Cross Reference to Related Applications

This application claims the benefit of provisional application AU 2021903191 and application PCT/AU2021/050282 the entire disclosures of which are incorporated herein by reference.

Field of the invention

The invention relates to cancer therapy, especially to cytotoxic agents and chemo-sensitizing and radio-sensitising agents, particularly isoflavonoids, and to improving the bioavailability of same.

Background of the invention

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Plant-derived phenolic isoflavonoids have been the subject of considerable scientific research since the late-1980s. Many of these compounds have auxin or hormonal functions in plants and also display biological activities in human tissues. One of the most extensively studied plant isoflavones is genistein, remarkable for its pleiotropic actions across carcinogenesis, inflammation, cardiovascular function, and insulin resistance.

The anti-cancer activities of genistein appear to stem in part from its ability to block the phosphorylation of protein tyrosine kinases, resulting in mitotic arrest, terminal differentiation, and apoptosis of human cancer cells. Genistein also is anti-angiogenic. The anti-cancer effects of genistein also extend to epigenetic modifications of cancer cells through modulation of DNA methylation, miRNA-mediated regulation and histone modifications and to inhibition of proteasome activity. Importantly, isoflavonoids have been found to be useful as cytotoxic agents, and as sensitising agents for sensitising cancer cells to cytotoxic signals from chemical or radiation insult. Some have also been shown to reverse chemo-resistance.

Despite these potentially valuable therapeutic opportunities of genistein in particular and a wide range of other related plant isoflavonoids in general, those opportunities have failed to date to be translated into the clinic. There are a number of reasons for this. One is that their biological functions are not sufficiently potent to be drug-like. Another is that there is a question as to their susceptibility to various degrees to Phase 1 and Phase 2 metabolic processes with resulting decrease in potency and bio-availability, although the extent to which these process influence therapeutic potential has not been completely understood.

Some have attempted to address these deficiencies through the synthesis of analogues of the naturally-occurring isoflavonoids by creating new chemical entities with greater biological potency and/or being less susceptible to metabolic processes.

Idronoxil is an analogue of genistein. Idronoxil (phenoxodiol; dehydroequol; Haginin E (2H-1-Benzopyran-7-0,1,3-(4-hydroxyphenyl) is about 10x more potent as an anti-cancer agent compared to genistein, inducing cytostasis and cytotoxicity in a wide range of cancer cell types. Its biological effects include inducing apoptosis, cell cycle arrest, inhibition of angiogenesis, immune modulation and neuro-protection.

Idronoxil has proved to have better drug-like qualities compared to its parent isoflavone compound, genistein, particularly in having greater in vitro anti-cancer activity and in not being particularly susceptible to Phase 1 metabolic processes. However, idronoxil, in common with members of the isoflavone family, is likely susceptible to Phase 2 metabolic processes, and it is this phenomenon that is believed to account for the lack of meaningful clinical efficacy observed with this family of compounds to some extent.

Isoflavonoid molecules are highly insoluble in water. In common with other water- insoluble xenobiotics as well as water-insoluble internal hormones (steroidal hormones, thyroxine) and bile acids, the body seeks to convert these compounds into a water- soluble form that is excretable via the kidneys. Excretion can occur via the bile, but the rate of biliary excretion is slow compared to urinary excretion, leading the body to seek to convert as much of the xenobiotic into a water-soluble form that is possible.

Compounds such as idronoxil with an underlying phenolic structure share this feature with other phenolic drugs (eg. propofol, paracetamol, naloxone).

One mode of detoxification may involve a family of UDP-glucuronyl transferase enzymes that attach the xenobiotic to the sugar, glucuronic acid, to produce a water- soluble glucuronide conjugate. A secondary, less common detoxification process involves sulfotransferase enzyme activity that yields a water-soluble sulfated conjugate.

These two families of detoxifying enzymes are located principally in the liver and the gut mucosa.

Orally administered idronoxil is completely converted into water-soluble conjugates as a combined effect of transferase activity in the gut mucosa and first-pass liver metabolism; intravenously administered idronoxil also is completely conjugated, with a low level of unconjugated drug being drug retained within the cyclodextrin carrier.

The rate of conjugation has a significant impact on the bio-availability of isoflavonoid drugs to target tissue. Isoflavonoid glucuronyl and sulfate conjugates lack anti-cancer activity in vitro and require the action of glucuronidase and sulphatase enzymes to liberate the active drug candidate.

Most normal tissues generally express relatively high levels of glucuronidase and sulfatase activities, whereas tumour tissues are far more variable in their expression. That is, conjugated isoflavonoid drugs, as well as the broader family of phenolic drugs, are bio-available to healthy tissues because they possess the ability to deconjugate the drug, whereas their bio-availability to cancer tissue is far less certain and in some cases, non-existent.

It is even more concerning that some cancers express elevated levels of glucuronosyltransferases, which might explain the insensitivity of colo-rectal cancer cell lines (eg. HT-29, CaCo-2) to idronoxil. In this setting it could be seen that the administration of isoflavonoids such as idronoxil in the oral and intravenous dosage formulations used to date, with their high levels of exposure of those isoflavonoids to Phase 2 metabolic processes, would be disadvantageous to the bio-availability of those compounds to the target tumour tissue.

Various xenobiotic detoxification systems have been observed throughout the gastro intestinal tract. The observations have been of interest and relevant to the hypothesis of a relationship between high rates of carcinogenesis in mucosa having lower detoxification potential. In more recent studies, the distribution and operability of detoxification systems has been observed to be highly dynamic, variable and complex.

Given hydrophobicity, where trialled previously, the overwhelming approach to the delivery of isoflavonoids has been to make them less hydrophobic, that is so as to improve solubility in body fluids, in an attempt to deliver them to blood and to decrease the likelihood of detoxification and excretion. Examples of these formulations include PEG and cyclodextrin conjugates.

To date a significant number of clinical trials involving isoflavonoids have been undertaken. None of these trials have demonstrated consistent efficacy. For example, Idronoxil has held an IND from the US FDA since about 2000 in both oral and intravenous dosage formulations and in that form has undergone over 12 Phase 1, Phase 2 and Phase 3 clinical studies in over 300 patients with late-stage cancers. Instances of clinical response (complete response, partial response, stable disease) have been observed, but neither dosage formulation has delivered a consistent, clinically meaningful anti-cancer effect.

The mechanism(s) underpinning that lack of consistent efficacy are not completely understood. A lack of bioavailability is clearly a concern but there is an absence of a complete understanding of the mechanisms underpinning the limited bioavailability. Confounding the point is that, quite apart from conjugation, some isoflavonoid drugs are metabolized in the liver into inactive metabolites.

To date, idronoxil has not obtained a marketing approval. In fact, the two last clinical studies involving isoflavonoids have failed to provide any evidence of clinical benefit. One study involving idronoxil was a Phase 3 study in patients with late-stage ovarian cancer that was abandoned following the recruitment of 142 women because of lack of any clinical benefit. Another study in patients with late-stage cancers and involving a compound known as ME-143 (4,4'-(7-hydroxychroman-3,4-diyl)diphenol) also failed to show any clinical benefit and its clinical development was halted by its owners.

Given the amount of effort over the last 25 years that has gone into the clinical development of isoflavonoids as human therapeutics based on their potent anti-tumour effects in pre-clinical studies, it is remarkable that no isoflavonoid has come to market, perhaps pointing to a general acceptance of the research community that the clinical efficacy of isoflavonoids is limited by the inherent hydrophobicity of the molecules.

There remains a need for new and/or improved cancer therapies.

Summary of the invention The invention seeks to address some of the above discussed needs or limitations and in one aspect provides a composition including:

- a lipophilic base for use in a device for rectal, vaginal or urethral application;

- a surfactant;

- a compound of general formula (I) wherein

R ¹ is H, C1-10 alkyl, C1-10 haloalkyl, OH, OR ^A or 0C(0)R ^A where R ^A is C1-10 alkyl, C _1-10 haloalkyl or an amino acid;

R ² is H, OH, or R ^B where R ^B is an amino acid or COR ^A where R ^A is as previously defined; R ³ is H, halo or CMO alkyl.

A and B together with the atoms between them form a six membered ring selected from the group wherein

R ⁴ is H, COR ^D where R ^D is H, OH, C1-10 alkyl or an amino acid, C0 ₂ R ^c where R ^c is C1-10 alkyl, COR ^E where R ^E is H, Cno alkyl or an amino acid, COOH, COR ^c where R ^c is as previously defined, or CONHR ^E where R ^E is as previously defined;

R ⁵ is H, C0 ₂ R ^C where R ^c is as previously defined, or COR ^c OR ^E where R ^c and R ^E are as previously defined, and where the two Rs groups are attached to the same group they are the same or different;

R ⁶ is H, C0 ₂ R ^C where R ^c is as previously defined, COR ^c OR ^E where R ^c and R ^E are as previously defined, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; X is O, N or S;

Y is selected from the group wherein R ₇ is H, or C _1-10 alkyl; C _1-10 haloalkyl, halo, OR ^F where R ^F is H, C _1-10 alkyl, C _1-10 haloalkyl, or 0C(0)R ^A where R ^A is as previously defined;

R ⁸ is H, halo or COR _D where RD is as previously defined; and “ ===== ” represents either a single bond or a double bond.

Typically the compound of Formula I is:

In another aspect, the invention provides a composition including:

- a lipophilic base for use in a device for rectal, vaginal or urethral application; - a surfactant

- a compound of general formula (II) wherein

Ri is H, or R _A CO where R _A is C _1-10 alkyl or an amino acid; R ₂ is H, OH, or RB where RB is an amino acid or CORA where RA is as previously defined;

A and B together with the atoms between them form the group: wherein

R ₄ is H, CORD where RD is H, OH, C _1-10 alkyl or an amino acid, CO ₂ R _C where R ^c is C _1-10 alkyl, , CORE where RE is H, C _1-10 alkyl or an amino acid, COOH, CORc where Rc is as previously defined, or CONHRE where RE is as previously defined;

R ₅ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;

X is O, N or S;

Y is where R ₇ is H, or C _1-10 alkyl; and

“ == ” represents either a single bond or a double bond.

