Use of rimcazole to treat cancer

Abstract

The invention provides the use of an inhibitor of the dopamine transporter (also known as a dopamine reuptake inhibitor), including but not limited to rimcazole (cis-9-[3-(3,5-dimethyl-1- piperazinyl)propyl]carbazole), in the treatment of cancer and particularly to the use of said compound in the treatment of spontaneous cancer (i.e. cancer that arises naturally in an authentic patient, in particular a human or canine patient).

Description

[0001] USE OF RIMCAZOLE TO TREAT CANCER

[0002] Field of the Invention

[0003] The present invention relates generally to the use of an inhibitor of the dopamine transporter (also known as a dopamine reuptake inhibitor), including but not limited to rimcazole (c / s-9-[3- (3,5-dimethyl-1-piperazinyl)propyl]carbazole), in the treatment of cancer and particularly to the use of said compound in the treatment of spontaneous cancer (i.e. cancer that arises naturally in an authentic patient, in particular a human or canine patient). The present invention also relates to the use of rimcazole (c / s-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]carbazole), or other compounds of the invention, in combination with an anti-inflammatory agent. The present invention also relates generally to new clinical applications of rimcazole (c / s-9-[3-(3,5-dimethyl- 1-piperazinyl)propyl]carbazole) or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof.

[0004] Background

[0005] Rimcazole (cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]carbazole) is a well-known sigma-1 receptor antagonist which has been investigated for a variety of medical applications, including for the treatment of cancer.

[0006] Studies on the potential of rimcazole as a cancer treatment have been based on investigations in laboratory model systems, including cultured tumour cells and laboratory animals bearing engrafted tumour cells from other species such as humans (W02000000599; Spruce et al (2004); Rybczynska et al (2013)).

[0007] Although these models appeared to demonstrate the potential use of rimcazole in the treatment of cancer, the results obtained demonstrated that inhibition of tumour growth by rimcazole - both in vitro and in vivo - occurred only at relatively high (micromolar) concentrations (W02000000599; Spruce et al (2004); Rybczynska et al (2013); and WO2001074359).

[0008] For example, Spruce etal (2004) described, using a number of models, that the in vivo tumour cell killing action of rimcazole (in murine xenograft models) is mediated at blood (plasma) concentrations in the micromolar range. This is consistent with in vitro data that also demonstrates growth inhibitory effects of rimcazole at micromolar concentrations and with the reported micromolar binding affinities of rimcazole for the sigma-1 receptor.

[0009] The same publication displays (see Figure 3B) in vitro growth inhibitory data for rimcazole across the NCI 60 tumour cell line panel (supplied by the National Cancer Institute, USA). The panel consists of established tumour cell lines derived from a range of tumour types, propagated for extended periods of time in growth factor rich media. These data are reproduced below (Table 1): TABLE 1 The NCI 60 human tumour cell line panel is considered by those skilled in the art to be representative of the full range of human tumour types. The NCI (US National Cancer Institute) further recognizes the close similarities between human and canine cancer to the extent that it has “comparative oncology programmes” to inform oncology clinical development that is relevant to both dogs and humans (National Cancer Institute - Helping Dogs — and Humans — with Cancer). It is therefore valid to extrapolate information acquired in human tumour model systems to canines.

[0010] It can be seen from Table 1 (data from assays of viable cell number after 48 hours of exposure to rimcazole) that across the entire NCI 60 panel, the lowest IC50 value (meaning a 50% reduction in viable cell number) is 1.89 microM and the highest IC50 value is 37.6 microM. Therefore, there appears to be a minimum threshold for effectiveness that is above 1 micromolar.

[0011] In in vivo studies in mice bearing tumour xenografts, plasma concentrations after a single dose of rimcazole 40 mg / kg, administered to mice either intraperitoneally (IP) or orally, peaked at 2-3 microM (Spruce et al., 2004), demonstrating that rimcazole was reaching levels in the systemic circulation that were consistent with micromolar binding affinity of rimcazole for sigma-1 sites and also with the in vitro tumour cell line data . Higher plasma concentrations would in turn be expected with daily administration, rising until steady state was reached. There was no observable toxicity in mice at such blood levels. By extrapolation, in the study reported by Rybczynska et al (2013) in which mice were treated with rimcazole at a daily dose of 26 mg / kg IP, plasma concentrations would also reach levels in excess of 1 microM.

