and in particular transdermal administration, the formulation will comprise penetrants including either or both chemical penetrants (CPEs) and peptide-based cellular penetrating agents (CPPs) that encourage transmission across the dermis and/or across membranes including cell membranes, as would be the case in particular for administration by suppository or intranasal administration, but for transdermal administration as well. Particularly suitable penetrants especially for those that contain at least one agent other than buffer include those that are described in the above-referenced W02016/105499 and W02017/127834. In addition to formulations with penetrants, transdermal delivery can be effected by mechanically disrupting the surface of the skin to encourage penetration, or simply by supplying the formulation applied to the skin under an occlusive patch.

[0037] Briefly, the penetrants described in the above-referenced PCT applications are based on combinations of synergistically acting components. Many such penetrants are based on combinations of an alcohol, such as benzyl alcohol to provide a concentration of 0.5-20% w/w of the final formulation with lecithin organogel present in the penetrant to provide 25-70% w/w of the formulation. Typically, these formulations are either essentially polar e.g. aqueous formulations that comprise sufficient nonionic surfactant in order to be present at 1-25% w/w of the final formulation and an excess of polar solvent or, alternatively anhydrous formulations wherein the additional components of the penetrant include sufficient bile salts to provide 1-15% w/w of the formulation and a surfactant present to provide 20 60% w/w of the formulation. These penetrants are also useful when the anti-metastasis agent is a buffer, but less lecithin organogel may be required - e.g. 1-50% byweight when the buffer is present at high concentration. While lecithins are exemplified, other zwitterions could also be used.

[0038] Alternatively, or in addition, the penetrant is such that the resulting formulation comprises at approximately 1:1:1 equimolar mixture of bile salt:lecithin and a completion component as well as one or more electrolytes sufficient to impart viscosity and viscoelasticity, one or more surfactants and benzylalcohol or an analog thereof The completion component can be a polar liquid, a non-polar liquid or an amphiphilic substance; however an amphiphilic substance is highly preferred.

[0039] The details concerning the behavior of this type of composition, in particular in aqueous solvent, are disclosed in Chen, C-Y, et al, Langnir (2014) 30:10224-10230, specifically incorporated herein by reference for its disclosure of methods to prepare advantageous micellular compositions for transdermal administration of active agents. As described, mixtures of lecithin and bile salt in an approximately 1:1 molar ratio, and in particular in the presence of an electrolyte, form viscous wormlike micelles in water that are stable. These micelles are helpful in effecting transfer of active agents, especially high molecular weight agents across skin. While a 1:1 molar ratio is optimal, applicants have found that variation from this ideal is workable - i.e. from 1:0.5 to 1:5 when the completion component in the ":1:11" system described above is a fatty acid ester, such as isopropyl palmitate. Thus, an "approximately 1:1:1 molar ratio" refers to this range.

[0040] In any case, the penetrant may further comprise a keratinolytic agent effective to reduce thiol linkages, disrupt hydrogen bonding and/or effect keratin lysis and/or a cell penetrating peptide (sometimes referred to as a skin-penetrating peptide) and/or a permeation enhancer. The additional components as listed above can be included in both the penetrant that provides 0.5-20% benzyl alcohol and lecithin organogel at 25-70% w/w of the final formulation and the above penetrant that comprises sufficient bile salt:ecithin:completion component to provide a 1:1:1 equimolar ratio of these to the formulation.

[0041] In an alternative to administering topically to intact skin, the surface of the skin may also be disrupted mechanically by the use of spring systems, laser powered systems, systems propelled by Lorentz force or by gas or shock waves including ultrasound and may employ microdermabrasion such as by the use of sandpaper or its equivalent or using microneedles or electroporation devices. Simple solutions of the agent(s) as well as the above-listed formulations that penetrate intact skin may be applied using occlusive patches, such as those in the form micro-patches. External reservoirs of the formulations for extended administration may also be employed.