Preferably the lipophilic base is an esterified oil or an oil fraction or derivative or fat or synthetic version thereof. More preferably the lipophilic base has a mono, di and/or triglyceride profile substantially the same as, or identical to the profile of Suppocire CM.

The surfactant promotes the absorption of the compound in vivo , eg via the rectum. Typically the surfactant is a non-ionic surfactant. Preferably, the surfactant is a polyethylene glycol stereate, eg distereate and/or monostearate, more preferably the surfactant is polyethylene glycol 8-monosterate.

In another aspect there is provided a suppository pessary or intra- urethra I device formed from a composition described above.

In another aspect there is provided a method of treating or preventing cancer, comprising administering to a person in need thereof a suppository, pessary or intra- urethral device described above.

In another aspect there is provided a use of a composition described above in the preparation of a suppository, pessary or intra-urethral device for the prevention and/or treatment of cancer.

In another aspect there is provided a suppository, pessary, or intra-urethral device formed from a composition described above for use in preventing or treating cancer.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Concentration profile of Idronoxil in rat plasma a formulation of Idronoxil administered as a solid suppository with surfactant (formulation of Example 1A) and without surfactant (formulation of Example 1B).

Figure 2. Concentration profile of Idronoxil in rat plasma a formulation of Idronoxil administered as a solid suppository with MBK formulation (circles, formulation of Example 1D) and with Suppocire CM and Mryj8 formulation (squares, formulation of Example 1A). Figure 3. Schematic of the Caco-2 cell monolayer permeability assay set up.

Figure 4. Graph of the SAXS results showing structures formed by suppository formulations of idronoxil with surfactants TPGS (top line), Myrj S8 (middle line) and TPGS (bottom line).

Figure 5. Schematic of the experimental set up of the capillaries in the SAXS/WAXS beamline.

Figure 6. Graph of the SAXS scattering curves for the initial y-scan of the Tween 80 suppository containing idronoxil.

Figure 7. Graph of the SAXS scattering curves for the suppository formulation containing Tween 80 + idronoxil over 30 min upon addition of SRF.

Figure 8. Graph of the SAXS scattering patterns of the suppository formulated with Myrj S8 + idronoxil (NexGen formulation) over 30 min upon addition of SRF.

Figure 9. Graph of the SAXS scattering patterns of the suppository formulation containing Kolliphor EL + idronoxil over 30 min upon addition of SRF.

Figure 10. Graph of the SAXS scattering patterns of the suppository formulation with TPGS + idronoxil over 30 min upon addition of SRF.

Figure 11. Graph showing the concentration of idronoxil in the lipid and water phases after dispersal in SRF.

Figure 12. CryoSEM image of the placebo in SRF after 20 minutes of mixing. The overview of the imaging area is shown in (a). The two insets (b) and (c) on the right show the features at higher magnification. The scale bars represent 100 pm.

Figure 13. CryoSEM of the formulation 1A in SRF showing (a) and (b) the sample frozen 5 min after mixing and (c) 45 min after mixing. Scale bars represent 250 pm.

Figure 14. CryoSEM pictures with cathodeluminscience (CL-SEM) of (a) the placebo equilibrated in SRF and (b) formulation of idronoxil, Myrj S8 and Suppocire equilibrated in SRF. Figure 15. CryoSEM images of suppositories of the formulation 1A mixed with simulated rectal fluid with a scale bar of 500 pm after left 5 minutes and right 22 minutes of mixing.

Figure 16. CryoSEM images of suppositories formulated with Kolliphor EL surfactant with a scale bars of 100 pm imaged after (left) 15 minutes and (right) 45 minutes of mixing.

Figure 17. CryoSEM images of suppositories formulated with Kolliphor EL surfactant with a scale bars of 1 mm imaged after (left) 15 minutes and (right) 45 minutes of mixing

Figure 18. CryoSEM images of suppositories formulated with TPGS surfactant with a scale bars of 500 pm imaged after (left) 6 minutes and (right) 30 minutes of mixing.

Figure 19. CryoSEM images of suppositories formulated with TPGS surfactant with a scale bars of 1 mm imaged after (left) 6 minutes and (right) 30 minutes of mixing.

Figure 20. CryoSEM images of suppositories formulated with Tween 80 surfactant with a scale bars of 500 pm imaged after (left) 7 minutes and (right) 29 minutes of mixing.

Figure 21. CryoSEM images of suppositories formulated with Tween 80 surfactant with a scale bars of 1 mm imaged after (left) 7 minutes and (right) 29 minutes of mixing.

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text. All of these different combinations constitute various alternative aspects of the invention.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Various features of the invention are described with reference to a certain value, or range of values. These values are intended to relate to the results of the various appropriate measurement techniques, and therefore should be interpreted as including a margin of error inherent in any particular measurement technique. Some of the values referred to herein are denoted by the term “about” to at least in part account for this variability. The term “about”, when used to describe a value, may mean an amount within ±25%, ±10%, ±5%, ±1% or ±0.1% of that value.

The term “and/or” can mean “and” or “or”.

The term “(s)” following a noun contemplates the singular or plural form, or both.

Unless the context requires otherwise, all percentages referred to herein are percentages by weight of the composition.

Unless the context requires otherwise, all amounts referred to herein are intended to be amounts by weight. The use of the term “about” includes and describes the value or parameter per se. For example, “about x” includes and describes “x” perse. In some embodiments, the term “about” when used in association with a measurement, or used to modify a value, a unit, a constant, or a range of values, refers to variations of ±10%. For example, “about 400” in some embodiments includes 360 - 440.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

Similarly, the general chemical terms used in the formulae described herein have their usual meaning.

As described herein, the inventors have sought to improve the clinical efficacy of isoflavonoids, especially isoflavonoids for the treatment of cancer. The inventors have investigated hereto-unexplored approaches for the clinical application of these compounds.

The inventors have recognised that it is possible to obtain an improved sustained release profile of isoflavonoids when a surfactant is present in a lipophilic formulation. In one example, the plasma concentration of an isoflavanoid (e.g. idronoxil) reached a high level, and did so for longer, when released from a lipophilic formulation including a surfactant compared to a lipophilic formulation that did not include a surfactant. In addition, a lipophilic formulation including a surfactant resulted in an advantageous increase in the plasma level of isoflavanoid compared to a lipophilic formulation but without surfactant over longer time scales.

On the basis of the findings described herein, the inventors have recognised the applicability of isoflavonoids for treatment of cancer or sensitisation of cancer cells to chemo- or radiotherapy when given in the form of a formulation having a hydrophobic or lipophilic base.

In one aspect, therefore, the present invention provides a composition including: - a lipophilic base for use in a device for rectal, vaginal or urethral application;

- a compound of general formula (I) or (II), as defined herein; and

- a surfactant.

The compounds of general formula (I) or (II) may be defined as isoflavonoids. A. Isoflavonoids

The isoflavonoids for use in a composition according to the invention described are shown by Formula (I) wherein R ¹ is H, C _1-10 alkyl, C _1-10 haloalkyl, OH, OR ^A or 0C(0)R ^A where R ^A is C _1-10 alkyl,

C _1-10 haloalkyl or an amino acid;

R ² is H, OH, or R ^B where R ^B is an amino acid or COR ^A where R ^A is as previously defined;

R ³ is H, halo or C _MO alkyl. A and B together with the atoms between them form a six membered ring selected from the group

wherein

R ⁴ is H, COR ^D where R ^D is H, OH, C _1-10 alkyl or an amino acid, C02R ^c where R ^c is C _1-10 alkyl, COR ^E where R ^E is H, C _1-10 alkyl or an amino acid, COOH, COR ^c where R ^c is as previously defined, or CONHR ^E where R ^E is as previously defined;

R ₅ is H, C0 ₂ R ^C where R ^c is as previously defined, or COR ^c OR ^E where R ^c and R ^E are as previously defined, and where the two R ⁵ groups are attached to the same group they are the same or different;

R ⁶ is H, C02R ^C where R ^c is as previously defined, COR ^c OR ^E where R ^c and R ^E are as previously defined, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;

X is O, N or S;

Y is selected from the group wherein

R ⁷ is H, C _1-10 alkyl, C _1-10 haloalkyl, halo, OR ^F where R ^F is H, C _1-10 alkyl, C _1-10 haloalkyl, or 0C(0)R ^A where R ^A is as previously defined;

R ⁸ is H, halo or COR _D where R _D is as previously defined; and “ ===== ” represents either a single bond or a double bond.

Preferably, X is O.

In preferred embodiments, the compound of formula (I) is selected from the group consisting of

wherein

Rs is H or CORD where RD is as previously defined;

Rg CO ₂ R _C or CORE where Rc and RE are as previously defined; Rio is CORc or COR _C ORE where Rc and RE are as previously defined;

Rii is H or OH;

Ri2 is H, COOH, CO2R _C where Rc and is as previously defined, or CONHR _E where RE is as previously defined; and “ ===== ” represents either a single bond or a double bond.

Preferably, the compound of Formula (I) is wherein Rn and R ₁₂ are as defined above.

Even more preferably, the compound of Formula (I) is otherwise known as idronoxil (also known as phenoxodiol; dehydroequol; Haginin E (2H-1-Benzopyran-7-0,1,3-(4-hydroxyphenyl)).

In another aspect, the isoflavonoids for use in a composition according to the invention described are shown by Formula (II):

(II) wherein

Ri is H, or RACO where RA is C _1-10 alkyl or an amino acid;

R2 is H, OH, or RB where RB is an amino acid or CORA where RA is as previously defined;

A and B together with the atoms between them form the group: wherein

R ₄ is H, CORD where RD is H, OH, C _1-10 alkyl or an amino acid, CO ₂ R _C where R ^c is C _1-10 alkyl, CORE where RE is H, C _1-10 alkyl or an amino acid, COOH, CORc where Rc is as previously defined, or CONHRE where RE is as previously defined;

R ₅ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;

X is O, N or S;

Y is where R ₇ is H, or C _1-10 alkyl; and

“ ===== ” represents either a single bond or a double bond. In one preferred embodiment, Rs is aryl substituted with an alkoxy group. Preferably, the alkoxy group is methoxy. In another preferred embodiment, Rs is hydroxy.

