Compositions comprising polyethylene glycol in the therapy of Head and Neck

Squamous Cell Carcinoma

Field of the invention.

The present invention concerns compositions for use in the prophylaxis and/or treatment of Head and Neck Squamous Cell Carcinoma (HNSCC). The present invention also concerns methods for preventing and/or treating such carcinomas. Other aspects, objects and advantages of the present invention will be apparent from the description below.

Background of the Invention.

Head and neck cancer is estimated to have caused approximately 7900 deaths in the U.S. in 2010 (Pfister D.G et al; J. Natl. Compr. Cane. Netw. 2011 ; 9:596-650). Common risk factors for developing this disease include the smoking or chewing of tobacco, consumption of alcohol, chewing of betel nut and/or infection with human papillomavirus (HPV).

Head and neck squamous cell carcinoma accounts for approximately 3 percent of all cancers in the United States. As used herein, the term "HNSCC" includes any cancer of the head and neck that begins in squamous cells.

HNSCC is associated with high rate of death and morbidity. This aggressive epithelial malignancy implicates the mucosal lining of the upper aerodigestive track including the oral cavity, oropharynx and larynx.

Even following curative treatment, patients still have an increased lifetime risk of developing new second cancers, usually in the head and neck or lung regions. An early risk factor for developing HNSCC is oral leukoplakia, while approximately 30% of survivors develop second malignancies. Even those patients who do not experience recurrence of the primary cancer have a high risk of developing a second malignancy as a result of antecedent premalignant cancer field, also known as "field cancerization". Chemoprevention of HNSCC with, for example, nonsteroidal anti-inflammatory drugs

(NSAIDS), retinoic acid, beta-carotene have to-date demonstrated minimal efficacy and are marred with toxicity. Despite extensive treatment options, the 5-year survival rate for HNSCC has not greatly improved over the last 4 to 5 decades. Furthermore, as a consequence of the disease severity and the ensuing radical surgical management, HNSCC patients have a worsening quality of life due to debilitating changes in facial appearance, speech, swallowing and breathing.

As such, there still exists a need for improving the prevention and/or treatment of this disease.

In PCT/US2012/035434 there is disclosed, inter alia, methods for preventing and/or treating HNSCC comprising the step of administering an effective amount of polyethylene glycol (PEG).

Summary of the Invention.

The present invention concerns compositions and methods for preventing and/or treating HNSCC.

In accordance with the present invention there is provided a composition for use in preventing and/or treating HNSCC in a subject, the composition comprising polyethylene glycol (PEG) at a concentration of 30mg/ml or greater wherein the PEG has a weight average molecular weight (M _w ) of about 800 daltons or greater.

In accordance with the present invention there is provided a method for preventing and/or treating HNSCC in a subject, the method comprising administering to the subject an effective amount of the composition of the invention. Compositions for use in such a method are also provided. The composition of the invention comprises (or consists essentially of) PEG at a concentration of 30mg/ml or greater, wherein the PEG has a M _w preferably of 800 daltons or thereabout to 20,000 daltons or thereabout, for example 2000 daltons or thereabout to 15,000 daltons or thereabout, more preferably 3000 or thereabout to 12,000 or thereabout, for example, 3000 or thereabout to 10000 or thereabout, most preferably 3200 or thereabout to 9000 or thereabout (e.g. 3000 or thereabout to 4000 or thereabout, 3500 or thereabout to 7000 or thereabout or 7000 or thereabout to 9000 or thereabout). In most preferred embodiments, PEG has a M _w of 3350 or thereabout, 4000 or thereabout, 8000 or thereabout, 10000 or thereabout, 15000 to 20000 or thereabout. Of these, PEG having a M _w of 8000 or thereabout or 10000 or thereabout is particularly preferred.

In another embodiment, the invention provides a composition for use in the prophylaxis and/or treatment of HNSCC as described in various aspects and embodiments of the invention herein, the composition comprising (e.g. as its sole therapeutically active constituent) PEG having a M _w as described herein at a concentration of 30mg/ml or greater.

In an embodiment, the present invention provides a method for preventing and/or treating HNSCC in a subject comprising topically administering to said subject an effective amount of the composition of the invention. Compositions for use in such a method are also provided.

In accordance with another embodiment, the present invention provides a method for reducing or suppressing HNSCC initiation and/or proliferation in a subject comprising topically administering to the region of the subject afflicted with pre-existing HNSCC and/or a pre-malignant lesion and/or oral leukoplakia, an effective amount of the composition of the invention. Compositions for use in such a method are also provided.

In accordance with another embodiment, the present invention provides a method for preventing and/or treating HNSCC in a subject comprising locoregionally administering to the subject an effective amount of the composition of the invention. Compositions for use in such a method are also provided. The present invention further provides a composition for locoregional use in the prevention and/or treatment of HNSCC in a subject afflicted with said disease wherein the composition comprises PEG as described herein. It is further contemplated that in an embodiment of the present invention, a method for preventing and/or treating HNSCC in a subject, wherein an effective amount of the composition of the invention is administered to the subject from 1 to 5 time(s) a day, preferably 2 to 4 times a day, more preferably 3 times a day. Compositions for use in such a method are also provided.

In an embodiment, the present invention provides a method for reducing or suppressing HNSCC proliferation in the head and neck of a subject comprising administering to the subject (preferably topically administering) an effective amount of the composition of the invention to one or more of: the lip, oral cavity (including the tongue, buccal mucosa, alveolar ridge, retromolar trigone, gums, floor of mouth, hard palate), salivary glands, nasal cavity (including nasopharynx), paranasal sinuses, pharynx (including oropharynx such as the base of tongue, soft palate, tonsillar pillar and fossa) hypopharynx (including pyriform sinus, lateral pharyngeal wall, posterior pharyngeal wall, postcricoid pharynx), and larynx

(including supraglottis (e.g. false cords, arytenoids, epiglottis, arytenoepiflottic fold), glottis, subglottis of the subject.