[0042] The active buffer component is any mildly basic compound or combination that will result in a pH of 7-8 in the microenvironment of the tumor cells. Such buffers, in addition to or in lieu of bicarbonate salts, include lysine buffers, chloroacetate buffers, tris buffers (i.e., buffers employing tris (hydroxymethyl) aminoethane), phosphate buffers and buffers employing non-natural amino acids with similar pKa values to lysine. These may be, for example, the enantiomers of native forms of such amino acids or analogs of lysine with longer or shorter carbon chains or branched forms thereof. Histidine buffers may also be used. Typically the concentration of buffer in the compositions is in the range of 1-40% wt/wt. More typical ranges for sodium bicarbonate or sodium carbonate or both as pH adjusters are 5-35% by weight. The lack of inflammation side effects at these concentrations is surprising as it is noted in US 5,176,918 that uses lower concentrations to treat other conditions such as arthritis.

[0043] In all of the foregoing cases, additional anticancer components for example chemotherapeutic agents such as irinotecan, floxuridine, daunorubicin, cytarabine, and the like may be used.

[0044] As noted above, it is understood that some tumors do not respond to treatment with buffer, but apparently metastasize byvirtue of elevated levels of proteases that attack the extracellular matrix surrounding the tumor. In any event, breakdown of the ECM would encourage metastasis. Therefore, an additional active agent that is optionally included in the compositions of the invention is one or more appropriate protease inhibitor. Particularly important are inhibitors of cathepsins, for example of cathepsin B, and inhibitors of matrix metalloproteinases (MMPs). These components are active alone or augment the effect of buffer for tumors that are not resistant to buffer treatment.

[00451 Another active agent is Withaferin A. Withaferin A inhibits tumor metastasis and manifests other anti-cancer activities, e.g., inhibition of the neovascularzation associated with carcinoma, as well as cell proliferation. Withaferin A is also a leptin sensitizer with strong anti-diabetic properties that could induce healthy weight loss and beneficial effects on glucose metabolism.

[00461 In addition to straightforward buffering to adjust pH, the unfavorable extracellular pH- problem can be addressed using the alternative target DNA/-f exchanger I (NI-E1) protein can be the target for anti-metastasis agents. Other anti-metastatic agents include inhibitors of the src homology region 2-containing protein tyrosinase phosphatase (Shp2). A

multiplicity of inhibitors of this activity is known, including Fumosorine, PHPS (NSC 87877) and NSC-117199, phenyhydrazonopyrazolone sulfonate (PHPS1). DCA, cryptotanshinone, 11-1308 and #220-324, metaloproteinases-2 and -9 (MMP-2 and MMP-9) and certain cathepsins, in particular B, D and L.

[0047] Other agents include inhibitors of E-cadherin and of epidermal growth factor receptor (EGFR). Known inhibitors include erlotinib, an anti-integrin drug (cilengitide), cariporide, eniporide and amiloride.

[0048] The formulations may include other components that act as excipients or serve purposes other than active anti-tumor effects. For example, preservatives like antioxidants e.g., ascorbic acid or a-lipoic acid and antibacterial agents may be included. Other components apart from therapeutically active ingredients and components that are the primary effectors of dermal penetration may include those provided for aesthetic purposes such as menthol or other aromatics, and components that affect the physical state of the composition such as emulsifiers, for example, Durasoft@ (which is a mixture of thermoplastic polyurethane and polycarbonate). Typically, these ingredients are present in very small percentages of the compositions. It is understood that these latter ancillary agents are neither therapeutically ingredients nor are they components that are primarily responsible for penetration of the skin. The components that primarily effect skin penetration have been detailed as described above. However, some of these substances have some capability for effecting skin penetration. See, for example, Kunta, JR. et al, J. Pharm.Sci. (1997) 86:1369-1373, describing penetration properties of menthol. That said, their presence in the formulations useful in the invention is not required to achieve transdermal transport of the active agent(s), and they may be omittedfrom the formulation.

[0049] in some embodiments, the final formulation for transdermal administration may be in the form of a micellular composition. Typical micelle dimensions are in the range of 10-100nm. Methods for providing micellular forms are known in the art. Micelleformation is encouraged by appropriate levels of components that are capable of micellular formation, as well as exertion of mechanical agitation. (See Cheng, C-Y et al. (2014) supra). Various devices for encouraging micelle formation are available commercially.

[0050] For example, formation of micelles is enhanced bymilling. The level of enhancement is determined by the pressure and speed at which milling occurs as well as the number of passes through the milling machine. As the number of passes and speed and pressure are increased, the level ofmicelle formulation is enhanced.