[0012] The sole situation - exemplified in Spruce et al in W02000000599 - in which rimcazole was revealed to induce apoptosis at submicromolar concentrations was in genetically engineered tumour cell lines in which the wild-type (functional) tumour suppressor gene p53 (also known as TP53) was either transiently transfected under the control of a strong, constitutive promoter (Figure 14 c, 293 human embryonic kidney cells) or stably transfected with p53 under the control of a tetracycline inducible promoter which leads to very high levels of p53 protein in the cells in response to administration of a tetracycline analogue, doxycycline (Figure 14b, human osteosarcoma cells). These are both very artificial systems that express high levels of a functional form of the p53 protein - a protein that is well known to act as a universal brake on the development of cancer. Cancer therefore cannot develop if p53 is functioning normally and this is particularly so if functional p53 is expressed at higher than normal levels. Indeed, the p53 tumour suppressor protein is widely recognised to be altered in a way that either leads to a reduction in the level of the p53 protein or to a direct or indirect functional inactivation of the p53 protein in most, and possibly all, cancers (Vousden and Lane, 2007). It is therefore a critical brake on cancer.

[0013] From this, it can be readily understood that the only systems hitherto associated with the induction of apoptosis by rimcazole at submicromolar concentrations are not compatible with the clinical situation of naturally occurring spontaneous cancer in a real patient, (i.e. a patient treated according to the present invention).

[0014] The teaching in the art is hence consistent that, in order for rimcazole to achieve anti-tumour effects, it needs to reach concentrations in the micromolar range in the extracellular milieu (the extracellular environment). The extracellular milieu is comprised of the tissue culture media in the case of in vitro studies of tumour cells cultured in the laboratory; or in the case of in vivo studies involving experimental animal models of cancer, it comprises the extracellular - interstitial - fluid with which the blood (plasma) concentrations are in equilibrium.

[0015] Unfortunately, however, prior art studies also consistently found that plasma concentrations of rimcazole in the necessary micromolar range for efficacy are also associated with severe adverse effects.

[0016] For example, a range of Phase I and Phase II trials of rimcazole (known as BW234U) were conducted in the 1980’s in healthy volunteers and schizophrenic patients (Davidson et al, (1985); Chouinard and Annable (1984); Gilmore et al, (2004)). A grand mal seizure was reported in a healthy volunteer administered with a single oral dose of BW234U (800 mg) which was associated with plasma concentration - blood level - of 682 ng / ml (2.12 microM). Healthy volunteers administered 300 mg twice daily (600 mg total dose) had peak blood levels that ranged to a maximum of 600 ng / ml (1.86 microM). Two schizophrenic patients had grand mal (major epileptic) seizures at 600 mg / day (associated with peak blood levels up to 1.86 microM). Chouinard et al. (1984) also reported an episode of convulsions in a schizophrenic patient administered with 550 mg daily. When the median daily dose was reduced to 125 mg, mean peak blood levels were 59 ng / ml (0.18 microM i.e.180 nanoM) and were associated with no adverse effects. Phase II trials were conducted for periods up to one year with doses of BW234U 200 mg twice daily, with no overt toxicity but also no clear evidence of anti-psychotic efficacy - which led to the programme being discontinued.

[0017] Clinical trials of rimcazole were subsequently conducted in 2008 and 2010 (as reported by Rybczynska et al (2013)). It is important to note that these trials are incorrectly referred to as ‘cancer trials’ by Rybczynska, whereas in fact they were conducted in healthy volunteers (this is clarified in the 2011 published report of the Irish Medicines Board (Irish Medicines Board. Rimcazole Clinical Trial at Shandon Clinic. Final Report (2011). Rimcazole was administered in an ascending single dose phase and then in repeat dosing at 400 mg twice daily (800 mg total daily dose). The clinical study revealed severe toxicity in the healthy volunteers with three out of 12 volunteers experiencing major adverse events (convulsions) at a dose of 400 mg twice daily. This led to a termination of the study and a complete cessation of rimcazole clinical development in humans.