The present invention further provides methods for preventing and/or treating HNSCC in a subject as described in various aspects and embodiments of the invention herein, wherein the HNSCC has afflicted one or more of: the lip, oral cavity (including the tongue, buccal mucosa, alveolar ridge, retromolar trigone, gums, floor of mouth, hard palate), salivary glands, nasal cavity (including nasopharynx), paranasal sinuses, pharynx (including oropharnyx such as the base of tongue, soft palate, tonsillar pillar and fossa) hypopharnyx (including pyriform sinus, lateral pharyngeal wall, posterior pharyngeal wall, postcricoid pharynx), and larynx (including supraglottis (e.g. false cords, arytenoids, epiglottis, arytenoepiflottic fold), glottis, subglottis of the subject, the method comprising administering (preferably topically administering) to the afflicted area of the subject, an effective amount of the composition of the invention. Compositions for use in such methods are also provided. A preferred embodiment of the invention is a method for preventing and/or treating HNSCC in a subject wherein the HNSCC has afflicted the oral cavity (or anatomical site thereof) comprising administering (for example locoregionally and/or topically administering) to the oral cavity an effective amount of the composition of the invention. Compositions for use in such a method are also provided.

In accordance with a further embodiment, the present invention provides a method for reducing or inhibiting Epidermal Growth Factor Receptor (EGFR) surface expression and/or phosphorylation of the receptor in the squamous cells of the head and/or neck of a subject, the method comprising administering to the subject an effective amount of the composition of the invention. Compositions for use in such a method are also provided.

In another embodiment, the present invention comprises a method for preventing and/or treating HNSCC in a subject which method comprises co-administering to the subject an effective amount of the composition of the invention with an effective amount of one or more additional therapeutic agent(s). Compositions for use in this method optionally further comprising the one or more additional therapeutic agent(s) are also provided.

In another aspect of the invention there is provided a method for preventing and/or treating HNSCC in a subject which method comprises administering to said subject:

(a) an effective amount of a therapeutic agent such as an anti-EGFR agent (for example an anti-EGFR antibody such as cetixumab);

(b) an effective amount of a composition of the invention. In some embodiments of this aspect of the invention, step (a) occurs before step (b). In other embodiments of this aspect of the invention, step (a) occurs after step (b). In further embodiments of this aspect of the invention, step (a) and step (b) occur concurrently.

In another embodiment of the invention, there is provided a method for preventing and/or treating HNSCC in a subject in remission of HNSCC, the method comprising administering to the subject an effective amount of the composition of the invention. The subject may be in partial or complete remission. Compositions for use in such a method are also provided. In another embodiment of the invention there is provided a method for ameliorating (such as preventing) the recurrence of HNSCC in a subject in remission of that disease, the method comprising administering to the subject an effective amount of the composition of the invention. Compositions for use in such a method are also provided.

In another embodiment of the invention there is provided a method for preventing and/or treating metastatic HNSCC in a subject which method comprises administering to the subject an effective amount of the composition of the invention. Compositions for use in such a method are also provided.

In another embodiment of the invention there is provided a method for preventing and/or treating locally advanced HNSCC in a subject which method comprises administering to the subject an effective amount of compositions of the invention. Compositions for use in such a method are also provided.

In another aspect of the invention there is provided a method for regressing a HNSCC carcinoma in a subject which method comprises (a) administering an effective amount of a composition of the invention. Compositions for use in such a method, such as described herein, are also provided. Preferably, the HNSCC carcinoma over-expresses EGFR. The EGFR expression status of the HNSCC carcinoma may be determined according to standard methods, techniques and kits such as described herein.

In an embodiment of this aspect of the invention, the method further comprises (b) resecting and/or ablating the HNSCC carcinoma and/or administering an effective amount of a therapeutic agent. In this embodiment of the invention, step (b) may occur after step (a). By regressing the HNSCC carcinoma, the present invention may reduce the degree of trauma or treatment related adverse events to the subject resulting from subsequent therapeutic procedures. In another aspect of the invention, there is provided a method for preventing and/or treating HNSCC in a subject comprising

(a) resecting and/or ablating one or more HNSCC carcinomas;

(b) administering to the subject an effective amount of the composition of the invention. In one embodiment of this aspect of the invention, step (a) occurs before step (b). Preferably the composition of the invention is administered to the resected/ablated region of the subject. In another embodiment, step (b) occurs before step (a). In another embodiment, step (a) and step (b) occur concurrently.

The invention further provides the use of the PEG having a M _w of 800 or greater and a concentration of 30mg/ml or greater in the manufacture of a medicament for the treatment and/or prophylaxis of HNSCC, as described in various aspects and embodiments herein.

The invention further provides the use of PEG having a M _w of 800 or greater and a concentration of 30mg/ml as described in various aspects and embodiments herein in the manufacture of a medicament, for regressing a squamous cell carcinoma such as HNSCC.

Description of the Figures. Figure 1:

Inhibition of cellular proliferation by PEG-3350 in SCC-25 cells: The cells were seeded in 96-well plates and after 24 hours treated with different concentrations (0 - 200 mM) of PEG- 3350 for 24 hours. The cellular proliferation was determined by WST-1 assay. All the experiments were performed with n = 5 and total number of determinations were 18. The data are presented as mean percent of control (no PEG treatment) + S.E.M. One way ANOVA was applied for statistical evaluation; ** indicates p<0.0l compared to 0 mM.

Figure 2: Inhibition of cellular proliferation by PEG-8000 in SCC-25 cells: The cells were seeded in 96-well plates and after 24 hours treated with different concentrations (0 - 50 mM) of PEG- 8000 for 24 hours. The cellular proliferation was determined by WST-1 assay. All the experiments were performed with n = 5 and total number of determinations as 18. The data are presented as mean percent of control (no PEG treatment) + S.E.M. ; One way ANOVA was applied for statistical evaluation; ** indicates p<0.0l compared to 0 mM.

Figure 3 :

Inhibition of cellular proliferation by PEG-10000 in SCC-25 cells: The cells were seeded in 96-well plates and after 24 hours treated with different concentrations (0 - 30 mM) of PEG 10000 for 24 hours. The cellular proliferation was determined by WST-1 assay. All the experiments were performed with n = 5 and total number of determinations as 18. The data are presented as mean percent of control (no PEG treatment) + S.E.M.; One way ANOVA was applied for statistical evaluation; ** and * indicates p<0.0l and /?<0.05, respectively, compared to 0 mM.