[0051] When the milling machine is a Dermamill 100 (Blaubrite) marketed by Medisca, typical speeds include any variation between I to 100, where I is the slowest and 100 is the fastest. The pressure is selected from I to 5, where I is the highest and 5 is the lowest. Multiple passes, for example,2-10 or more passes, are contemplated in some embodiments. Intermediate values are also included. The speed and pressure can be varied for each pass.

[0052] Alternative milling machines could also be used, and comparable speeds, pressures and numbers of passes are replicated by comparison to the equivalents on the Dermamill 100. Alternatively, nicelle densities can be compared microscopically to assure equivalent results to those obtained with Dermamill 100. In some embodiments, the micelle density is at least 20% and in many cases at least 30%, or more.

[0053] Typically, the dimensions of the micelles are in the nanometer range - e.g., 10 150nm, including intermediate values.

[00541 This form of the formulation is particularly significant in those formulations that contain bile salts. In embodiments where a bile salt is added to the combination of benzyl alcohol and lecithin organogel in lieu of topping off with an aqueous medium, micelles that would have been relatively spherical may become elongated and worm-like thus permitting superior penetration of the stratum corneum of the epidermis. The worm like formation of the micelles is particularly helpful in accommodating higher molecular weight therapeutic agents. As is known, bile salts are facial amphiphiles and include salts of taurocholic acid, glycocholic acid, taurochenodeoxycholic acid, glycochenodeoxvcholic acid, cholic acid, deoxycholic acid. Detergents are also useful in lieu of bile salts, and include Tween) 80 and Span@ 80.

[0055] The inclusion of bile salts thus facilitates the ultradeformability of micelles which, in turn, facilitate passage of low and high molecular weight drugs and other active agents, such as nucleic acids and proteins. These compositions overcome the skin penetration barrier by squeezing themselves along the intercellular sealing lipid thereby following the natural gradient across the stratum corneum. This facilitates a change in membrane composition locally and reversibly when pressed against or attracted to a narrow pore.

100561 Thus, bile salts in combination with lecithin organogel facilitate the factors of micellar stability, enhanced viscosity and visco-elasticity that are critical in transdermal drug delivery. Both thermodynamic and kinetic stability are enhanced by the addition of background electrolytes, such as sodium chloride and sodium citrate. These electrolytes can more effectively increase viscosity and visco-elasticity of micelles and screen the repulsion between bile salt anions at a minimal concentration.

[0057] In some embodiments, the molar ratio of bile salt to lecithin is 1:1, and the concentration of electrolyte is determined by titration of the solution to transparency and enhanced viscosity as determined when the solution container is inverted.

[0058] Formulations designed for transdermal administration of the active ingredients specifically buffering agents and other therapeutically effective compounds and any ancillary components can be tested in a variety of known protocols for their nature and for their effectiveness in effecting transit across the skin. For example, the level of micellular formation can be determined by rheological study on a commercially available rheometrics RDA-3 strain controlled rheometer. Levels of collagen and proteoglvcan components of the extracellular matrix in skin from subjects exposed tothe penetrating composition can be measured histologically, for instance with H & E and Alician blue. Skin models have been prepared as three dimensional constructs using human derived epidermal keratinocytes and cultured on normal human derived dermal fibroblasts to create a multi-layered highly differentiated model of human dermis and epidermis. Water loss measurements may also be pertinent and these can be measured using the commercially available dermalab evaporimetry system marketed by Cortex Technology, Hadsun, Denmark. Collagen message levels may also be measured using standard techniques and the permeability of the human epidermis may be measured by determining the electrical conductivity. Monitoring electrical conductivity of skin exposed to various formulations will identify the most efficient formulations in increasing permeability. Tracer elements such as copper and iron can also be used to ascertain the effectiveness of penetration as can chromatographic analysis of components that transit skin layers.

[0059] For illustration only, the following is an example of an integrative cooperative CPE formulation directed to the extra-cellular matrix.