In preferred embodiments, the compound of formula (II) is

As used herein the term "alkyl" refers to a straight or branched chain hydrocarbon radical having from one to ten carbon atoms, or any range between, i.e. it contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl group is optionally substituted with substituents, multiple degrees of substitution being allowed. Examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, and the like.

As used herein, the term "C1-10 alkyl" refers to an alkyl group, as defined above, containing at least 1, and at most 10 carbon atoms respectively, or any range in between (e.g. alkyl groups containing 2-5 carbon atoms are also within the range of Ci- io).

Preferably the alkyl groups contain from 1 to 5 carbons and more preferably are methyl, ethyl or propyl.

As used herein, the term "aryl" refers to an optionally substituted benzene ring. The aryl group is optionally substituted with substituents, multiple degrees of substitution being allowed.

As used herein, the term "heteroaryl" refers to a monocyclic five, six or seven membered aromatic ring containing one or more nitrogen, sulfur, and/or oxygen heteroatoms, where N-oxides and sulfur oxides and dioxides are permissible heteroatom substitutions and may be optionally substituted with up to three members. Examples of "heteroaryl" groups used herein include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxo- pyridyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazyl, pyrazinyl, pyrimidyl and substituted versions thereof.

A "substituent" as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a "ring substituent" may be a moiety such as a halogen, alkyl group, or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term "substituted," as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterised and tested for biological activity.

The terms "optionally substituted" or “may be substituted” and the like, as used throughout the specification, denotes that the group may or may not be further substituted, with one or more non-hydrogen substituent groups. Suitable chemically viable substituents for a particular functional group will be apparent to those skilled in the art.

Examples of substituents include but are not limited to:

C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy, C ₁ -C ₆ hydroxyalkyl, C ₃ -C ₇ heterocyclyl, C ₃ -C ₇ cycloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylsulfanyl, C ₁ -C ₆ alkylsulfenyl, Cr C6 alkylsulfonyl, C ₁ -C ₆ alkylsulfonylamino, arylsulfonoamino, alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, mercapto, amino, acyl, carboxy, carbamoyl, aminosulfonyl, acyloxy, alkoxycarbonyl, nitro, cyano or halo.

The term "isoflavonoid" as used herein is to be taken broadly and includes isoflavones, isoflavenes, isoflavans, isoflavanones, isoflavanols and similar or related compounds. Some non-limiting examples of isoflavonoid core structures are shown below:

wherein represents either a single bond or a double bond.

Some of the compounds discussed above may be referred to by the names dihydrodaidzein (compound 1 where Rs is H), dihydrogenestein (compounds 2 and 5), tetrahydrodaidzein (compound 8) and equol and dehydroequol (compound 10).

Methods for synthesis of the above described compounds are described in W01 998/008503 and W02005/049008 and references cited therein towards the synthesis, the contents of which are incorporated herein by reference in entirety.

B. Bases for forming suppository, pessary or urethral devices As described herein, the inventors have found that a lipophilic base in combination with a surfactant enable an improved therapeutic effect of an isoflavonoid, compared with lipophilic bases without surfactant.

In the disclosure below, ‘base’ may refer to a substance commonly used as a carrier in a suppository, pessary or intra-urethral device. Generally the base has a solvent power for the isoflavonoid enabling at least partial, preferably complete dispersion of the isoflavonoid in the base.

The base may be comprised of, or consist of an oil or fat. The base may be a blend of oils or fats.

The oil or fat may be esterified with glycerol. The oil or fat may be interesterified. The base may be formed or derived from a hydrogenated oil or fat. The oil or fat may be substantially or completely hydrogenated. The base may be derived from a synthetic oil. The base may derived from a natural or synthetic oil or fat. The base may be substantially comprised of mono-, di-, tri- glycerides, preferably substantially tri glycerides.

The base may be a hard fat. Hard fats can be made from substantially hydrogenated oils through esterification with glycerol, or by interesterification. The oil may be completely hydrogenated, or partially hydrogenated. Hard fats are characterised by their melting point, hydroxyl value and saponification value. Hard fats are typically solid at room temperature. The base may comprise mono, di and/or triglycerides. Preferably the base substantially comprises triglycerides. The base may be derived from natural oils such as canola oil, palm oil, palm kernel oil, soya bean oil, vegetable oil, castor oil, and combinations thereof. Oils derived from these sources may be fractionated to obtain oil fractions containing saturated fatty acids.

The base may be formed from synthetic oils or fats, examples including Fattibase, Wecobee, Witepsol (Dynamit Nobel, Germany), Suppocire (Gattefosse, France), Hydrokote and Dehydag.

A base may comprise a hard fat comprising esterified or non-esterified fatty acid chains. Preferably the base is substantially esterified. Preferably the base consists essentially of esterified fatty acid chains. Most preferably the base is completely esterified. The fatty acid chains may be in the form of mono, di and/or trigycerides, preferably C10-C18 triglycerides. The tri-ester fraction may be predominant.

The triglyceride fraction may comprise about 50%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.9% w/w of the base. The triglyceride fraction may be from about 70%-99.9% w/w of the base. The hard fat may consist substantially of triglycerides. The hard fat may consist essentially of triglycerides.

The base may be completely or substantially insoluble in water.

The base may be a hard fat, and have a defined hydroxyl value. The hydroxyl value of a hard fat is a measure of the context of free hydroxyl groups in a chemical substance, and is typically expressed in units of milligrams of KOH per gram of substance equivalent to the hydroxyl content. The base may have a hydroxyl value of less than 15. The hydroxyl value indicates the amount of mono and/or di glycerides in the base. A hydroxyl value close to 0 indicates the hard fat is comprised essentially of triglycerides. Preferably the base may have a hydroxyl value of less than 5. Preferably the base may have a hydroxyl value of about 5. Preferably the base may have a hydroxyl value of 5. Preferably the base may have a hydroxyl value of close to 0.

The base may be a hard fat and have a defined melting point. The base may have a melting point of approximately body temperature. The base may have a melting point of about 30 to about 40 °C or any range therein, including about 30 °C, about 35 °C, about 37 °C or about 40 °C. The base may have a melting point of about 35 °C to about 40 °C. Preferably the melting point of the base is about 38 °C to about 40 °C. More preferably the melting point of the base is about 38 °C to about 39 °C.

The base has a saponification value. The saponification value of the base is a measure of the number of milligrams of potassium hydroxide required to saponify 1g of the base. The saponification value of the base may be from about 210 to 260.

In one preferred embodiment the base is Suppocire CM. Suppocire CM consists of mono-, di and triglyceride esters of fatty acids, the tri-ester fraction being predominant. Suppocire CM consists substantially of triglycerides. Suppocire CM has a melting point approximately equal to body temperature.

The proportion of the lipophilic suppository base in the final product is a function of the dosage of active pharmaceutical ingredient and the presence of other pharmaceutical or inert ingredient (if any) but may be provided by way of example in an amount of about 1 to 99% w/w formulation.

In some embodiments the formulation comprises at least 40 %, at least 45 %, 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% w/w of base. The formulation may comprise from about 40% to about 95% w/w of base or any range therein. The formulation may comprise from about 60% to about 90% w/w base. The formulation may comprise from about 70% to about 85% w/w base. In preferred embodiments the formulation may comprise about 81.5% w/w base. C. Surfactant

The surfactant used may comprise one or more surfactants.

A surfactant is an amphiphilic molecule, it may be comprised of a head and a tail or it may be comprised of multiple blocks wherein each block has a different hydrophobicity or hydrophilicity. The different solubilities of the parts of the molecule allow the surfactant to preferentially arrange itself at the interface between two phases and lower the surface tension, or interfacial energy.

Preferably the surfactant is non-ionic.

The surfactant has a hydrophobic-lipophilic balance. The hydrophilic-lipophilic balance is also called HLB. Those skilled in the art will be familiar with the HLB. The HLB is an estimation of the relative size and strength of the head and the tail of the surfactant on a scale from 0 to 20.

The HLB of the surfactant may be between about 6 and about 13 or any range therein. The HLB of the surfactant may be 6, 7, 8, 9, 10, 11, 12 or 13. The HLB of the surfactant may be about 6, about 7, about 8, about 9, about 10, about 11, about 12 or about 13. Preferably the HLB of the surfactant may be from about 7 to about 11. More preferably the HLB of the surfactant is about 10. A person skilled in the art will understand that wherein the surfactant comprises two or more surfactants, the surfactants collectively provide the defined HLB range.

The surfactant may show inverse aqueous solubility characteristics with increasing temperature.

The surfactant used may be a polyethylene glycol stearate. The surfactant may be a monosterate, or a distearate or a mixture of both. The surfactant may include free glycols. The average molecular weight of the polymer chain, may be indicated in the name of the specific substance e.g. Polyethylene-glycol-100 stearate is a polyethylene glycol of approximately 100 g/mol attached to a stearic acid. The average number of ethoxylated groups attached in the polymer chain may be indicated in the name of the specific substances, e.g. Polyethylene-glycol-8 is a polymer chain with an average of 8 ethoxy groups. Polyethylene glycol may be abbreviated as PEG. The surfactant may have an average number of PEG groups of about 5 to about 15 or any range within. The surfactant may have an average number of 5 PEG groups, 6 PEG groups, 7 PEG groups, 8 PEG groups, 9 PEG groups, 10 PEG groups, 11 PEG groups, 12 PEG groups, 13 PEG groups, 14 PEG groups, or 15 PEG groups. Preferably the surfactant has 8 PEG groups.