Figure 4: Inhibition of cellular proliferation by PEG-BPA in SCC-25 cells: The cells were seeded in 96-well plates and after 24 hours treated with different concentrations (0 - 6 mM) of PEG- BPA for 24 hours. The cellular proliferation was determined by WST-1 assay. All the experiments were performed with n = 5 and total number of determinations as 18. The data are presented as mean percent of control (no PEG treatment) + S.E.M.; One way ANOVA was applied for statistical evaluation; no statistically significant differences were observed.

Figure 5 :

Inhibition of cellular proliferation in the human SCC-9 cell-line after 24 hours incubation with (A) PEG-3350, (B) PEG-8000 and (C) PEG- 10000. The effects of the PEG compounds were calculated and plotted as a percentage of the maximum effect induced by 10 μΜ doxorubicin. Data are presented as the individual cuves for each of n=3 (in triplicate) experiments, +/- standard error of the mean (SEM).

Figure 6 Inhibition of cellular proliferation in the human SCC-9 cell-line after 72 hours incubation with (A) PEG-3350, (B) PEG-8000 run 1 and 2, (C) PEG-8000 run 3, (D) PEG-10000 run 1 and 3 and (E) PEG-10000 run 2. The effects of the PEG compounds were calculated as a percentage of the maximum effect induced by 10 μΜ doxorubicin. Data are presented as the individual curves for each of n=3 (in singlicate) experiments.

Detailed Description of the Invention.

Compositions of the Invention.

Compositions of the invention may comprise PEG as its sole therapeutically active constituent or may contain one or more therapeutic agents (particularly anti-cancer agents) as described in more detail below. In preferred embodiments, compositions of the present invention are aqueous compositions. Compositions of the present invention, depending, in particular, on the intended preparation (described in more detail below), may be in the form of a solid, semi-solid (e.g. slurry or paste) or liquid.

The term "polyethylene glycol" (PEG), otherwise known as poly(oxyethylene) or poly(ethylene oxide) (PEO), refers to a polymer of ethylene oxide as well known to those skilled in the art. The polyethylene glycol (PEG) used in the invention typically has the general formula H-(OCH ₂ CH ₂ ) _n OH. In some other embodiments of the invention as described throughout this specification, other polyethylene glycol compounds may be used such as end-capped structures and polyoxyethylenes that include minor amounts of alkylene oxide units other than ethylene oxide.

PEG used in compositions of the invention preferably has a weight average molecular weight (M _w ) in a range wherein the lower limit of the range is selected from the group consisting of: 800,1000, 2000, 3000, 4000, 6000; and an upper limit of the range is, selected independently, from the group consisting of: 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12,000, 15,000, 20,000. Preferred ranges are wherein the lower limit is 3000 or 4000 and the upper limit is, selected independently, 5000, 6000, 7000, 8000, 9000, 10000, 12000. Some preferred particular ranges have been set forth previously. It will be apparent of course to the skilled reader that the upper limit referred to throughout this specification although selected independently, must necessarily have a higher value than the selected lower limit. For example, the PEG may be 'PEG 3350', 'PEG 4000', 'PEG 8000', 'PEG 10000' as defined in some national or regional pharmacopeias. Further examples of suitable PEGs recognized in some national or regional pharmacopoeias include Macrogols, for example Macrogol 3350, Macrogol 4000, Macrogol 8000, Macrogol 10000. Macrogol 8000 is particularly preferred. Preferably, the PEG is not systemically absorbed to any significant extent when topically administered to the subject.

Compositions of the present invention comprise PEG at a concentration of 30mg/ml or greater. In preferred embodiments, compositions of the present invention comprise PEG at a concentration in the range of 30mg/ml up to, and including, the maximum solubility available in that composition, e.g. an aqueous solution (for example, at 25°c) for the PEG concerned (having regard, amongst other things, to its M _w ). It is recognized that the maximum solubility available of PEG may be affected by factors such as the prevailing temperature when the composition is prepared, what other constituents (such as described herein) are present in the composition, pH and the like. Such matters are routine considerations for the skilled artisan. In general, it is preferred that compositions of the present invention comprise PEG at a high concentration with respect to the maximum solubility available, i.e. 0.5 fold or greater, 0.6 fold or greater,0.7 fold or greater,0.8 fold or greater, or 0.9 fold or greater of the maximum solubility available for the PEG concerned in that composition (having regard to the matters described herein).

Suitably, compositions of the present invention comprise PEG having a concentration of at least l lOmg/ml or greater, at least 120mg/ml or greater, preferably, at least 130mg/ml or greater, e.g. 140mg/ml or greater such as at least 150mg/ml or greater, at least 200mg/ml or greater or 300mg/ml or greater; having regard to the maximum solubility available for the PEG concerned in the composition.

Throughout this specification, the concentration of PEG is referred to in units of mg/ml. It is well within the purview of the skilled artisan to convert this unit into equivalent molarity (such as mM).

In some embodiments, compositions of the present invention comprise (or consist essentially of) PEG having a concentration in a range wherein the lower limit is 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mg/ml and the upper limit, selected independently, is 100,110, 120, 130, 140, 150, 160, 170,180,190, 200, 300, 350, 400mg/ml, or the upper limit is the maximum solubility available for the PEG concerned in the composition, having regard to the matters described herein. For example, compositions of the present invention may comprise PEG at 120mg/ml to 200 or 300mg/ml, 130mg/ml to 200 or 300mg/ml, 140mg/ml to 200 or 300mg/ml, 150mg/ml to 200 or 300mg/ml, 160mg/ml to 200 or 300mg/ml, or 160mg/ml up to, and including the maximum solubility available for the PEG concerned in the composition. Preferred ranges for PEGs having a particular M _w are set forth below.