- Cetyltrimethyl ammoniumbromide (from about 2.0% to about 10.0 %) - Sodium cholate : Lecithin (96% pure ):Isopropyl myristate (equi-molar 1:1:1 from about 10% to about 40.0 %) Sodium citrate (titrate to transparency/incr. viscosity of #2.) - Benzyl alcohol (from about 2.0% to about 30.0 %) - Cis-Palmitoleic acid (from about 20.0% to about 30% of BA) - Methyl pyrrolidone (0.4%) /Dodecyl pyridinium (1.1%) (from about 0.5% to about 5.0%) Pluronic 127 (qs to 100%) An example of a CPP formulation directed to the cellular component of the SC permeability barrier is: - ACSSSPSKHCG, [ alanine-cysteine-serine-serine-serine-proline-serine-lysine-hisitidine cysteine-glycine ] identified as TD-] Thioglycolic Acid (TGA) (from about 2.0 % to about 7.0 % concentration) [may be substituted by other reducing agents] - Proteinase K (from about 5mg/mL to about 15 mg/mL)

100601 The aspect of the invention that includes administering buffers so as to raise the pH locally at the environment of a solid tumor or in the vicinity of gout or melasma, expands the indications to which the methods of the invention are applicable. It has been found, generally, that the requirements for effective penetration of the skin in the case of buffers as active agents are less restrictive than those required for alternative agents useful in preventing cancer metastasis. While the penetrants described above in detail are useful, a wider range of concentrations of, for example, lecithin organogel is effective in these cases. In some cases, extra lecithin in addition to that in an organogel is included. In addition, although for these indications delivery to the locus of the solid tumor, including melanoma, or melasma or gout is desirable, effective systemic pH alteration can be used as a way to diagnose the effectiveness of penetration when topical administration is employed. Therefore, in some of the examples below, elevation of the pH of urine in a model system such as a mouse can be used to verify the effectiveness of the penetrants in the formulation although the effect on pH is desirably local to the tumor, melasma or gout.

100611 The following examples are intended to illustrate but not to limit the invention.

Example 1 Specific Formulations for Administration of Buffering Agents

[0062] The following compositions have been prepared and are found useful in the methods of the invention. In the tables below, "LIP" represents lecithin organogel comprised of a 1:1 molar mixture of soy lecithin containing 96% phosphatidyl choline and isopropyl palmitate; "BA" represents benzyl alcohol; PLU-F127 represents the detergent poloxamer F127 granules; PLU-Water represents PLU-F127 dissolved in deionized water. (Alternatively, commercially available Pluronic F127 30%gel could be used) and Durasoft@ is a commercially available form of emulsifier.

[0063] in the tables below, sodium bicarbonate and sodium carbonate were supplied as such. Tris buffer at pH 8.1 was used. The phosphate "buffer" was supplied as Na H2 PO 4 .

The percentages are wt/wt i.e., weight percentages.

Table 1 - Bicarbonate Formulations

LB IIB LIP 30.00% 25.00% Ethanol 1.50% 0.00% BA 1.00% 1.00% Menthol 0.50% 0.90% NaBicarbonate 33,50% 5.76% PLU-F127 10.05% 10.50% Water in gel with P U 23 45% 24.50% AdditionalWater 30.34% Durasoft 2-00%

TOTAL 100.00% 100.00%

Table 2 - Carbonate Formulations

IC IC IIIC IVC LIP 25.00% 25.00% 30.00% 40.00% Ethanol 0,00% 0.00% 1.50% 1.50%o BA 1.00% 1.00% 1.00% -1.00% Menthol 0.90% 0.90% 0.50% 0.50% Sodium Carbonate 9.36% 16.10% 33.50% 11.00% PLU-F127 10.50% 10.00% 10.05% 13.50% Water in gel with PLU 2450% 23.33% 23.45% 31.53% Additional Water 26.74% 22.67% 0.00% 0.00% Durasoft 2.00% 1.00% 0.00% 1.00%

TOTAL. 100.00% 100.00% 100.00% 100.00%

Table 3 - Tris Formulations

____________________ IT IFT IT IVT LIP 25.00% 25.00% 30.00% 38.40% Ethanol 0.00% 0.0% 1.50% 1.50% BA 1.00% 1.00% 1.00% 1.00% Menthol 0.90% 0.90% 0,50% 0.50% TRIS buffer, pH 8.1 13.37% 23.00% 33.50% 14.93% PLU-F127 10.50% 10.00% 10,05% 12.80% Water-in el-with PLU 24.50% 23.33% 23.45% 9.87% Additional Water 22.73% 15.77/0 0.00% 0.00% Durasoft 2.00%N 1.00% 0.00%N 1.00%