The surfactant may be a fatty acid ethoxylate, such as polyethylene glycol esterified with caprylic acid, capric acid, lauric acid, lyristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid or cerotic acid, or a blend thereof. The surfactant may be a monoester or a di ester or a mixture of mono and di esters. Preferably the surfactant is a stearic ester. In one embodiment the surfactant is Polyglycol-8 stearate. PEG-8-stearate may be a mono-stearate of a distearate, referred to respectively as PEG-8-MS or PEG-8-DS. Polyethylene glycol stearates may be obtained from various commercial sources, in various grades, under various tradenames, for example Cithrol4DS (PEG-8-DS, Croda) and Myrj S8 (PEG-8-MS, Croda).

Preferably the surfactant is PEG-8-MS.

The dispersion of the isoflavone in the lipophilic base with the surfactant advantageously improves the release profile of the isoflavone. The release of the isoflavone from the base is influenced by the surfactant used. The surfactant may be chosen such that the release profile of the active is substantially the same as the release profile obtained with PEG-8-MS.

Without wishing to be bound by theory it is proposed that the surfactant improves absorption of the isoflavone in vivo by forming an emulsion in the presence of water in vivo which aids the transfer of the active across the mucosal membrane. The surfactant aids or improves the drug absorption due to the interaction of the surfactant between the water and the oil phase.

Wthout wishing to be bound by theory it is proposed that in some embodiments the surfactant does not substantially form micelles but rather forms an emulsion structure in the presence of rectal fluid in vivo. The surfactant may be chosen such that it is capable of forming or aiding the formulation of an emulsion with the liquefied lipophilic base and water within the rectum.

In some embodiments the surfactant is a linear surfactant. In some embodiments the surfactant is not a branched surfactant. In some embodiments the surfactant is not an aryl-surfactant. In some embodiments the surfactant is not a polysorbate.

In some embodiments the composition does not comprise an alcohol co surfactant, such as diethylene glycol ethyl ether.

In some embodiments the composition does not include an additional phenolic compound. In some embodiments the composition does not include tocopherol.

The composition may comprise any amount of surfactant effective to improve the transfer of the isoflavone across the mucosal membrane. The proportion of the surfactant in the final product is a function of the dosage of active pharmaceutical ingredient and the presence of other pharmaceutical or inert ingredient (if any). The proportion of the surfactant in the final product may be expressed as a percentage of the base, may be from about 2 to about 50 % w/w of the base or any range therein including about 2%, about 5%, about 8%, about 10%, about 15%, about 20%, about 22%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w/w of the base. The proportion of the surfactant in the final product expressed as a percentage of the base, may be from 2 to 50% w/w of the base or any range therein including 2%, 5%, 8%, 10%, 15%, 20%, 22%, 25%, 30%, 35%, 40%, 45%, or about 50% w/w of the base. For example, a formulation comprising 81.5 % w/w base and 18.5% w/w active, wherein the base comprises 50% surfactant, comprise 18.5 % w/w active, 40.75 % w/w surfactant and 40.75 % w/w base. Alternatively, each component of the formulation may be expressed as a percentage of the weight of the total formulation. For example, a formulation comprising 18.5 % w/w active, 40.75 % w/w surfactant and 40.75 % w/w base.

In one embodiment the surfactant used is PEG-8-MS. The proportion of the PEG- 8-MS in the base is from about 5% to about 20%. Preferably the proportion is about 8%. Preferably the proportion is 8%. In another embodiment the surfactant used is PEG-8-DS. When the surfactant used is PEG-8-DS the proportion of the surfactant in the base is from about 15% to about 50%, preferably about 22%. Preferably the proportion is 22%. In another embodiment the proportion of surfactant in the base is preferably about 50%.

In another preferred embodiment, the composition provides an apparent permeability coefficient (P _a A-B) of greater than 25 or 26 or 27 of a compound of formula (I) across a cell monolayer from an apical chamber to a basolateral chamber as measured in a Caco-2 permeability assay. Preferably, the apparent permeability is measured after approximately 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes.

CaCo-2 permeability assays have been used to predict intestinal absorption with a good correlation between apparent permeability (P _app A-B), and in-vivo absorption in the intestine. Skolnik, S et al; Journal of Pharmaceutical Sciences, Vol 99 No. 1, July 2010. Low apparent permeability correlates with low intestinal absorption. Accordingly, it is proposed that higher apparent permeability is predictive of better intestinal absorption.

D. Manufacture

The isoflavonoid suppository, pessary and devices for urethral application of the invention may be prepared as follows. The isoflavonoid is contacted with a suppository base (as described above) in molten form in conditions enabling at least partial, preferably complete or substantially complete dispersion of the isoflavonoid in the base. This solution is then poured into a suitable mould, such as a PVC, polyethylene, or aluminium mould. For example, the isoflavonoid may be contacted with the base at a temperature of from about 35° C to about 60° C and preferably from about 55° C to about 60° C. Preferably the isoflavonoid is contacted with the base at a temperature of 20° C above the melting point of the base. The isoflavonoid can be milled or sieved prior to contact with the base.

It will be understood that the method for manufacture of the formulation and devices formed from same of the invention require a dispersion of the isoflavonoid in the suppository base so that the isoflavonoid is at least partially dispersed therein. In one embodiment, the conditions provided for manufacture, and formulation or device formed from same, enable at least, or provide at least, 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90%, preferably 95% of the isoflavonoid for a given dosage unit to be dispersed in the dosage unit. In these embodiments, no more than 50% of the isoflavonoid for a given dosage unit, preferably no more than 40%, preferably no more than 30%, preferably no more than 20%, preferably no more than 10%, preferably no more than 5% of isoflavonoid for a given dosage unit may be in admixture with, (i.e. undispersed in) the suppository base of the dosage unit.

In a preferred embodiment the base used for the manufacture of the suppository is a hard fat, and is solid at room temperature. The hard fat is melted to a maximum temperature of approximately 20°C above the melting temperature of the fat. The melting temperature of the fat is preferably body temperature. The temperature is controlled during the manufacturing process such that the base is kept at the lowest possible temperature when the active and surfactant is introduced. Following introduction and stirring of the active the composition is cooled to the maximum possible cooling temperature. The control of the temperature advantageously reducing the post hardening of the suppository. Control of the temperature of the manufacturing process also increases homogeneous distribution of the active within the base. Overheating of the hard fat base modifies the crystal structure of the fat which also impacts the distribution of active and potential release profile of the active from the composition.

The manufactured suppository may be in a glassy solid state or a semi-crystalline state, preferably glassy solid state. The solid form of the base may be characterised by DSC or XRD.

In a preferred embodiment, all of the isoflavonoid added to a dosage unit is dispersed in the base. The dosage unit may contain the isoflavonoid from about 0.1 to about 50% w/w. The dosage unit may contain the isoflavonoid from about 5% about 6% about 7% about 8% about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about

21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about

28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about

35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about

42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about

49%, about 50% w/w. Preferably the dosage unit may be from about 15 to about 20% w/w or any range therein. The dosage unit may contain the isoflavonoid from about 15.5%, about 16.5%, about 17.5% about 18.5%, or about 19.5% w/w. In this embodiment, no isoflavonoid is left in admixture with the suppository base. This is believed to increase the likelihood of the uptake of all of the isoflavonoid given in the dosage unit.

It will be understood that the objective of the manufacture process is not to admix, or to mingle, or to blend the suppository base with the isoflavonoid as generally occurs in pharmacy practice of admixing components, as it is believed that the resulting admixture would have a lower likelihood of providing therapeutic benefit. In this context, it is particularly important that any other excipient, carrier or other pharmaceutical active does not adversely interfere with the dispersion of the isoflavonoid in the base, for example as may occur if the isoflavonoid forms a complex with a charged molecular species (other pharmaceutical active, carrier or excipient), the result of which would be to decrease the propensity of the complex, and therefore the isoflavonoid contained in it, to disperse in the suppository base.

Optionally the suppositories, pessaries or intra-urethral devices may be coated, prior to packing, for example with cetyl alcohol, macrogol or polyvinyl alcohol and polysorbates to increase disintegration time or lubrication or to reduce adhesion on storage.

One or more sample suppositories, pessaries, or intra-urethral devices from each batch produced are preferably tested by a dissolution method for quality control, for example a method as described in Example 5. Preferably, the one or more sample suppositories, pessaries, or intra-urethral devices exhibits a dissolution profile that is comparable to the MBK formulation of Example 1D. Alternatively, a sample from each batch is tested in vitro, preferably using a method described herein, to determine whether greater than 75% of the isoflavonoid dissolves from the suppository, pessary, or intra-urethral device within 6.5 hours.

Typically the suppository, or pessary device according to the invention is substantially hydrophobic or lipophilic throughout and does not contain a hydrophilic substance such as hydrophilic carrier or pharmaceutical active, or hydrophilic foci or region formed from the ligation or complexing of the isoflavonoid to or with another pharmaceutical compound, carrier or excipient.

Preferably the formulation for forming the suppository, pessary and devices for urethral application does not include a further pharmaceutical active, cytotoxic or chemotherapeutic agent. In this embodiment, the only active is the isoflavonoid and the formulation does not include a platin, taxane or other cytotoxic or chemotherapeutic agent.

E. Physical characteristics

The total weight of the suppository preferably ranges from about 2000 to about 3500 g and more preferably from about 2200 to about 3300 mg. According to one embodiment, the suppository has a total weight ranging from about 2200 mg to about 3300 mg.

The suppository or pessary is preferably smooth torpedo-shaped.

The melting point of the suppository or pessary is generally sufficient to melt in the patient's body, and is typically no more than about 37° C.

In one particularly preferred embodiment there is provided:

- a kit including: a plurality of suppositories sufficient in number to provide an individual with a suppository once daily, or twice daily, for a period of 30 to 90 days, preferably 30 to 60 days, preferably 30 days each suppository including:

400mg or 600 mg of idronoxil; a suppository base in the form of an oil or fat; wherein the suppository base is provided in an amount of 1-99% w/w of the suppository,

- the kit further including : written instructions to provide the suppository once daily, or twice daily for a period of 30 to 90 days, preferably 30 to 60 days, preferably 30 days, preferably for use in treatment of cancer, more preferably for sensitising cancer cells to cytotoxic effect of a chemo- or radiotherapy, preferably where the cancer is prostate cancer.