[0018] The clinical data in humans therefore suggested that it was implausible to consider rimcazole as a safe and effective treatment for cancer, because the micromolar blood levels predicted to be needed for anti-tumour effectiveness are overtly toxic. The similarity between dogs and humans in disposition to epilepsy predicts that dogs will be equally susceptible to a risk of epileptic seizures at micromolar blood (plasma) concentrations of rimcazole. As described above, it is well known, and reported in the literature, that canines and humans have similar prevalence rates for idiopathic epilepsy. Indeed, the similarity is such that dogs are proposed as a natural animal model of human epilepsy (Loscher, 2022).

[0019] By way of illustration, historical data from a 3 month oral toxicity study of rimcazole in healthy beagle dogs are reproduced below; these supported the clinical development of rimcazole (BW234LI) in schizophrenia in the 1980's (Davison et al, 1982; Guy et al, 1983; Choiunard et al, 1984; Gilmore at al, 2004; and elsewhere). Table 2 sets out mean values for peak plasma concentrations of rimcazole in healthy beagle dogs following daily administration of oral rimcazole at doses of 10 mg / kg, 20 mg / kg and 40 mg / kg per day for 3 months (personal communication, Glaxo Wellcome). Blood samples were taken 2 and 4 hours (2h and 4h) after the first daily dose (to coincide with the peak blood level, Cmax). Mean blood levels are expressed as nanograms per ml with the corresponding molar equivalent concentrations shown below (nanomolar - nM - and micromolar - microM, respectively), based on a molecular weight for rimcazole dihydrochloride (BW234LI) of 321.5 g / mol.

[0020] TABLE 2

[0021] There was no evidence of overt, dose-limiting clinical toxicity at any of these dose levels. Overt signs of toxicity including ataxia (incoordination of movement), convulsions (epileptic seizures), vomiting, and excessive salivation were observed when doses of 50 mg / kg / day and above were administered. These adverse events are reflective of unacceptable clinical toxicity.

[0022] The beagle dog pharmacokinetic data illustrate that peak blood levels remain consistently in the sub-micromolar (below 1 micromolar) range in beagle dogs receiving oral rimcazole at 10 mg / kg / day and 20 mg / kg / day for three months. At 40 mg / kg / day, plasma concentrations in some cases exceed 1 micromolar; however, there is no overt clinical toxicity. At the next dose level above this (50 mg / kg / day) severe clinical toxicity emerged in some dogs including convulsions (epileptic fits). At this dose level, peak plasma concentrations of rimcazole are consistently in the micromolar range.

[0023] Therefore, as predicted, the seizure threshold in canines is the same as in humans, with seizures occurring when plasma concentrations of rimcazole are consistently in the micromolar range. Canine cancer patients therefore faced the same technical challenge as human patients: micromolar plasma concentrations of rimcazole were taught to be required for anti-tumour efficacy but these would be toxic in both species.

[0024] Based on all available data, it appears that to accommodate an acceptable margin of safety, 1 micromolar would be considered to be a conservative ceiling for rimcazole plasma concentration, in order to avoid drug levels rising above the toxicity threshold. Therefore, according to consistent teaching in the prior art, the drug can only be given safely to both humans and dogs at doses where rimcazole plasma concentrations are consistently below 1 micromolar. However, as explained above, no anti-tumour activity for rimcazole would reasonably be expected at concentrations below 1 micromolar.

[0025] In light of the data discussed above, it was hence the clear understanding in the art that the doses of rimcazole - and corresponding micromolar blood (plasma) concentrations - which would be required in order for rimcazole to realise its potential as a cancer treatment were implausible as a safe and effective clinical treatment for cancer (i.e. as a treatment of actual human and canine patients). This is because such concentrations in the bloodstream would cause severe toxicity (epileptic seizures) in both canines and humans.