TOTAL 100.00% 100.00% 100.00% 100.00%

Table 4 - Phosphate Formulations

III, H1fIP IVP LIP 25.00% | 25.00% 30.00% 37.25% Ethanol 0.00% 0.oo% 1.50% 1.50% BA 1.00% 1.00% 1 00% 1.00% Menthol 0.90% 0.90% 0.50% 0 50% Monosodium phosphate 16.02% 27.55% 33.50% 17.36% PLU4-F27 10.50% 10.00% 10.05% 12 42% Water in gel with PLU 24.50% 2____3.33% 23.45% 28.97% Additional Water 20.08% 1.22% 0.00% 0.00% Du rasof 2.00 i000/0 00~o% 1.000%/

TOTAL 100.00% 100.00% t 0.00% 100.00%

Table 5 Carbonate, TRIS, Phosphate Combination Formulations

Phos - low Phos - high II IV LIP 25.00% 25.00% 30.00% 3.25% Ethanol 0.00% 0.00% 1 .50% 1.50% BA 1.00% 1.000% 1.00% 1.00% Menthol 0.90% 0.90% 0.50% 0.50% Sodium Carbonate | 5.30% 5.30% | 5.30% 5.30% TRIS (121.14) 6.00% 6.00% 6.00% 6.00% Monosodium phosphate 6.00% 6.00% 6.00% 6.00% PLU-F127 10.50% 10.00% 1005% 1.42% Water in gel with PLU 24.50% 23.33% 23.45% 28,97% Additional Water 12.14% 21.47% 16.20% 0.06% DURASOFT 2.00% 1.00% 0.00% 1 00%

TOTAL 100.00% 10000/. 100.0% 100.00%

Table 6 - Alternative Buffers

25 28 29 A(2) B(2) C(2) LIP 6.00% 12.00% 12.00% 14.00% 15.00% 18.00% BA 1.00% 1.00% 1.00% 1.00% 1.00% 1.00% Menthol 0.50% 0.50% 0.50% 0.25% 0.25% 0.50% Durasoft 1.50% 1.50% 1.00% Pluronic Granules 4.20% 4.20% 4.20% 5.40% 2.10% 3.60% Water 37.80% 37.80% 40.30% 31.60% 29.65% 41.90% Sodium Carbonate 7.00% 7.00% 7.00% - - 3.00% Sodium Bicarbonate 28.00% 28.00% 28.00% 32.50% 32.50% 15.00% propylene glycol 3.00% 3.00% 6.00% 10.00% 5.00% almond oil 3.00% 3.00% 3.00% 4.00% 3.00% 4.50% zinc oxide 0.25% 0.50% cetyl alcohol 2.00% 2.00% 2.00% 2.00% 3.00% 3.00% lecithin 3.00% cetiolultimate (mixture oftridecane and undecane) 3.00% ethanol 1.50% 1.50% 1.50% 1.50% 1.50% 1.50% EGTA 0.50% 1.00% Sodium Decanoate 1.00% TOTAL 100.00% 100.00% 100.00'% 100.00% 100.00% 100.00%

Table 7 - Anyhrous Formulations

menthol 0.20% 0.20% 0. 50% Ethanol 2.00% 2.00% 1.50% Benzyl Alcohol 8.00% 8.00% 100% Cetyl Alcohol 2.00% Almond Oil 5.00% 5.00% 3 00% Lecithin 15.90% 15.90% 6.00% Lipmax 15.90% 15.90% 6.00% Th21yl ne IrCo 3.00% 3.00%,/ 0.00%// F127 Pluronic Powder 16.00% 16.00% 4.20% Water 39.80% Sodium Bicarbonate 26.00% 0.00% 28.00% Sodium Carbonate 7.00% 33.00% 7.00% Durasoft 1.00% 1.00% 1.00% 100.00% 100.00% 100.00%

Example 2 Topical Administration of Bicarbonate

[0064] 24 NCR nude 5 week old male mice were used in this study, divided into four groups of six mice each. Topical compositions were applied to the back of each mouse from hip to shoulder three times per day for 8 successive days for a total of 24 applications. A control group was administered sodium bicarbonate in water by mouth. The groups are as follows:

GroupI1 Sodium bicarbonate in H 2 0 Group 2. transdermal bicarbonate Dose #1 (30pL)(10pLx3 doses) Group 3. transdermal bicarbonate Dose#2 (220L)(73Lx3 doses) Group 4. transdermal bicarbonate Dose #3 (1110pL)(2x185mLx3 doses)

[0065] The transdermal formulations comprise penetrants to result in the formulations as follows: LIP-30.0%, EtOH-1.5%, BA-I.0%, menthol-0.5%, sodium bicarbonate-33.5%. PLU-F127-10.1%, PLU-water-23.5%;i.e., Formulation IB in Table 1. The concentration of sodium bicarbonate in the control group was 200 mM and the consumption was adlibidum. The concentration of sodium bicarbonate in the transdermal formulations was 33.5% wt/wt.