F. Methods of treatment

The formulations according to the invention in suppository, pessary, or intra- urethral device form are useful for improving the bioavailability of isoflavonoids in a range of therapeutic applications.

In one particularly preferred embodiment, the formulations are useful for treatment of cancer, whereby the isoflavonoid is used as a cytotoxic monotherapy, or as a chemo-sensitising agent for another cytotoxic molecule.

Thus in one embodiment there is provided a method of treating or preventing cancer in an individual, including administering to a person in need thereof a suppository, pessary or intra- urethral device formed from a formulation according to the invention.

In one embodiment there is provided a use of a formulation according to the invention in the preparation of a suppository, pessary or intra- urethral device for the prevention or treatment of cancer.

In another embodiment there is provided a suppository, pessary or intra- urethral device formed from a formulation according to the invention for use in preventing or treating cancer.

Methods for applying a suppository are well known in the art. Generally the methods involve inserting the suppository to a point aligned with the inferior and medial haemorrhoid veins, thereby enabling the release of the drug to the inferior vena cave.

Methods for applying a pessary, or for urethral application of a pharmaceutically active ingredient are well known in the art. 'Treatment' generally refers to both therapeutic treatment and prophylactic or preventative measures.

Subjects requiring treatment include those already having a benign, pre- cancerous, or non-metastatic tumor as well as those in which the occurrence or recurrence of cancer is to be prevented.

The objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e. , slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.

Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and/or quality of life.

In one embodiment, the method is particularly useful for delaying disease progression.

In one embodiment, the method is particularly useful for extending survival of the human, including overall survival as well as progression free survival.

In one embodiment, the method is particularly useful for providing a complete response to therapy whereby all signs of cancer in response to treatment have disappeared. This does not always mean the cancer has been cured.

In one embodiment, the method is particularly useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.

"Pre -cancerous" or "pre -neoplasia" generally refers to a condition or a growth that typically precedes or develops into a cancer. A "pre -cancerous" growth may have cells that are characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by markers of cell cycle.

In one embodiment, the cancer is pre-cancerous or pre -neoplastic. In one embodiment, the cancer is a secondary cancer or metastases. The secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain.

Other examples of cancer include blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SGLG), non-small cell lung cancer (NSGLG), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, salivary gland carcinoma, kidney or renal cancer, prostate cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophagael cancer, tumors of the biliary tract, as well as head and neck cancer.

"A condition or symptom associated" with the cancer may be any pathology that arises as a consequence of, preceding, or proceeding from the cancer. For example, where the cancer is a skin cancer, the condition or relevant symptom may be microbial infection. Where the cancer is a secondary tumor, the condition or symptom may relate to organ dysfunction of the relevant organ having tumor metastases. In one embodiment, the methods of treatment described herein are for the minimisation or treatment of a condition or symptom in an individual that is associated with a cancer in the individual.

In the above described embodiments, the formulation according to the invention may be useful for preventing doubling time of the cancer cells or otherwise inhibiting tumour growth, either through cytotoxic effect on the tumour cells or otherwise by generally inhibiting cell replication. In these embodiments it will be understood that the suppository formulation provides an anti neoplastic “monotherapy” effect. In another embodiment, the method of treatment described above further includes the step of administering cytotoxic chemotherapy or radiotherapy to the individual.

In yet another embodiment there is provided a method of sensitising a cancer to chemo or radiotherapy including the steps of:

- providing an individual having a cancer in need of chemo or radiotherapy;

- administering to the individual a suppository, pessary or intra- urethral device formed from a formulation according to the invention;

- administering chemo or radio-therapy to the individual.

In another embodiment, the treatment provides for sensitisation of the tumour to radiotherapy, especially stereotactic radiotherapy. In one embodiment the treatment may provide for a reduction in tumour size utilising a sub-optimal radiation dose. It will be understood that a suboptimal radiation dose is one incapable of reducing tumour size in the absence of isoflavonoid formulation treatment.

In another embodiment, the treatment provides for sensitisation of the tumour to chemotherapy. In one embodiment, the treatment provides for a reduction in tumour size utilising a sub-optimal chemotherapy dose. It will be understood that a suboptimal chemotherapy dose is one incapable of reducing tumour size in the absence of isoflavonoid formulation treatment.

In one embodiment, the isoflavonoid formulaton treatment is provided either as a cytotoxic monotherapy, or as a radio or chemosensitising therapy according to a variable dosing regime, prior to, or at the time of radio or chemotherapy. The variable dosing regime may include an increasing dose of isoflavonoid treatment during a run in period prior to radio or chemotherapy and/or an increasing dose during radio or chemotherapy. In one example, the isoflavonoid is provided in a dose of about 600 mg once daily for a period of 1 to 2 weeks and increased to 800 mg once daily for a period of 1 to 2 weeks or 1 month or longer, and further increased to 1600 mg (2x800 mg) once daily for a period of 1 to 2 weeks or 1 month or longer. Actual amounts will be influenced by disease status, age, weight, gender and other pharmacologically relevant variables. The isoflavanoid may be administered in an amount of 400-800 mg.

In one embodiment, the treatment provides for an inhibition of increase in prostate specific antigen (PSA) score, or for inhibition of tumour growth. In one embodiment the treatment provides for a reduction in PSA score, preferably a 50%, 60%, 70%, 80%, 90% or 100% reduction in PSA score.

It will be understood that the formulation may also be applied in the form of a device adapted for urethral application enabling the treatment of transitional epithelial carcinoma of the bladder.

Examples

Example 1 : Formulations

A. Formulation of Idronoxil in Suppocire CM with Myrj S8

74.98 g of Suppocire CM pellets and 6.52 g of Myrj S8 were weighed into a beaker, and heated to 45-55 °C on a heater stirrer plate. When the excipients started melting a stirrer bar was added and the mixture was slowly mixed. Once the base was fully melted, 18.50 g of idronoxil was added very slowly, allowing each portion to be completed dispersed prior to the addition of a second portion. The mixture was then stirred vigorously, maintaining the temperature of 45-55 °C for 15 minutes. The mixture was checked to ensure there was no formation of agglomerates.

Using a 20 ml_ syringe the mixture was transferred into a mold for forming suppositories of about 2.2 g. The mixture was cooled and solidified and the container sealed. The suppositories were stored at 2-8 °C.

As used herein, this formulation may be referred to as formulation 1A or formulation of Example 1A.

B. Formulation of Idronoxil in Suppocire CM

81.5 g of Suppocire CM pellets were weighed into a beaker, and heated to 45-55 °C on a heater stirrer plate. When the excipients started melting a stirrer bar was added and the mixture was slowly mixed. Once the base was fully melted, 18.50 g of idronoxil was added very slowly, allowing each portion to be completed dispersed prior to the addition of a second portion. The mixture was then stirred vigorously, maintaining the temperature of 45-55 °C for 15 minutes. The mixture was checked to ensure there was no formation of agglomerates.

Using a 20 ml_ syringe the mixture was transferred into a mold for forming suppositories 2.2 g suppositories. The mixture was cooled and solidified and the container sealed. The suppositories were stored at 2-8 °C.

As used herein, this formulation may be referred to as formulation 1B or formulation of Example 1B.

C. Formulation of Idronoxil in Suppocire CM with PEG-8-DS

63.57 g of Suppocire CM pellets and 17.93 g of PEG-8-DS (22%) were weighed into a beaker, and heated to 45-55 °C on a heater stirrer plate. When the excipients started melting a stirrer bar was added and the mixture was slowly mixed. Once the base was fully melted, 18.50 g of idronoxil was added very slowly, allowing each portion to be completed dispersed prior to the addition of a second portion. The mixture was then stirred vigorously, maintaining the temperature of 45-55 °C for 15 minutes. The mixture was checked to ensure there was no formation of agglomerates.

Using a 20 ml_ syringe the mixture was transferred into a mold for forming suppositories of 2.2g. The mixture was cooled and solidified and the container sealed. The suppositories were stored at 2-8 °C.

As used herein, this formulation may be referred to as formulation 1C or formulation of Example 1C.

D. Formulation of Idronoxil dispersed in MBK

81.5 g of MBK pellets (PCCA Base MPK™) were weighed into a beaker, and heated to 45-55 °C on a heater stirrer plate. When the excipients started melting a stirrer bar was added and the mixture was slowly mixed. Once the base was fully melted, 18.50 g of idronoxil was added very slowly, allowing each portion to be completed dispersed prior to the addition of a second portion. The mixture was then stirred vigorously, maintaining the temperature of 45-55 °C for 15 minutes. The mixture was checked to ensure there was no formation of agglomerates.

Using a 20 mL syringe the mixture was transferred into a mold for forming suppositories of 2.2 g. The mixture was cooled and solidified and the container sealed. The suppositories were stored at 2-8 °C.

The MBK base (PCCA Base MPK™) consists of methylbutylketone in a hydrogenated vegetable oil and PEG-8-DS at approximately 50%w/w, or in a fatty acid base.

As used herein, this formulation may be referred to as formulation 1D or formulation of Example 1D.

Example 2: Rat pharmacokinetic studies following administration of a suppository containing idronoxil

The plasma pharmacokinetic studies were performed in conscious rats that were rectally administered with the suppositories at a dose of 35 mg/kg in different suppository lipid bases. The results for the formulation of Example 1A (18.5% IDX in 8% Myrj 8S and Suppocire CM base) and Example 1D (18.5% IDX in MBK™) were compared.