In some embodiments, compositions of the present invention comprise (or consist essentially of) PEG having a weight average molecular weight (M _w ) in a range wherein the lower limit of the range is selected from the group consisting of: 800,1000, 2000, 3000, 4000, 6000; and an upper limit of the range is, selected independently, from the group consisting of: 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12,000, 15,000, 20,000. Preferred ranges are wherein the lower limit is 3000 or 4000 and the upper limit is, selected independently, 5000, 6000, 7000, 8000, 9000, 10000, 12000, the composition having a PEG concentration in a range wherein the lower limit is 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mg/ml and the upper limit, selected independently, is 100,

110,120,130,140,150,160,170,180,190, 200, 300, 350, 400mg/ml, or the upper limit is the maximum solubility available for the PEG concerned in the composition, having regard to the matters described herein. For example, compositions of the present invention may comprise PEG having a Mw of 3000 to 12,000, preferably 3000 to 10000, such as 3350, 4000, 8000 or 10000 and a concentration of 120mg/ml to 200 or 300mg/ml, 130mg/ml to 200 or 300mg/ml, 140mg/ml to 200 or 300mg/ml, 150mg/ml to 200 or 300mg/ml, 160mg/ml to 200 or

300mg/ml, or 160mg/ml up to, and including, the maximum solubility available for the PEG concerned in the composition.

Compositions of the present invention may comprise PEG having a M _w of 3350 ('PEG 3350') at a concentration of 30mg/ml to 670mg/ml. Preferred concentration ranges of PEG 3350 are wherein the lower limit is 30, 40, 50, 60, 70,80,90, 100, 120, 150, 200mg/ml and the upper limit, selected independently, is 150, 200, 250,300, 400,500,600 or 670mg/ml. More preferred ranges are 150mg/ml to 670mg/ml, for example, 167.5mg/ml to 335mg/ml or 167.5mg/ml to 670mg/ml or 335mg/ml to 670mgl/ml. Particularly preferred concentrations are 167.5mg/ml, 200mg/ml 335mg/ml and 670mg/ml or thereabout. Of these, Compositions of the invention comprising (or consisting essentially of) PEG 3350 at a concentration of 200mg/ml is particularly preferred.

The terms "thereabout" and "about" as referred to in this specification is intended to indicate that strict compliance with the referenced value is not necessary. Minor variations of the referenced value, that do not substantially alter the properties conferred, are considered within the ambit of that referenced value.

Compositions of the present invention may comprise PEG having a M _w of 10000 ('PEG 10000') at a concentration of 30mg/ml to 300mg/ml. Preferred concentration ranges of PEG 10000 are wherein the lower limit is 30, 40, 50, 60, 70, 80, 90, 100, 150mg/ml and the upper limit, selected independently, is 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300mg/ml. Particularly preferred PEG 10000 ranges are 70 to 400mg/ml, 100 to 300mg/ml, 150 to 300mg/ml. Particularly preferred PEG 10000 concentrations are 75mg/ml, 150mg/ml and 300mg/ml. Of these, 300mg/ml is most preferred. Compositions of the invention comprising (or consisting essentially of) PEG 10000, at a concentration of

300mg/ml or thereabout is a particularly preferred embodiment of the invention as it combines favorable efficacy and formulation properties.

Compositions of the present invention may comprise PEG having a M _w of 8000 ('PEG 8000') at a concentration of 30mg/ml to 400mg/ml. Preferred concentration ranges of PEG 8000 are wherein the lower limit is 30, 40, 50, 60, 70, 80, 90, 100, 150mg/ml and the upper limit, selected independently, is 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400mg/ml. Particularly preferred PEG 8000 concentrations are 50,100, 200 and 400mg/ml or thereabout. Compositions of the invention comprising (or consisting essentially of) PEG 8000, at a concentration of 200mg/ml or thereabout is also a particularly preferred embodiment of the invention as it combines favorable efficacy and formulation properties.

Compositions of the present invention may further comprise other constituents such as another therapeutic agent (see below), and one or more excipients. Examples of excipients include one or more electrolytes such as sodium chloride, potassium chloride, sodium bicarbonate, sulphate such as sodium sulphate. In one embodiment, compositions of the invention comprise sodium chloride and potassium chloride and optionally sodium bicarbonate. Compositions of the invention may comprise one or more sweetener(s) (such as aspartame, acesulfame potassium (acesulfame K), sucralose and saccharine and combinations thereof) and one or more flavouring(s) (such as orange, lemon-lime, lemon, citrus, chocolate, tropical fruit, aloe vera, tea, strawberry, grapefruit, blackcurrant, pineapple and vanilla). Compositions may further comprise ascorbate and/or citrate. Compositions of the invention may further comprise preservatives and other additives such as, for example, antimicrobials, anti-oxidants, pharmaceutically acceptable carriers, chelating agents, and inert gases and the like as known and called for by acceptable pharmaceutical practice.

Compositions of the invention may be presented in a variety of preparations. For example, compositions of the invention may be prepared in the form of an aqueous solution or suspension (for example a mouthwash), emulsion, paste (e.g. toothpaste), cream, balm (e.g. lip balm), ointment, foam, paint, sponge, gel, chewing gum, spray, lozenge, troche, syrup (e.g. viscous syrup), linctus, slurry, film (e.g. orodispersible film), tablet (e.g. orodispersible tablet), capsule (e.g. liquid-gel capsule), granule, caplet, buccal patch. Particularly preferred preparations of compositions of the invention include solutions, suspensions, linctus and paints.

Depending on the intended use of the composition of the invention, certain preparations may be particularly apt. For example, where it is desired to administer a composition of the invention to the lip, the composition may be prepared as a lip balm or cream. Where it is desired to administer the composition to an area of the buccal mucosa, a mouthwash, spray or lozenge may be particularly appropriate. Where the preparation is a mouthwash, it maybe gargled or swirled around the mouth before swallowing or expelling. Compositions of the invention may be prepared as an aerosol preparation, particularly for administration to the nasal, buccal and oropharynx. Compositions of the invention may also be prepared as non-pressured preparations, such as in a nebulizer or an atomizer. In some embodiments, the composition of the invention may be a linctus preparation, a preparation form well known to those skilled in the art. This form may assist in increasing the contact time between PEG and the target tissue such as the pharynx and/or larynx mucosa and may be particularly appropriate in the prophylaxis and/or treatment of HNSCC at these anatomical sites. In one embodiment, compositions of the invention are administered topically to the subject. Topical administration, in the context of the present invention, refers to the application of a composition of the invention to a surface of the subject's body. Topical administration includes application to an internal surface of the subject, for example the buccal mucosa. Compositions of the present invention may be administered to the target area (e.g. the carcinoma) and in the local region thereof (sometimes referred to herein and in the art as "locoregional administration"). Typically, compositions of the present invention are applied to a region of the carcinoma that is accessible at the surface of subject's body, for example, surface exposed HNSCC that has afflicted the lip, tongue, buccal mucosa (e.g. buccal floor and/or roof), nasal cavity, pharynx, larynx and anatomical sites thereof. Topical

administration may include per os administration of compositions of the invention. For example, topical administration may include swallowing a composition of the invention. Accordingly, contact between the composition of the invention and the surface exposed region of the carcinoma (if present) is enabled as the composition passes into the stomach via the pharynx.