[0066] Urine samples were collected at one hour, three hours and six hours after the first drug application and stored at 4°C for subsequent pH determination. On days 2-12 urine was collected twice daily-prior to the first application and 15 minutes after the last application. The mice were sacrificed one hour after the last drug application on day 8 and the back skin was harvested and placed on bibulous paper.

[0067] To set a base line for pH without dosing, prior to beginning the protocol, urinary pH was measured at three time points during a single day in seven mice at 0900, five mice at 1300 and four mice at 1630. The pH of the urine had an overall mean value of 5.57 which did not vary over this time period.

[0068] As shown in Figure 1, all of the groups showed an increase in urine pH over the first six hours of treatment. The most significant increase occurred in Group 3 which was administered 220 microliters of the formulation.

[0069] Although the study was designed to be conducted for two weeks (Mon-Fri), it was terminated after day eight because the members of Group 2 (1,110 microliter) developed skin irritation; this was shown even in the low dose group receiving 30 microliters, i.e., Group 2.

100701 Figure 2 shows the urine values of pH over the course of the eight day study. Although there was some variation, the group receiving the highest dosage (Group 4) was able to maintain a high pH over the course of the study.

[00711 The study was repeated using the formulations 25, 28 and 29 in Table 6 with the results shown in Table 8 and using the formulations in Table 7 with results shown in Figure 3.

Table 8 - Mean urine pH at two collection time points on day one and overall.

urine1I urine 2 overall SB water 6.30 6.03 6.15 25 6.36 6.77 6.62 28 6.92 6.86 6.89 29 6.82 7.31 6.97

[00721 As shown, transdermal administration was more effective than oral administration.

Example 3 Transdermal Absorption in Humans

[0073] Healthy human Subjects Aged 18-60 were enrolled in a double-blinded, placebo controlled, randomized and cross-over in designed study. The subjects applied 0.6g/kg of body weight of formulation of Table 9, per randomization group, as follows: legs from ankle to top of thighs; arms from wrist to shoulder (including the deltoids) or drank 0.13 ounces of water per kg bodyweight (equates to about 8 ounces for a 140 pound subject) at 15 min, 1 hr 15 min, 2 hours and 15 min, 3 hours and 15 mins to control for dilution of urine.

Table 9

LIP 30.0% Ethanol 1.5% BA 1.0% Menthol (or limonene for 0.5% lipophilic) NaBicarbonate 33.5% PLU-F127 101% PLU-Water 23.5%

100741 At hourly intervals (at 1, 2, 3, and 4 hours) from start time of application, subjects collected 10-20 ml (approximately a Tablespoon) of urine and the pH was determined. The results are shown inTable 10.

Table 10 - Average Urine pH values for each treatment and time point

Product Baseline 1 hour 2 hours 3 hours 4 hours (n=20) (n=20) (n=20) (n=19) (n=20) Control (oral) 5.75 5.95 6.01 6.08 6.01 Formulation 5.86 6.11 6.27 6.24 6.23

[0075] No adverse effects were shown and the transdermal formulation out-performed oral administration.

Example 4 Treatmentof iout in Humans

[0076] A randomized, double-blinded, placebo controlled study of the efficacy and safety of a topical alkalinizing treatment for reducing pain associated with an acute gout flare was conducted to determine if topical application of sodium bicarbonate and menthol in a transdermal delivery system can effectively and safely reduce pain associated with an acute gout flare and if time to resolution is shortened.

[0077] 40 subjects with a past history of gout and presenting in the clinic with a gout attack that started within 36 hours and who had been prescribed cochicine, aged 18+ were employed randomized into groups of 20.