Surgical procedures

Male Sprague-Dawley rats (n=4-8 per group, body weight 300 ± 30 g) were used for the studies. Rats were fed a “soy-free diet” from Specialty Feeds (WA, Australia) for 7-10 days before the study. The rats were anaesthetised (using isoflurane anaesthesia) and placed on a heated pad at 37°C. Marcaine (0.5 % bupivacaine) was administered to the surgical incision sites above the carotid artery and at the nape of the neck. The carotid artery was exposed and a BASi Culex® carotid catheter (Bioanalytical Systems Inc. (BASi®), West Lafayette, IN 47906 USA) was first tunnelled from the nape of the neck before being inserted into the carotid artery (for blood collection). The incision sites at the site of cannulation and at the nape of the neck were sutured and post-surgery the rats were placed in individual BASi Culex Nxt™ Raturn cages and the catheter attached to a BASi Culex Nxt™ automatic sampler. Rats then regained consciousness. Food was withheld during the overnight recovery, i.e. post-surgery, and until 8 hours after dosing but water was available ad libitum. Animals were conscious throughout the study (i.e. post-surgery, dosing and sampling) except during rectal dosing as is described below.

Preparation of idronoxil suppository formulations for rectal dosing in rats

Suppositories were prepared via the formulations described in Example 1A and 1D above. The suppositories were prepared in suitable sizes as described below.

The 1A solid suppositories were melted at 50°C, mixed and then filled into a 1 ml_ syringe with an attached 18 G needle (also heated to 50°C). The formulation was then dispensed into individual 0.5 ml_ plastic tubes making a single suppository dose. The filled mass was allowed to solidify at room temperature and it was then placed in the fridge. This solid mass was extruded out of the 0.5 ml_ tubes for dosing.

For the MBK formulation (1D), melted solid suppository was aspirated into a 1 ml_ pasteur pipette, allowed to solidify and then extruded out. Formulations were then cut to an approximate mass which equated to a required dose for each rat.

Dose administration

The animals were temporarily anaesthetised using isoflurane anaesthesia and the appropriate mass of the solid suppository (e.g. ~65 mg of formulation for a 300 g rat receiving a 35 mg/kg dose of idronoxil) was dosed into the rectum by first dilating the rectum with straight forceps, and then advancing the dose ~1 cm with a plunger from a 31 G insulin syringe. The animals were kept under anaesthesia for approximately 20 minutes to prevent leakage of formulation out of the rectum. After this time period rats were then placed back in their cages and allowed to regain consciousness.

Plasma collection

Blood samples were collected at set time points for up to 24 h following dosing into BASi Culex® borosilicate glass sampling tubes containing 5 pl_ of 1,000 lll/mL heparin. Collection tubes were kept at 4°C during sampling. The samples were then placed in a chilled centrifuge set at 4°C and spun down at 2250 g for 5 min. Aliquots of plasma (20 mI_) for each sample collected were transferred into labelled 1.5 ml_ Eppendorf tubes. Remaining plasma was transferred into labelled 1.5 ml_ Eppendorf tubes as a reserve of samples. All samples were stored at -20°C until analysis.

Sample analysis

Plasma concentrations of idronoxil were measured by the HMST rust-lab using a validated HPLC-MS/MS method.

Non-compartmental pharmacokinetic (PK) analysis

The elimination rate constant (slope, k) and half-life (i.e., 0.693/k) were calculated from the elimination phase of the concentration versus time profiles. The area under the plasma concentration time profiles from time zero to 8 hr (AUC0-8hr), zero to 24 hr (AUCO-24 hr) and zero to infinity (AUC0- ) were calculated using the linear trapezoidal rule. Bioavailability was calculated via comparison to the AUC0- obtained previously following IV dosing of 3.5 mg/kg idronoxil in a lipid-free co-solvent formulation.

Statistical analysis

Statistics including mean, standard deviation (SD), standard error of the mean (SEM), and unpaired Students t-tests were analysed using GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA, USA). A P value less than 0.05 was considered statistically significant.

Example 3: Plasma pharmacokinetics of the Myrj S8 and Suppocire suppository formulation (1A) and MBK suppository formulation (1D) after rectal administration Plasma concentrations of idronoxil over time following rectal administration of the suppository formulation of Example 1A to conscious rats are shown on linear-linear scale in Figure 2 and the plasma pharmacokinetic data are summarized in Table 1.

Table 1. Pharmacokinetics of Myrj S8 and MBK formulations

Table 1: Mean plasma pharmacokinetic parameters of idronoxil after rectal administration of 35 mg/kg idronoxil to conscious rats in 1A and 1D formulations. Bioavailability is calculated via comparison to data from a previous 3.5 mg/kg IV dose of idronoxil in a lipid-free cosolvent formulation. Data shown for 1A and 1D formulations represents mean ± SEM for n=8 and n=4 rats, respectively, unless stated otherwise. Statistical differences were determined using an unpaired Student’s t-test. a Half-life could only be estimated in rat 1, 3, 7 and 8 as there was no clear first order elimination profile in all other rats. b Half-life could only be estimated in rat 1, 3 and 4 as there was no clear first order elimination profile in rat 2. c Results were significantly greater ( P < 0.05) than the 1A formulation using an unpaired Student’s t-test. Although the plasma concentrations and AUC appeared slightly lower for the 1A formulation compared to the 1D formulation, the Cmax, T _ma x, AUCo-sm·, AUCo-24hr and Bioavailability %F for 0-8 h after dosing the 1A formulation were not significantly different to the 1D formulation (Table 1). The plasma AUC and bioavailability from 0- infinity were, however, significantly lower after administration of the 1A formulation when compared to the 1D formulation (Table 1). As the terminal elimination rate constant (slope, k) was highly variable for the 1A formulation the half-life was also variable and was not significantly different to the 1 D formulation with the MBK base.

The overall plasma pharmacokinetics and bioavailability were relatively similar for the 1A formulation compared to the 1D formulation. However, the AUC and bioavailability from 0- were statistically lower for the 1A formulation compared to the 1D formulation. Overall the two suppository bases containing a surfactant yielded relatively similar plasma exposure and pharmacokinetics despite differences in composition and/or preparation.

Example 4: Comparison between a suppository containing surfactant (1A) and without surfactant (1B)

A suppository was prepared with Idronoxil dispersed in Suppocire CM (Example 1B). A second suppository was prepared according to Example 1A, where idronoxil was dispersed in a base containing Suppocire CM and 8% of PEG-8-MS (Myrj S8). The pharmacokinetic and sustained released profile of the active was measured using the protocols described in Example 2. The results of the PK profile upon rectal administration of the suppositories in rats are shown in Figure 1. The suppository comprising both the base and the surfactant showed superior sustained release profile, with release of the active being measured for approximately 25 hours. Active release was still measured at almost twice the length of time of the release of active from the plain Suppocire base. This demonstrates the superior formulation and benefit of the addition of the surfactant to the suppository.

More advantageously, not only was the release sustained over longer periods of time, but concentration of Idronoxil measured in plasma was significantly higher across the first 10 hours of the experiment. This shows the dramatic improvement and benefit of the formulation described in Example 1A, compared to dispersion of the active in the base without the surfactant (Example 1 B).

Example 5: Dissolution profile of suppository formulations

The dissolution profiles of the suppository formulations were measured using USP apparatus 2 (Paddle Apparatus) using 900 ml_ of 5% (w/w) Tween in water (pH 7.2 ± 0.2) as dissolution medium. The medium was heated at 37 °C and stirred at 50 rpm. Suppositories were placed in the medium filled vessels. Aliquots of sample were taken at scheduled time points and assayed HPLC to determine the concentration of released active. The HPLC assay was performed using isocratic reverse phase HPLC method with UV detection at 333 nm using a YMC-Pack Pro C18 RS, 250 mm column, a flow rate of 1.0 ml/min, temperature at 25°C and a mobile phase of 10 mM potassium dihydrogen phosphate:acetonitrile (55:45). Reference standards and drug product sample solutions are prepared at 0.1 mg/ml_ in 10 mM potassium dihydrogen phosphate:acetonitrile (30:70) and The determination of assay is achieved by quantitation against an external reference standard of idronoxil.

Example 6: Comparative dissolution and PK profile of different formulations

The dissolution behaviour was measured as described in Example 5. The Pharmacokinetic behaviour of the formulation was measured as described in Example 2 for different formulations. The results are summarized below in Table 2. Results were given a comparable value compared to the MBK formulation.

Table 2: Comparative results of dissolution and PK concentrations in rats for idronoxil formulated in different bases compared to MBK formulation (1D)

The results in Table 2 shows the comparative results for formulations of suppositories containing 18.5% Very poor is <10%, poor <25% and comparable is similar to formulation 1D. Idronoxil, of dissolution behaviour and PK concentration measured in rats, compared to the formulation containing MBK. The results show that multiple different lipophilic bases can be used with different surfactants, present at different % w/w, to achieve the desired dissolution and PK profile.

Example 7: Caco-2 permeability assay

The permeability of idronoxil in the presence of a surfactant was compared to the permeability of idronoxil alone using the Caco-2 permeability assay. The Caco-2 permeability assay approximates the permeation of idronoxil in the rectum when administered as a suppository.

The assay was performed using a monolayer of Caco-2 (colon) cells suspended inside a dual chamber. A schematic of the set-up is shown in Figure 3. The apical chamber, or inner chamber of the cell represents the intestinal tract, and the basolateral chamber, or outer chamber, represents the blood. The assay measures the permeability of idronoxil across the cell monolayer in the presence or absence of a surfactant.

The study was performed by adding the compound of interest (idronoxil) to the apical compartment for 90 min. Subsequently the compound concentrations in both A (Apical) and B (Basolateral) chambers were measured. The concentrations of compounds were used to calculate the apparent permeability coefficient (P _{a p} A-B). The higher the number, the greater the permeability. The assay was then repeated but the compound was instead added to the basolateral chamber, allowing the calculation of the permeability in the opposite direction as P _app B-A. A P _app B-A/ P _app A-B ratio >2 indicates the compound is an efflux substrate.

Experimental Procedure

The Caco-2 permeability assay was performed following the procedure outlined below.