An effective amount of the composition of the invention to be employed therapeutically will depend, for example, upon the therapeutic and treatment objectives (e.g. prophylaxis or treatment, or both), the route of administration, the age, body mass, condition of the subject undergoing treatment or therapy (for example, by assessing the subject's performance status and/or other co-morbidities ), stage and/or aggressiveness (e.g. TNM score) of the carcinoma (if present), any auxiliary or adjuvant therapies being provided to the subject, and on the subjects previous response (if appropriate) to therapy with compositions of the invention. The duration of therapy with compositions of the invention depends, in part, on the considerations given above. Such considerations are within the purview of the attending physician or healthcare professional.

An effective amount of the composition of the invention may be determined, at least in part, by the desired reduction in EGFR surface expression in the target tissue (for example, a HNSCC carcinoma). An effective amount of the composition of the present invention may produce in the target tissue a reduction in EGFR expression of at least 30% (or thereabout), for example, at least 40%, compared to the expression observed prior to contact with the composition.

The degree of EGFR reduction may be determined using methods described in the examples herein. In particular, the degree of EGFR reduction may be determined using flow cytometry, as described, for example, in PCT/US2012/035434 or otherwise well known to the skilled artisan.

The EGFR expression status of a target tissue may be determined using standard methods and kits (for example EGFR pharmDx™, available from Dako Denmark A/S, Glostrup,

Denmark).

In some embodiments, the composition of the invention is topically administered to the subject so as to coat the target tissue (e.g. the surface exposed region of a HNSCC carcinoma, and preferably the local tissue region thereof). Compositions of the present invention may be repeatedly administered to the target tissue of the subject to maintain or renew the coating of the target tissue. Thus the present invention provides a method of preventing and/or treating HNSCC in a subject as described in various aspects and embodiments described herein, the method comprises coating the target tissue of the subject with the composition of the invention. In preferred embodiments, the method comprises coating the target tissue so as to produce a PEG concentration at the target tissue/composition interface of greater than 30mg/ml, preferably greater than 50mg/ml, more preferably greater than lOOmg/ml such as 150mg/ml or greater, e.g. 200mg/ml or greater. Since PEG at the M _w described herein is known to be generally well tolerated, even at relatively high concentrations, with a low risk of any serious adverse events, the subject, attending physician or healthcare professional can readily administer an effective amount of the composition of the invention to meet or exceed the PEG concentration at the target tissue/composition interface as set forth herein.

In particularly preferred embodiments, the method comprises coating the target tissue (e.g. surface exposed carcinoma and preferably the local region thereof) with a composition comprising PEG 8000 or PEG 10000 having a concentration of 150mg/ml to 300mg/ml, preferably 200mg/ml to 300mg/ml. Particular examples of compositions of the invention comprise PEG 8000 at a concentration of 200mg/ml or thereabout, or PEG 10000 at a concentration of 300mg/ml.

An effective amount of PEG for use in the prophylaxis of HNSCC may differ from an effective amount of PEG for use in the treatment of HNSCC. In one embodiment, in a prophylaxis setting, a lower amount of PEG is required than typically used in a treatment setting.

Therapeutic agents.

Compositions of present invention may be used in conjunction with one or more therapeutic agents for the prophylaxis and/or treatment of HNSCC. For example, compositions of the invention may be co-administered with the one or more therapeutic agent(s). The term "coadministered" means the coordinated administration of compositions of the invention with one or more therapeutic agents to prevent and/or treat HNSCC. Such coordinated administration between compositions of the invention and one or more therapeutic agent(s) may be simultaneous, sequential or separate.

Examples of therapeutic agents that may be used in conjunction with compositions of the invention include radiation therapy and anti-cancer agents. The term "anti-cancer agent" means a therapeutic agent that is capable of inhibiting the initiation and/or proliferation of cancer and/or promoting cell death (e.g. by apoptosis) in cancer cells such as squamous cell carcinomas, particularly those of the head and neck. Such therapeutic agents include those approved by national or regional regulatory authorities for such use.

Examples of anti-cancer agents include agents that target EGFR expression and/or function (for example by inhibiting functional signaling of the EGFR), herein referred to as "anti- EGFR" agents". Examples of anti-EGFR agents include anti-EGFR antibodies such as cetixumab, panitumumab, zalutumab, nimotuzumab. Other anti-EGFR agents include Erlotinib, Gefitinib, Lapatinib, BIBW-2992. Examples of anti-cancer agents include agents that target VEGFR expression and/or function ("anti-VEGFR agents"). Examples of anti-VEGFR agents include bevacizumab, sorafenib, sunitinib, vandetanib. Examples of anti-cancer agents include agents that target IGF-IR expression and/or function ("anti-IGF-lR agents"). Examples of anti-IGF-lR agents include figitumumab and cixtumumab.

Examples of anti-cancer agents include agents that inhibit the mammalian target of rapamycin ("mTOR agents"). Examples of such mTOR agents include Temsirolimus, Everolimus.

Further examples of anti-cancer agents include platinating agents such as cisplatin and carboplatin; taxanes such as paclitaxel and docetaxel; folate anti-metabolites such as pemetrexed; fluorouracil, methotrexate.

Examples of other anti-cancer agents include Dasatinib, Ionafarnib and Bortezomib.

In some embodiments, compositions of the present invention comprise the PEG as described herein together (for example in intimate physical admixture) with an effective amount of one or more therapeutic agents, e.g. anti-cancer agents, such as described above. Preferably, the composition comprises PEG that is not conjugated to the therapeutic agent such as an anticancer agent. The reader of this specification may assume that each combination of PEG as described herein together with one or more of the anti-cancer agents described above is individually and specifically contemplated as an embodiment of the invention.