100781 The formulations used are shown in Table 11.

Table 11 - Gout Formulations

Menthol 0.00% 0.50% Ethanol 1.50% 1.50% Benzyl Alcohol 1.00% 1.00%o Cetyl Alcohol 2.00% 2.00% Almond Oil 3.00% 300% Lecithin 10.00% 7.00% IPP 10.00% 7 00% Profvlene Glycol 5.00%N 5.0 0%0/ Poloxamer/Pluronic Powder 9.00% 5.40% Water 57.50% 33.60% Sodium Bicarbonate 0.00% 33.00% Durosoft PK-SG 1.00% 1.00%o _0000% 100.00%

[00801 Subjects applied 10 ml of Control or Active cream to the entire limb of each affected joint three tires a day One identified "target joint" and up to 2 additional joints were followed.

[0081] The subjects reported joint pain as measured using a II-point scale (0-10, with 0 as "no pain" and 10 as "worst possible pain"). The reduction in pain in the target joints for the Active group (two subjects) was -3.0 points both after 30 minutes and 24 hours and -3.5 points at 4 days and 6 days Subjects in the ControlGroup showed no reduction at 30 minutes and 2 days and only -1 point after 24 hours and at 4 and 6 days. The maximum reduction in pain in a secondary joint for the Active group was -2 points, versus less or no reduction in pain for the Control group.

[00821 The reduction in pain associated with Active product use was observed as early as 15 minutes after product application.

Example 5 Treatment of Melasma in Humans

[0083] A randomized, double-blind study was conducted evaluating tranexamic acid in a transdermal delivery system, alone or with skin turnover agents for improvement of melasma to evaluate the efficacy and safety of 1) tranexamic acid (TA), 2) TA and 5% glycolic acid (GA) and 3)TA used in combination with retinoic acid (RA.) to control for the improvement of melasma. All the formulations including Control are alkaline. The formulations used are shown inTable 12.

[0084] Table 12 - Melasma Formulations

Ingredient Control Active (TA) Active (TA+GA) Tranexamic Acid 0.000% 6.000% 6.000% Glycolic Acid 0.000% 0.000% 5.000% Sodium Carbonate 1.060% 1.000% 1.000% Triethanolamine 0.000%_ _ 0.000% _ 1.000% Lecithin 13.300% 12.500% 12.500% IPP 13.300% 12.500% 12.500% Benzyl Alcohol 1.060% 1.000% 1.000% Durosoft PK-SG 1.060% 1.000% 1.000%/ Poloxamer/Pluronic 9.255% 8.700% 6.900% Powder Water 60.745% 57.100% 52.900% Crisp Fruit Fragrance 0.220% 0.200O 0.200% 100.000% 100.000% 100.000%

[0085] 36 women aged 18-65 with moderate to severe melasma were divided into groups of 12 each, and the subjects applied the formulations twice daily at the site of the melasma. The (Groups are as follows: Group 1: Control; Group2: Tranexamic acid (TA) 6% Formulation; Group 3: TA 6%+Glycolic Acid 5/ (GA) Combined Formulation; Group 4: TA 6% Formulation and Retinioic Acid 0.25% (RA) (separate products, with TA applied immediately before RA).

[0086] Outcome measures were by Standardized 2D photography, 5 views-frontal, 45 and 90 degrees and included blinded Investigator Global Assessment of Improvement compared to baseline; subject Self-Assessment of Improvement compared to baseline and VISIA System Photography (one site). Adverse events were evaluated as well.

100871 Follow-ups were by a 3 day phone call and visits at 2, 4, 8 and 12 weeks. In all these groups including Control had 50%-65% of subjects showed reduction in pigment after 30 days both by independent assessment and self-assessment. The results were generally improved for Groups 3 and 4 compared to Group 2 - at 30 days independent assessment found 50% of Group 2 subjects improved, and 60% and 65% of subjects in Groups 3 and 4 respectively. (Self-assessment results showed little differences all at about 60%.)

Claims (21)

1. A method for treating gout or reducing a symptom of gout, the method comprising topically administering to a subject in need thereof a transdermal formulation comprising a buffering agent and menthol, wherein the buffering agent comprises sodium bicarbonate or sodium carbonate.