[0026] For these reasons, rimcazole was discontinued from its clinical development path. It was not thought possible to provide a rimcazole treatment for cancer which was both effective and safe. It is hence widely understood in the art that rimcazole is not a suitable clinical candidate.

[0027] Needless to say, however, there remains a need in the art for safe and effective cancer treatments for both human and canine patients.

[0028] Summary of the Invention

[0029] Despite the widespread technical prejudice in the art against the suitability of rimcazole as a clinical candidate, the present inventors have hypothesised, and subsequently exemplified, that in the clinical condition of spontaneous cancer (malignant neoplasia that arises naturally in an authentic patient, as distinct from an animal model engineered to develop cancer in the laboratory), rimcazole can in fact be administered at doses that are well tolerated and are effective in treating or managing cancer in patients, in particular canine and human patients.

[0030] The present invention has been reached as a result of new reasoning, by the inventors that a known pharmacological property of rimcazole - inhibition of dopamine reuptake by high affinity (nanomolar) binding to the dopamine transporter- would enhance the anti-tumour action of rimcazole in the clinical situation of spontaneous cancer such that it would, against all prior expectation, be capable of treating the disease in a safe and effective manner - at plasma (blood) concentrations substantially below the toxic threshold. This would be achieved in two ways:

[0031] Firstly, by high affinity (nanomolar) binding of rimcazole to the dopamine transporter which can result in anti-tumour action of its own through modulation of dopamine pathways.

[0032] Secondly, the high affinity binding of rimcazole to the dopamine transporter was reasoned, in the specific situation of spontaneous cancer in a patient, to secondarily enhance the binding affinity of rimcazole to a second target - the sigma-1 receptor - through a cooperative allosteric interaction between the two molecular targets when physically complexed in vivo. The basis for this aspect of the hypothesis was that an inter-molecular association between the dopamine transporter and the sigma-1 receptor (a molecular chaperone) is known to occur and that this molecular association alters protein conformation and enhances ligand binding affinity for the molecular complex (Hong et al. 2017). Activities of rimcazole attributable to its antagonist action at the sigma-1 receptor would therefore be revealed at unexpectedly low (nanomolar, non-toxic) concentrations in certain situations.

[0033] The aforementioned explains why agents of the invention that are useful to treat spontaneous cancer are not restricted to rimcazole but more generally include compounds that combine the property of dopamine transporter inhibition with antagonism of the sigma-1 receptor.

[0034] The inventors have proceeded, by way of clinical exemplification, to demonstrate that rimcazole does indeed achieve highly meaningful anti-tumour action at safe doses associated with plasma concentrations that are below the toxic, micromolar blood level.

[0035] The action of rimcazole as an inhibitor of the dopamine transporter (DAT) is widely considered in the art to be an undesirable side-activity in the context of its potential as a treatment for cancer. Indeed, as stated by Kim and Maher in the book chapter Sigmal Pharmacology in the Context of Cancer, 2017: “A concern with using doses of rimcazole that may be required for anti-tumor activity is that rimcazole is also a potent dopamine transporter (DAT) inhibitor. Rimcazole binds Sigmal with low affinity and binds DAT with high affinity." The view has continued to be widely held that the higher affinity of rimcazole for DAT, compared to the sigma- 1 receptor, is undesirable and is responsible for off-target, undesirable effects that teach against its use in the context of cancer treatment.

[0036] Contrary to this generally accepted teaching, however, the present inventors reasoned and subsequently demonstrated that rimcazole’s action as an inhibitor of the dopamine transporter is, in fact, critical to its anti-tumour activity.

[0037] As described for the first time herein, the dopamine-modulatory action of rimcazole enables it to substantially inhibit tumour growth at doses of drug leading to plasma concentrations that are consistently in the sub-micromolar (below 1 micromolar) range, thus avoiding the risk of toxicity. Inhibition of cellular uptake of dopamine by rimcazole leads to a rise in extracellular dopamine that exerts a dopaminergic effect at cell surface dopamine receptors.