It will be apparent that an "effective amount" of one or more therapeutic agent(s) need not necessarily be the same weight amount as an effective amount of PEG. It will also be apparent that when considering the term "effective amount" in relation to radiation therapy, an appropriate dose unit (for example gray (gy) or rad) should be deployed. In other embodiments of the invention there is provided a kit comprising a composition of the invention together with at least one composition of one or more anti-cancer agents such as described above, optionally together with instructions for use.

Methods of treatment and compositions for use in such methods.

The present invention provides a method for preventing and/or treating HNSCC in a subject comprising (or consisting essentially of) administering an effective amount of the composition of the invention. The present invention also provides compositions for use in the prophylaxis and/or treatment of HNSCC in a subject.

Compositions of the present invention may be of particular use in treating HNSCC that over- express surface EGFR. The EGFR expression status of a squamous cell carcinoma in a subject may be determined according to standard methods and kits (for example EGFR pharmDx™, available from Dako Denmark A/S, Glostrup, Denmark).

In one embodiment, the method is for preventing HNSCC. In another embodiment, the method is for treating HNSCC. In a further embodiment, the method is for preventing and treating HNSCC.

The synonymous terms "prophylaxis" and "preventing" and grammatical variations thereof means inhibiting the initiation of HNSCC and/or inhibiting the progression of a pre- malignant pathology of the epithelium (such as oral leukoplakia) and/or early stage carcinoma to a later stage carcinoma.

In some embodiments, prophylaxis methods of the invention are performed prior to a positive diagnosis of HNSCC. The term "treat" and grammatical variations thereof means inhibiting the proliferation of HNSCC and/or promoting cell death in HNSCC. In some embodiments, treat includes "cure", although the term cure does not necessarily mean the complete restoration of health with respect to the malignancy. Those skilled in the art recognize that a treatment may have varying degrees of curative effect and as such are encompassed by the term "treat".

The present invention further provides a method for preventing HNSCC in a subject in remission of that disease comprising (or consisting essentially of) administering to the subject an effective amount of a composition of the invention. The remission may be total or partial. Compositions for use in such a method are also provided.

The present invention further provides a method for treating locally advanced and/or metastatic HNSCC in a subject comprising (or consisting essentially of) administering to the subject an effective amount of the composition of the invention.

In another embodiment of the invention there is provided a method for preventing and/or treating HNSCC in a subject susceptible to developing HNSCC comprising (or consisting essentially of) administering to the subject an effective amount of the composition of the invention. Such subjects include those with a history and/or concurrent use of: tobacco (smoking and/or chewing), excessive alcohol intake, betel nut chewing. Such subjects include those that are infected with HPV (particularly with serotype HPV 16) and/or immunocompromised and/or with a prior or familial history of (or predisposition to) developing HNSCC. The invention further provides a method for preventing and/or treating HNSCC in a subject that is HPV negative comprising (or consisting essentially of) administering to the subject an effective amount of the composition of the invention.

Compositions for use in such methods are also provided. In another embodiment, there is provided a method for preventing HNSCC in a subject afflicted with oral leukoplakia and/or erythroplakia comprising (or consisting essentially of) administering to the subject an effective amount of the composition of the invention.

Compositions for use in such a method are also provided. In another embodiment, there is provided a method for preventing HNSCC in a subject afflicted with pre-malignant alterations to the epithelium of the upper aerodigestive tract comprising (or consisting essentially of) administering to the subject an effective amount of a composition of the invention. Pre-malignant alterations may arise as a result of "field cancerization". Field cancerization refers to a process whereby the epithelium undergoes alterations (which may be multiple and independent of one another) that primes the epithelium for transformation. These alterations may be evident in subtle changes to the epithelium vasculature, cellular dysplasia and other molecular changes to the epithelium. This embodiment may be particularly apt in preventing HNSCC in subjects with a prior history of (or predisposition to) HNSCC.

In another aspect of the invention there is provided a method for preventing and/or treating HNSCC in a subject which method comprises (or consists essentially of):

(a) administering to said subject an effective amount of a therapeutic agent such as radiation therapy and/or an anti-cancer agent, e.g. an anti-EGFR agent (for example an anti-EGFR antibody such as cetixumab);

(b) administering to said subject an effective amount of the composition of the invention.

In one embodiment of this aspect of the invention, step (b) and step (a) occur concurrently. In another embodiment step (b) occurs after step (a). In another embodiment, step (b) occurs before step (a). In another embodiment of this aspect of the invention, the method further comprises (or further consists essentially of) the step of (c) administering an effective amount of a therapeutic agent e.g. radiation therapy and/or an anti-cancer agent such as an anti-EGFR agent (for example, an anti-EGFR antibody such as cetixumab). In this embodiment, step (b) may occur after step (a) and before step (c). Therefore, an effective amount of the composition of the invention may be administered between cycles of treatment with other therapeutic agents, particularly radiation and/or anti-cancer agents. The therapeutic agents of step (a) and step (c) need not necessarily be the same. Where the therapeutic agents of step (a) and step (c) are the same, the posology followed in step (a) and step (c) need not necessarily be the same.

In another aspect of the invention, an effective amount of the composition of the invention may be used to treat HNSCC in a subject in conjunction with resection and/or ablation of a HNSCC carcinoma. Accordingly, there is provided a method for treating HNSCC in a subject comprising (or consisting essentially of) the steps of:

(a) resecting and/or ablating at least one HNSCC carcinoma;

(b) administering to the subject an effective amount of the composition of the invention. Step (a) may occur either before step (b) or after step (b). In some embodiments of this aspect of the invention, there may be a further step (c) comprising the step of administering an effective amount of a therapeutic agent as described herein. Step (c) may occur after step (a) and before, after or concurrently with step (b). Where step (b) occurs after step (a), it is preferred that the composition of the invention is administered to the resected/ablated region of the subject.