2. The method of claim 1, wherein the symptom of gout is pain, joint tenderness, reduced range of motion, reduced mobility, inflammation, and/or abnormal levels of uric acid crystals in one or more joints, tendons, or surrounding tissue

3. The method of claim 1 or claim 2, wherein treating gout or reducing the symptom of gout reduces pain, reduces joint tenderness, reduces inflammation, improves range of motion, increases mobility, improves the time to resolution, raises the pH in the vicinity where the subject is administered the transdermal formulation, and/or reduces the levels of uric acid crystals in the vicinity where the subject is administered the transdermal formulation.

4. The method of any one of claims 1 to 3, wherein the method reduces the symptom of gout by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative the symptom before administering the transdermal formulation.

5. The method of any one of claims 1 to 4, wherein the transdermal formulation reduces the effects of a disease resultant from gout.

6. The method of claim 5, wherein the disease resultant from gout is osteoarthritis, rheumatoid arthritis, or psoriatic arthritis.

7. The method of any one of claims I to 6, wherein the sodium bicarbonate or sodium carbonate is at a concentration from about 30% to about 35% w/w of the transdermal formulation.

8. The method of claim 7, wherein the sodium bicarbonate or sodium carbonate is at a concentration of about 33% w/w of the transdermal formulation.

9. The method of any one of claims 1 to 8, wherein the menthol is at a concentration from about 0.1% to about 5.0% w/w of the transdermal formulation.

10. The method of claim 9, wherein the menthol is at a concentration of about 0.5% w/w of the transdermal formulation.

11. The method of any one of claims I to 10, wherein the transdermal formulation is formulated as a cream, lotion, or ointment.

12. The method of any one of claims 1 to 11, wherein the method further comprises administering another therapeutic agent.

13. The method of claim 12, wherein the other therapeutic agent is an analgesic, an anti inflammatory, a corticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), an oral urate lowering therapy (ULT), a drug that lower uric acid, or a combination thereof.

14. The method of claim 13, wherein the other therapeutic agent is administered before, contemporaneously with, and/or after administering the transdermal formulation.

15. The method of claim 14, wherein the other therapeutic agent is colchicine.

16. The method of any one of claims 1 to 15, wherein the transdermal formulation further comprises a penetrant or a penetration enhancer comprising one or more of benzyl alcohol, cetyl alcohol, isododecane, isopropyl palmitate (IPP), isopropyl stearate, menthol, phosphatidyl choline, undecane, and lecithin, wherein the lecithin is selected from an egg lecithin, a soy lecithin, and a synthetic lecithin.

17. The method of any one of claims 1 to 16, wherein the transdermal formulation further comprises a source of fatty acids comprising one or more of an alkanoic acid, caprid acid, diacid, ethyloctadecanoic acid, hexanoic acid, lactic acid, lauric acid, a lecithin, linoelaidic acid, linoleic acid, linolenic acid, neodecanoic acid, oleic acid, palmitic acid, pelargonic acid, propionic acid, stearic acid, and vaccenic acid, wherein the lecithin is selected from an egg lecithin, a soy lecithin, and a synthetic lecithin.

18. The method of any one of claims 1 to 17, wherein the transdermal formulation further comprises a penetrant or penetration enhancer, a source of fatty acids, and a polar solvent, and one or more of a humectant, an emulsifier, a surfactant, and an emollient; and wherein the formulation is formulated as a cream, lotion, or ointment.

19. A method for treating gout or reducing a symptom of gout, the method comprising: topically administering to a subject in need thereof a transdermal formulation comprising a buffering agent and menthol, wherein the buffering agent comprises sodium bicarbonate or sodium carbonate, and

administering colchicine before, contemporaneously with, or after administering the transdermalformulation.

20. The method of claim 19, wherein the transdermal formulation comprises about 33% sodium bicarbonate or sodium carbonate and about 0.5% menthol.

21. The method of any one of claims 1 to 20, wherein the gout is one or more of a recurrent gout attack, gouty arthritis, hyperuricaemia, gout related cardiovascular disorders (e.g. hypertension, cardiovascular disease, coronary heart disease), Lesch-Nyhan syndrome, Kelley-Seegmiller syndrome, kidney disease, kidney stones, kidney failure, joint inflammation, inflammatory joint disease, arthritis, osteoarthritis, rheumatoid arthritis and psoriatic arthritis, urolithiasis, plumbism, hyperparathyroidism, psoriasis or sarcoidosis.