[0038] This mechanism is restricted to a clinical situation not previously studied in the art - namely, authentic patients with spontaneous cancer. The involvement of dopamine further teaches specific tumour types that will be particularly suitable for treatment with rimcazole, as well as new clinical populations.

[0039] The present invention in summary demonstrates - for the first time in the art - that rimcazole can after all be administered safely while at the same time achieving clinically important anti- tumour efficacy in authentic patients. This is in complete contradistinction from the teaching in the art, that it was not a viable clinical candidate for the treatment of cancer.

[0040] Accordingly, in one aspect, the present invention provides a method of treating spontaneous cancer in a human or canine patient by increasing the levels of extracellular dopamine in the cancer tissue.

[0041] In a further aspect, a method of treating spontaneous cancer in a human or canine patient is provided by an increase in the level of extracellular dopamine in the cancer tissue that acts coordinately, and cooperatively, with antagonism of the sigma-1 receptor.

[0042] In one aspect, the invention provides a method of treating spontaneous cancer in a human or canine patient comprising administering an effective amount of an inhibitor of the dopamine transporter (DAT), also known as a dopamine reuptake inhibitor, to the patient.

[0043] In some embodiments, the inhibitor of the dopamine transporter cooperates with sigma-1 receptor antagonism.

[0044] Accordingly, in a further aspect, the invention provides a method that administers an effective amount of an inhibitor of the dopamine transporter (DAT) alongside an effective amount of an antagonist of the sigma-1 receptor, to the patient.

[0045] In some embodiments, the two actions may be derived from a single compound that combines inhibition of the dopamine transporter (DAT) with antagonism of its molecular associate - the sigma-1 receptor - resulting in a cooperative allosteric interaction that enhances binding affinity. One such compound is rimcazole.

[0046] In some embodiments, the method comprises administering an effective amount of a compound which is rimcazole, or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof, to the patient.

[0047] In related aspects, the present invention also provides an inhibitor of the dopamine transporter (DAT), also known as a dopamine reuptake inhibitor, that may in one embodiment be combined with an antagonist of the sigma- 1 receptor, for use in said methods of treatment.

[0048] In a further embodiment, the invention provides a compound that combines inhibition of the dopamine transporter (DAT) with antagonism of the sigma-1 receptor in a single compound such as rimcazole, for use in said methods of treatment. Also provided is the use of an inhibitor of the dopamine transporter (DAT), also known as a dopamine reuptake inhibitor, that may be combined with an antagonist of the sigma-1 receptor, in the manufacture of a medicament for use in said methods of treatment.

[0049] In related aspects, the present invention also provides a compound which is rimcazole, or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof, for use in said methods of treatment. Also provided is the use of a compound which is rimcazole, or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof, in the manufacture of a medicament for use in said methods of treatment.

[0050] In a further embodiment, rimcazole, or other compound(s) of the invention, is given in combination with an anti-inflammatory drug.

[0051] Without wishing to be bound by theory, administration of rimcazole, or other compound(s) of the invention, in combination with an anti-inflammatory drug may improve anti-tumour efficacy by suppressing an inflammatory drive that opposes the effectiveness of the cancer treatment.

[0052] The anti-inflammatory drug treatment is preferably a non-steroidal anti-inflammatory drug (NSAID) that selectively inhibits the cyclo-oxygenase-2 (COX-2) enzyme in preference to other members of the COX family; such COX-2 inhibitors are commonly referred to as “coxibs”.

[0053] In the methods of the invention, advantageously, the compound(s) is / are administered such that the blood (plasma) levels of rimcazole are consistently below a concentration of 1 micromolar.

[0054] In some embodiments, treatment of spontaneous cancer comprises one or more of controlling (slowing) the growth of the disease (producing disease stabilisation); controlling the spread of the disease; extending the life of the patient; maintaining or improving the quality of life of the patient.