In some embodiments of this aspect of the invention, an effective amount of PEG may be administered at the site(s) and local region of the surgical resection/ablation e.g. locoregional administration. In some embodiments of the invention, an effective amount of the composition of the invention may be administered (e.g. locoregionally administered) to a lesion of the head and neck suspected of being afflicted with HNSCC. The present invention further provides methods for the multi-modal treatment of HNSCC

(that is treatment involving at least two different modalities for treating HNSCC, e.g. surgery, radiotherapy, chemotherapy) the method comprising administering an effective amount of the composition of the invention. In accordance with another embodiment, the present invention provides a method for reducing or inhibiting EGFR expression and/or phosphorylation in the squamous cells of the head and neck of a subject, by administering to the subject an effective amount of the composition of the invention. The dosages and duration of use is dependent on the amount of reduction of EGFR expression or phosphorylation desired. Preferably, in accordance with an embodiment of the present invention, the composition of the invention is administered to the subject at least 1 to 14 days, however PEG can be administered for a longer period, or until the reduction of expression and/or phosphorylation of EGFR in the target tissue is achieved.

In accordance with a further aspect of the invention there is provided a method for reducing the tumor burden (i.e. tumor number and/or volume) in a subject afflicted with HNSCC comprising administering an effective amount of the composition of the invention.

Compositions for use in such as method are also provided. The subject referred to in this specification is preferably a mammal and more preferably human.

Exemplification

Effect of various PEGs on the proliferation of SCC-25 cells. Cell Culture SCC-25 cells (CRL- 1628; American Type Tissue Culture, Rockville, MD, USA) were cultured in DMEM/F-12 media (containing 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate and 1200 mg/L sodium bicarbonate) supplemented with 400 ng/mL of hydrocortisone (Sigma/Aldrich Inc.,St.Louis, MO), 10% v/v fetal bovine serum (FBS), and 0.5% v/v Penicillin/Streptomycin (ATCC).

To assess the effect of PEG, these cells were treated with various concentrations of different molecular weights of PEG, or vehicle (phosphate buffered saline (PBS)), for 24 hours. Cells were then assessed for cell proliferation using the WST-1 cell proliferation assay, as described in more detail below. Test Formulations

Four different molecular weights of PEG were used in the studies:

1. PEG 3350 (catalogue number: P4338, Sigma-Aldrich)

2. PEG 8000 (catalogue number: P4463, Sigma-Aldrich)

3. PEG 10000 (catalogue number: P6667, Sigma-Aldrich)

4. PEG-BPA (catalogue number P2263, Sigma-Aldrich, PEG-BPA is PEG bisphenol A Epichlorohydrin copolymer, PEG 15000-20000).

Solubility Profile:

Before starting the in vitro antiproliferative experiments, maximal solubility of each PEG molecular weight was tested in cell culture media (PBS and DMEM/F12) as shown in Table 1.

Table 1 : Solubility of PEG at various M _w and concentrations in PBS and DMEM/F12 (final volume 5 mL). The concentration marked ^* was the highest tested solubility. Although tested separately, the results for PBS and DMEM-F12 were the same and are represented below as a single set of results. PEG Quantity Cone Molar PBS/ Notes M _w (g) (% w/v) Cone DMEM- (mM) F12

3350 0.5 10 29.85 soluble soluble at 25°C

1.0 20 59.70 soluble soluble at 25°C

2.0 40 119.40 soluble Heated at 37°C

3.0 60 179.10 soluble Heated at 37°C

4.0 80 238.80 soluble ^* Heated at 37°C

5.0 100 298.50 insoluble Very viscous

6.0 120 358.21 insoluble Very viscous

7.0 140 417.91 insoluble Very viscous

8.0 160 477.61 insoluble Very viscous

9.0 180 537.31 insoluble Very viscous

8000 0.5 10 12.5 soluble soluble at 25°C

1.0 20 25.0 soluble soluble at 25°C

1.5 30 37.5 soluble soluble at 37°C

2.0 40 50 soluble* viscous

2.5 50 62.5 insoluble Very viscous

3.0 60 75 insoluble Very viscous

3.5 70 87.5 insoluble Very viscous

4.0 80 100 insoluble Very viscous

4.5 90 112.5 insoluble Very viscous

5.0 100 125 insoluble Very viscous

10000 0.5 10 10 soluble soluble at 25°C

1.0 20 20 soluble soluble at 25°C

1.5 30 30 soluble* soluble at 25°C

2.0 40 40 insoluble High viscosity

2.5 50 50 insoluble High viscosity

3.0 60 60 insoluble Very viscous

3.5 70 70 insoluble Very viscous

4.0 80 80 insoluble Very viscous

4.5 90 90 insoluble Very viscous

5.0 100 100 insoluble Very viscous

15000- 0.1 2 1.14 soluble Heated at 37°C 20000

(PEG- BPA) 0.25 5 2.85 soluble Heated at 37°C

0.4 8 4.57 soluble Heated at 37°C

0.5 10 5.71 soluble* Heated at 37°C

0.75 15 8.57 insoluble Very viscous

1.0 20 11.43 insoluble Very viscous

1.25 25 14.28 insoluble Very viscous

1.5 30 17.14 insoluble Very viscous

1.75 35 20.0 insoluble Very viscous

2.0 40 22.86 insoluble Very viscous

Based on the data from Table 1, the following doses concentrations for each formulation molecular weight were tested for their in vitro antiproliferative activity (Table 2). Table 2:

Cell Proliferation Assay (WST-1)

Cell proliferation was assessed by measuring the cleavage of the tetrazolium salt WST-1 (4- [3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-l, 3-benzene disulfonate) to formosan according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN, USA). Briefly, cells were grown in 96 well plates in a final volume of 100 μΕ and then incubated with 10 μΕ of the WST-1 reagent at 37 °C for 30 minutes in a humidified 5% C0 ₂ incubator. Conversion of tetrazolium salt into formosan was determined spectrophotometrically at 440 nm absorbance (Molecular Devices, Sunnyvale, CA, USA).

Study Design:

To determine the comparative effect of different PEG molecular weights (M _w ) on cellular proliferation, 12.5 xlO ³ human HNSCC squamous carcinoma cells (SCC-25) were seeded in 96 well plates in a volume of 100 μΕ culture medium and incubated in a humidified 5% C0 ₂ incubator at 37°C for 24 hours. The cells (70-80% confluent) were then treated with different molecular weights (M _w ) of PEG [-3350, -8000, -10000 and PEG-BPA] using different concentrations based on their solubility profiles (Tables 1 and 2). The incubations were carried out in a humidified 5% C0 ₂ incubator at 37°C for 24 hours. The cellular proliferation (changes in cell number) was assayed using the WST- 1 assay kit as described above.