1. The HBSS Buffer was pre-warmed in a water bath to 37 °C.

2. The test compounds were removed from storage at -20 °C and sonicated for a few minutes (no less than 1 minute).

3. Donor solution buffer was prepared according to the Table 3. Table 3: Donor solution buffer preparation

Donor solution buffer:

For A-to-B direction:

HBSS buffer with 0.3% DMSO and 5 mM LY: add 150 pL DMSO and 50 pL LY (5 mM) into 50 ml HBSS buffer (pH 7.4).

HBSS buffer with 0.1% DMSO and 5 pM LY : add 50 pL DMSO and 50 pL LY (5 M) into 50 mL HBSS buffer (pH 7.4).

For B-to-A direction:

HBSS buffer with 0.3% DMSO: add 150 pL DMSO into 50 ml HBSS buffer (pH

7.4).

HBSS buffer with 0.1% DMSO: add 50 pL DMSO into 50 ml HBSS buffer (pH

7.4).

Receiver solution buffer:

For A-to-B direction:

HBSS buffer with 0.4% DMSO: add 200 pL DMSO into 50 ml HBSS buffer (pH

7.4).

For B-to-A direction:

HBSS buffer with 0.4% DMSO and 5uM LY: add 200 pL DMSO and 50 pL LY (5 mM) into 50 ml HBSS buffer (pH7.4).

Donor solution

The donor solution was prepared according to the Table 4.

Table 4: Preparation of donor solution

4. The TEER was measured. The cell culture plates were taken out of the incubator and the cell monolayers washed with HBSS buffer. Then the TEER values were measured at room temperature. 5. The compound solution (from step 3) was centrifuged at 4000 rpm for 5min prior to being loaded into the donor chambers.

6. The solutions were added to the chambers according to the below Table 5.

Table 5: Preparation of chambers for CaCo-2 assay 7. To determine LY concentration in the apical chember, 100 pl_ samples were taken from apical chambers and added into an opaque plate for LYTO.

8. The apical and basolateral plates were pre-warmed at 37°C for about 5 min, then transport was begun by placing the apical plate onto the basolateral plate. 9. The plates were kept in an incubator at 37°C for 90 min.

10. The apical plate was separated from the basolateral plate after 90min incubation.

11. 100 pL samples were taken from basolateral plate and added to an opaque plate as LYT90. LY concentrations for LYTO and LYT90 were measured by Fluorometer (at excitation of 485 nm/emission of 535 nm).

12. Donor or receiver samples were diluted by 0.4% DMSO HBSS, then mixed with ACN with IS (Osalmid or Imipramine) and measured using LC-MS/MS.

CALCULATIONS

The permeability coefficient for membrane transport of test compounds was determined using the following equation:

Papp (cm/sec) = (V _r /C ₀ ) (1/S) (dC/dt)

(Papp = apparent permeability, V _r = volume of medium in the receiver chamber, Co = Peak Area Ratio (PAR) of the test drug in the donar chamber, S = surface area of monolayer, dC/dt = drug PAR in the receiver chamber with time). Area of 24-well = 0.7 cm ²

Peak area ratio = Analyte peak area/IS peak area Results

Table 6: Permeability of idronoxil (IDX) with and without Myrj S8

Example 8: CaCo-2 assay of comparative surfactant formulations The Caco-2 assay was repeated using the different surfactant formulations as seen in Table 7.

Experimental Procedure

1. Caco 2 cell line obtained from ATCC was used for this permeability experiment. 2. Cells were splitted every other day at a split ratio of 1:3-1 :5 and grown in

Dulbecco's Modified Eagle Medium (GlutaMAX I, 4,500 mg/L D-glucose, sodium pyruvate.), supplemented with 10% FBS in the presence of antibiotics. Confluent Caco- 2 cells were seeded onto polycarbonate Transwell filter membranes (Millipore) at a density of 60,000 cells/well. 3. After 24 h post seeding, medium was changed and cultured for another 21 days before transport experiments.

4. For Idronoxil alone permeability experiment, donor solutions were prepared by diluting the stock solutions of Idronoxil in transport medium (HBSS buffer with 10mM HEPES, pH 7.4). Receiver solutions were the same HBSS buffer with 10mM HEPES, pH 7.4.

5. In Idronoxil along with surfactant experiments: MyrjS8, Cremophor EL, Tween 80, Glycerol Stearate and Isopropyl Myristate (0.87 pg/mL each in respective experiments) and Suppocire CM (9.9 pg/mL in each experiment) were added in apical side of both A B experiment and B A experiments. In Idronoxil along with no surfactant experiment, Suppocire CM (9.9 pg/mL in each experiment) was added in apical side of both A B experiment and B A experiments."

6. The transport of Idronoxil (10 pM) was measured in duplicate in two directions [apical to basolateral (A B) and basolateral to apical (B A)].

The quantification of Idronoxil in each chamber was performed using HPLC with a UV/VIS detector measuring at 300 nm and with a mobile phase of A: 10 mM Ammonium Acetate Buffer and B- Acetonitrile and a gradient elution of (A- 10 : B-90; v/v) as shown in Table 7; a flow rate of 0.8 mL/min; Injection volume: 20 pL; an Xterra phenyl (150 x 3.9 mm, 5 pm) column and using reserpine as the internal standard.

Table 7: Gradient elution conditions for HPLC analysis

CALCULATIONS

The permeability coefficient for membrane transport of test compounds was determined using the following equation:

Papp (cm/sec) = (V _r /C ₀ ) (1/S) (dC/dt)

(Papp = apparent permeability, V _r = volume of medium in the receiver chamber, Co = Peak Area Ratio (PAR) of the test drug in the donar chamber, S = surface area of monolayer, dC/dt = drug PAR in the receiver chamber with time). Area of 24-well = 0.7 cm ²

Peak area ratio = Analyte peak area/IS peak area

Results

The results of the Caco-2 assay with comparative formulations are shown in Table 8.

Table 8: Permeability of idronoxil (IDX) with comparative formulations

These experiments show that the formulation with Myrj S8 (PEG-8-MS) has an increased permeability coefficient compared to a suppository formulation Tween 80 or Isopropyl Myristate.

Example 9: Equilibrium structures of suppository formulations of idronoxil with different surfactants

Comparative formulations for studies. The investigation of the specific role of the surfactant in the effective delivery of drugs was done by substituting surfactants into the formulation (formulation 1A):

1. Suppocire CM (1.656 g)

2. Surfactant (0.144 g) 3. Idronoxil (0.400 g)

The suppositories were made using the mixing procedure provided by Noxopharm. The Suppocire CM was melted at no more than 55 °C and the surfactant was added and mixed at elevated temperature using magnetic stirring. The idronoxil (or other drug) was added and the stirring continued for 15-20 min. After this, the suppository formulation was ready to use, and could be stored in the fridge for later use.

The surfactants studied are shown in Table 9 below:

Table 9: Surfactant structures and physical properties

Studies of the behaviour of idronoxil in a simulated rectal environment used a simulated rectal fluid according to Table 10. The fluid corresponds to a fed state.

Table 10: Simulated rectal fluid (SRF) components for a 1 L solution

Component Mass (g)

Maleic Acid 3.96

Sodium Hydroxide 0.296

Bile Salt Extract 0.451

Phosphatidylcholine 0.37

Palmitic Acid 0.051 Bovine Serum Albumin 3.0 Sodium Chloride 2.0 Glucose 14.0

The equilibrium structures of the suppository formulations in a simulated rectal environment were analysed by small angle x-ray scattering (SAXS) and wide-angle x- ray scattering (WAXS).

The suppository formulations were loaded into glass capillaries with a 2.0 mm diameter. A schematic of the experimental set up is shown in Figure 5. As shown in Figure 5, the horizontal line across the capillary represents the fill line of the capillaries with the suppository formulation, which is the interface with the aqueous phase upon addition of the simulated rectal fluid prior to measurement. The capillaries were filled to a fill level of 2.2 cm from the bottom of the capillary. This was done to allow for the interface between the oily suppository phase and the aqueous SRF phase to be positioned in the X-Ray beam. The temperature control apparatus at the SAXS/WAXS beamline is as shown in Figure 5 with two heating plates (grey boxes) and a small gap 0.5 cm wide for the X-ray beam to pass through.

The scattering patterns of these samples are shown in Figure 5. As can be seen in Figure 5 both Tween 80 and TPGS formed micellar structures, no micellar structures were observed in the formulation with Myrj S8 at equilibrium. This indicates that the Myrj S8™ does not facilitate the dissolution of idronoxil via micellization.

Example 10: Dynamic sSAXS experiments of suppository formulations

Using the same suppository formulations described in example 9 dynamic sSAXS studies were done to investigate the first 30 min upon mixing the suppository formulations with SRF. The interface between the lipid formulation and the aqueous SRF was monitored for 30 min, with a scattering pattern acquired approximately every second.

The prepared capillaries were placed into the temperature block 30 min prior to the sample measurement to allow it to equilibrate to 37 °C. After equilibration, 37 °C SRF was added to the top of the suppository in the capillary. The sample was flicked and shaken to ensure a clean interface between the suppository and the SRF by removing any air bubbles. The position of the sample holder was visually adjusted to the centre of the capillary and near to the interface. The time between the addition of the SRF and when the first measurement could be taken was approximately 90 seconds.

The initial measurements were aimed at determining the position of the lipid-fluid interface by taking measurements down the capillary. Upon finding the interface (Figure 4, red), the sample holder was adjusted such that this position was in line with the beam. Measurements were taken at this position over 30 min after the addition of the SRF. One measurement was taken approximately every second. For the formulation containing idronoxil + Tween 80, Figure 6 shows the evolution of the SAXS scattering patterns over 30 min. Selected spectra are stacked on the right to clearly show differences over time. A final y-scan was performed for each sample at the end of the day to investigate the movement of the interface.

These results are shown in Figures 6 to 10.

The formulation 1A suppository (Myrj S8 + idronoxil) shows the rapid formation of vesicles, or large aggregates (Figure 8). The selected spectra are stacked on the right to clearly highlight differences between patterns. The arrows in the low q region highlight the formation of a broad peak in the low q region which rapidly moves to very low q, indicating formation of small self-assemblies that rapidly increase in size larger than can be detected with SAXS, which correlates nicely with the results of the equilibrium studies previously performed. The dynamic SAXS experiment shows that there is no micellar intermediate.