Each plate contained duplicate wells and in each experiment duplicate plates were included. 6 separate experiments were performed (n=6) on different days. However, experiment 1 (plate replicate A and B) and experiment 2 (plate replicate A) were considered outlier data and not included in the analysis. Therefore data for n=5 experiments are presented here.

Statistical Analysis:

Duplicate well and plate data were meaned and one-way analysis of variance (ANOVA) performed on the data using Minitab 16 (Minitab Ltd., Coventry, UK). Data was considered statistically significant when p<0.05 compared to 0 mM PEG.

Results:

The results present the effective concentration range of each PEG formulation molecular weight and identifies specific concentration that causes maximum inhibition of cellular proliferation in SCC-25 cells.

1 Dose course studies of PEG-3350 on cellular proliferation in SCC-25 cells (Figure 1). These studies data show that a maximal inhibition of proliferation of -88% was achieved at 50 mM PEG-3350 concentration.

2 Dose course studies of PEG-8000 on cellular proliferation in SCC-25 cells (Figure 2). These studies data show that a maximal inhibition of proliferation of -77% was achieved at 25 mM PEG-8000 concentration. 3 Dose course studies of PEG- 10, 000 on cellular proliferation in SCC-25 cells (Figure 3).

These studies data show that a maximal inhibition of proliferation of -82% was achieved at 30 mM PEG-10000 concentration.

4 Dose course studies of PEG-BPA on cellular proliferation in SCC-25 cells (Figure 4). These studies data show that a maximal inhibition of proliferation of -26% was achieved at 6 mM PEG-BPA concentration. Effect of various PEGs on proliferation of SCC-9 cells.

Cell Culture

SCC-9 cells (CRL-1629; ATCC), a human HNSCC cell line, were cultured in DMEM/F12 (1:1) medium (Thermo Scientific, Rockford, IL) supplemented with 10% FBS (Life

Technologies, Carlsbad, CA). The cells were cultured at 37°C and 5% C0 ₂ in a humidified incubator.

Test Formulations

Three different molecular weights of PEG were used in the studies:

1. PEG 3,350 (catalogue number: P4338, Sigma-Aldrich)

2. PEG 8,000 (catalogue number: P4463, Sigma-Aldrich)

3. PEG 10,000 (catalogue number: P6667, Sigma-Aldrich) Cell Proliferation Assay

Healthy cell number was assessed using the alamarBlue reagent (Invitrogen, Carlsbad, CA) and quantification of the metabolic cleavage from non-fluorescent resorufin to the fluorescent resorufin, following manufacturer's instruction. Briefly, cells were incubated with 12.5 μΕ alamarBlue reagent (10% [v/v]) for 20 minutes (24 hour PEG treatment) or 60 minutes (72 hour PEG treatment) at 37°C and 5% C0 ₂ in a humidified incubator. Fluorescence was determined by excitation at 540 nm and emission at 590 nm using a TECAN infinite M200 instrument (Tecan Group Ltd., Mannedorf, CH).

Study Design To assess the effect of PEG, cells were seeded in 96-well clear bottom black plates (Corning Lifesciences, Tewkesbury, MA) at 5000 cells per well in 100 μΕ growth medium. Cells were cultured overnight at 37°C, 5% C0 ₂ in a humidified incubator. Cells were incubated with different PEG molecules (PEG-3350, -8000 and -10000) at concentrations as indicated, or with 10 μΜ doxorubicin (Sigma- Aldrich) as a positive control (maximum effect), final volume 125 μΕ. Cells were incubated with the PEG molecules in triplicate for 24 hours or in singlicate for 72 hours and the experiments repeated for n=3.

Analysis

Concentration-response curves and associated IC ₅₀ values were calculated using GraphPad Prism (GraphPad Software, La Jolla, CA). See Figures 5 and 6. IC ₅₀ values and percentage effect values are quoted as the mean of n=3 experiments, + standard error of the mean (SEM).

Results

The results demonstrate formal potency (IC ₅₀ ) values for PEG-3350, -8000 and -10,000 in SCC-9 cells, with potencies of 30.3 (+ 6.0), 11.0 (+ 1.5) and 6.9 mM (+ 0.8), respectively, after 24 hours incubation (Figure 5). Maximum effects for PEG-3350, -8000 and -10,000 were all 106% of the effect induced with 10 μΜ doxorubicin. Furthermore, when the PEG molecules were incubated with SCC-9 cells for longer (72 hours incubation - Figure 6), the potencies remained broadly similar (PEG-3350, -8000 and -10,000 IC ₅₀ values of 25.7 [+ 7.0], 7.3 [+ 2.7] and 4.7 mM [+ 1.9], respectively), but superior efficacy was observed in comparison to 10 μΜ doxorubicin (PEG-3350, -8000 and -10,000 was 123, 124 and 124%, respectively).

Summary/Conclusions :

As discussed above, the goals of the current project were to further characterize the antiproliferative effect of PEG against the development of head and neck cancer (HNSCC). The study focus was to define PEG formulations (molecular weights) and concentrations that were maximally effective in causing an anti-proliferative effect in an in vitro cell culture model.

As shown in the results, except for PEG-BPA, all other PEGs were able to achieve a maximal inhibitory effect of -70-90% within the range of the concentrations studied, which translated to >100% of the inhibitory effect observed with 10 μΜ doxorubicin in the SCC-9 cells. However, the data shows that the molar potency of PEG 8000 and PEG-10,000 was superior compared to PEG-3350 (maximal inhibition of -80% can be achieved at lower dose for PEG- 8000 [25 mM] and/or PEG 10,000 [30 mM] compared to PEG- 3350 [50 mM] in SCC-25 cells and this rank order in potency was confirmed in formal IC ₅₀ studies in SCC-9 cells). However, it should be noted that on a gram-for-gram basis the potency of PEG-3350, PEG- 8000 or PEG-10,000 were broadly comparable. For PEG-BPA, the max concentration was limited by the solubility of this molecule - if a higher concentration could have been included it is possible that an increased inhibition would have been observed.

We therefore conclude that PEG-3350 (50 mM) PEG-8000 (25 mM) or PEG- 10,000 (30 mM) are all broadly equi-efficacious in inhibiting proliferation of SCC-25 and SCC-9 cells (HNSCC cell lines).