The rapid formation of vesicles or large aggregates appears to be via the formation of an emulsion, also observed while working with the samples in the partitioning experiments.

The Tween 80 + idronoxil suppository formulation shows the gradual formation of a broad peak in the low q region of the scattering patterns indicative of micellar structures which remain stable at equilibrium (Figures 6 and 7). Below, the images of the capillary are shown which correspond to the first, last and selected interface. The thin white cross indicates where the beam crosses the capillary. The opaque lower region is the solid capillary, while the bright upper region is the empty capillary, with some suppository residue on the walls. The scattering patterns indicate that Tween 80 is effective at dispersing the lipid as the lamellar peaks from the Suppocire CM decrease in intensity over time.

The Kolliphor EL + idronoxil suppository formulation (Figure 9) shows very similar scattering patterns to Tween 80, indicating the formation of some micellar aggregates that increase in size over time. Selected spectra are stacked on the right to demonstrate a change over time more clearly. In this time-series, the arrows in the low q region shows the formation of a broad peak, indicating the formation of self-assemblies. After 10 min, this peak shifts to lower q. The lamellar peaks of the Suppocire CM become less pronounced after 10 min, indicated by the downward pointing arrows.

The scattering patterns also show evidence that this surfactant aids in the dispersion of the lipid as the characteristic lipid peaks decrease in intensity. The idronoxil is mostly solubilised in the aqueous phase, likely through micellization of the Kolliphor EL.

The TPGS + idronoxil suppository shows the immediate formation of micelles of 9 nm in diameter which grow over time to 16 nm (Figure 10). The selected scattering patterns are shown in a stack on the right to demonstrate changes more clearly over time. The upwards pointing arrows highlight broad peaks in the low q region that indicate the formation of micelles almost immediately upon addition of SRF. These increase in size from 9 nm initially, to 16 nm after 30 min, as the scattering peak shifts to lower q values. These remain stable at equilibrium.

The formulation 1A suppository is likely forming an emulsion upon mixing. The other surfactants form less homogeneous emulsions that the mixture formed with the Myrj S8 surfactant.

The data shows that of the surfactants tested only Myrj S8 forms an emulsion upon mixing in the rectum. The formulation of the present disclosure has a high partitioning of idronoxil in the lipid phase in the presence of Myrj s8 relative to the other non-ionic surfactants.

Example 11 : Partioning of idronoxil in the lipid and water phases after dispersal in simulated rectal fluid The five surfactants of interest listed in Table 8 were used to formulate suppositories containing idronoxil. After mixing the melted suppositories with SRF, the mixture was phase separated using centrifugation and the aqueous and lipid phases were separated and the amount of idronoxil in the aqueous phase and lipid phase was measured using UV/VIS spectroscopy. The idronoxil measured in each sample is presented in Figure 11 , showing the relative amount of drug measured in the aqueous phase (in dark grey) and in the lipid phase (in light grey). The graph is presented named by the surfactant in the formulation, and these are ordered with increasing hydrophilicity from left to right as indicated by HLB values (in brackets).

The total soluble idronoxil in both the lipid and water phases is highest with surfactants with medium HLB values. The glyceryl stearate showed no significant soluble idronoxil in the aqueous phase, but a relatively large, albeit variable, amount soluble in the lipid phase. Preparing and utilising this formulation was very difficult; phase separation, poor homogeneity and sample smearing resulted in uneven sampling due to difficult handling. This resulted in a high amount of error in the measurement and indicates that this surfactant is not a suitable substitute for the suppositories of the present disclosure.

For the mid-range HLB value surfactants, Myrj S8™, Kolliphor EL and TPGS, the most overall soluble idronoxil was measured. The Myrj S8™ was able to solubilise the idronoxil into the lipid phase in a higher proportion than in the aqueous phase. In contrast, the Kolliphor EL and TPGS showed about equal solubility in the aqueous and lipid phases.

The highest HLB surfactant, Tween 80, showed a decreased amount of total idronoxil, with a higher proportion of idronoxil in the aqueous phase. In general, surfactants with a higher HLB value will be able to solubilise drugs in the aqueous phase more so than the lipid phase, which has limited the amount of idronoxil in solution in this situation.

The partioning study indicates that the formulation of the present disclosure, that is the suppository formulation using Suppocire and Myrj S8 facilitated a high amount of solubilised idronoxil, with the highest proportion of drug in the lipid phase which differentiates compared to the other surfactants. Example 12: Cryo SEM studies

In equilibrium studies of Veyonda formulations containing Myrj S8™ in SRF, the presence of micelles was not observed using SAXS analysis. To investigate the equilibrium state of this sample at a larger length scale, CryoSEM was employed. Two samples were analysed using this method: the placebo formulation of Suppocire CM and Myrj S8™ and the formulation of example 1 A which includes idronoxil.

For the CryoSem Studies suppositories were made according to the protocol of example 1A with:

Suppocire CM (1.656 g)

Idronoxil (0.400 g)

Surfactant (0.144 g)

The suppositories were melted at 40 °C and mixed with simulated rectal fluid (SRF, 1:1 m/m), shaken 20 x, and held at physiological temperature for the duration of the sampling. Samples were taken at approximately 5 minutes after shaking, and 25 minutes after shaking. The samples were pipetted into a brass puck, frozen in LN2 for 30 seconds, and subsequently fractured with a razor blade. The sample was immediately transferred into the SEM chamber and the chamber was evacuated. The imaging took place for up to 20 minutes after this, prior to the sample melting.

The Cryo SEM images of the placebo formulation are shown in Figure 12. Small, round oil-filled droplets are visible within the porous buffer network indicating the formation of an oil in water emulsion. The insets of Figure 12 (b) and (c) show how the oil droplets have smaller droplets near them, which have a size comparable to that of lipid vesicles.

The formulation of example 1A in SRF is shown in Figure 13. This sample was frozen at two time points: 5 and 45 min after mixing. After 5 min, the oil droplets appear to be dispersed within a buffer network, however the shapes are highly irregular. After 45 min, the irregularly shaped lipid droplets are no longer present, and smaller, rounder, and more regular oil spheres are observed. The two comparative samples (formulation of Example 1A and the placebo) were compared using cathodoluminscence-SEM (CL-SEM). CI-SEM is a technique that can measure light that is emitted from the sample as it is excited by the electron beam rastering across the surface. The method was used to observe the partitioning of idronoxil. Figure 14 (a) shows the placebo mixture imaged by CL-SEM on the left with the NOX66 mixture on the right. Notably, the placebo shows a weak emission from the lipids. In the NOX66 mixture (Figure 14(b)), the brightness of the signal coming from the idronoxil crystals saturates the signal obtained from the sample. The sample appears to be comprised of three components - oil droplets in an aqueous phase which contains the idronoxil drug crystals.

Further imaging of the microstructure of the suppositories according to formulation 1A is shown in Figure 15. The scale bars are 500 pm. The fractured structure of formulation 1A and simulated rectal fluid mixture shows an emulsion, where spherical oil droplets between 30 pm and 100 pm are visibly well distributed within the aqueous phase. The image also shows a surface that is not flat, which is an indication of a good emulsion. The similarity between the two images at the two time points shows stability in the emulsion.

Comparative suppositories were made using Kolliphor EL; TPGS and Tween 80 according to the formulation described in Example 9. The sample was melted at 40 °C and mixed with simulated rectal fluid (SRF, 1:1 m/m), shaken 20 x, and held at physiological temperature for the duration of the sampling. Samples were taken after shaking at approximately 15, 6 and 7 minutes for the first time point and 45, 30 and 29 minutes for the second time point. The samples were pipetted into a brass puck, frozen in LN2 for 30 seconds, and subsequently fractured with a razor blade. The sample was immediately transferred into the SEM chamber and the chamber was evacuated. The imaging took place for up to 20 minutes after this, prior to the sample melting. These images are shown in Figure 16 and 17 (Kollophor EL), 18 and 19 (TPGS) and 20 and 21 (Tween 80).

As shown in Figure 16 the fractured structure of the suppository containing the Kolliphor EL surfactant shows very few signs of oil droplets. It appears that the lipid is dissolved somewhat in the aqueous phase, forming a network within the aqueous buffer network. While some oil droplets are visible and present, their presence is minimal. This is also visible in the less magnified comparison of the two time points (Figure 17). The 5 minute sample here was frosted, so could not be used to determine the structure at exactly 5 min. There is no sign of a stable emulsion of oil droplets within an aqueous in these samples.

The Kolliphor EL behaviour in the suppository formulation and microstructure upon mixing with simulated rectal fluid is clearly not forming an emulsion, based on the absence of oil droplets in the cryoSEM images.

Figures 18 and 19 show that phase separation can be seen in the sample containing TPGS as a surfactant. While oil droplets are visible in the sample after both 6 minutes and 30 minutes, these droplets are not uniformly distributed with the aqueous buffer network. This indicates that there is phase separation and does not constitute a stable emulsion. This is also clearly seen in the less magnified overview images of Figure 18.

Figures 20 and 21 show the images of the mixture containing Tween 80. These mixtures do not show clear oil and aqueous phases, with the exception of a few large droplets. There are no oil droplets prevalent or uniformly distributed within the mixture. After 29 minutes, a large oil droplet is distinct, and some smaller oil droplets are well defined, indicating some phase separation over time. This is more clear in the less magnified image (Figure 21).

Based on the CryoSEM experiments and imaging it can be seen that only suppositories formulated with Myrj S8 form a stable emulsion when mixed with simulated rectal fluid.

The Tween 80 and TPGS surfactants show micelle formation in the SAXS data at low q, (example 10) indicating that the formation of micelles causes the surfactant to be less available for solubilizing the idronoxil, or stabilizing any emulsion structures. The absence of an emulsion structure at two time points in these samples confirms this (Figures 18-21).