[0055] In some embodiments, treatment of spontaneous cancer comprises the aim of producing curative benefit such that long-lasting remission or cure (eradication of disease) is achieved.

[0056] In some embodiments, treatment is of a canine patient with spontaneous cancer.

[0057] In some embodiments, treatment is of a human patient with spontaneous cancer.

[0058] In some embodiments, the human or canine patient is an immunocompetent patient i.e. the patient has a functioning immune system. This enables the immunotherapy action of rimcazole to be harnessed in the patient.

[0059] In some embodiments, the inhibitor of the dopamine transporter (e.g.. rimcazole) mediates antitumour action by an immunotherapy action.

[0060] In some embodiments, the immunotherapy action is achieved by the inhibitor of the dopamine transporter acting in concert with a sigma-1 antagonist that may optionally be delivered in the form of a single compound such as rimcazole. The cooperative association between two drug targets achieves sigma- 1 antagonist-mediated immunotherapy effects at plasma concentrations in a safe, non-toxic (submicromolar) range - in contradiction to the prior art teaching.

[0061] In some embodiments, the spontaneous cancer is an aggressive (i.e. rapidly growing) and / or advanced cancer. In some embodiments, the spontaneous cancer is aggressive and at an early stage.

[0062] In some embodiments, the spontaneous cancer is at an advanced (late) stage.

[0063] In some embodiments, the spontaneous cancer is a mast cell tumour.

[0064] In some embodiments, the spontaneous cancer is a squamous cell carcinoma (SCC). In some embodiments, the spontaneous cancer is a squamous cell carcinoma selected from nonmelanoma skin cancer; head and neck cancer including mouth and throat cancer; oesophageal cancer; non-small cell lung cancer (NSCLC); cancer of the cervix; vulval cancer; vaginal cancer; penile cancer; scrotal cancer; anal cancer; non-transitional bladder cancer; peritoneal (serous) cancer; and ocular (conjunctival) cancer.

[0065] In some embodiments, the spontaneous cancer is a malignant melanoma.

[0066] In some embodiments, the spontaneous cancer is mammary carcinoma.

[0067] In some embodiments, the spontaneous cancer is a tumour that is susceptible to immunotherapy.

[0068] In some embodiments, the spontaneous cancer is a fibrosarcoma.

[0069] In some embodiments, the spontaneous cancer is sarcoma of bone or soft tissue origin, melanoma, or haemangiosarcoma.

[0070] In some embodiments, the spontaneous cancer is selected from non-melanoma skin cancer; stomach cancer; colon cancer; anorectal cancer; bladder cancer; mammary cancer; kidney cancer; lung cancer; liver cancer; oral cancer; oesophageal cancer; B and T cell lymphoma; and blood cancer (leukaemia).

[0071] In some embodiments, the inhibitor of the dopamine transporter (e,g, rimcazole) is provided in an oral dosage form.

[0072] In some embodiments, the compound is administered to a canine patient with spontaneous cancer in a dose from about 5 mg / kg body weight daily up to about 30 mg / kg body weight daily.

[0073] In some embodiments, the compound is administered to a human patient with spontaneous cancer in a dose from about 1 mg / kg body weight daily up to about 5 mg / kg body weight daily.

[0074] In some embodiments, the inhibitor of the dopamine transporter (e,g, rimcazole) - is provided in a topical formulation.

[0075] In some embodiments, the compound of the invention - an inhibitor of the dopamine transporter that optionally is also an antagonist of the sigma-1 receptor (e,g, rimcazole) - is provided in a topical formulation. In some embodiments, the topical formulation is for application to the skin and is for local treatment of melanoma (melanocyte) cancer in a human or canine patient.

[0076] The present invention includes the combination of the aspects and preferred features described, except where such a combination is clearly impermissible or expressly avoided.

[0077] Summary of the Figures

[0078] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures:

[0079] Rimcazole as monotherapy delays disease progression:

[0080] Figures 1 -5 demonstrate the effectiveness of rimcazole to slow the progression of canine cancer when administered as monotherapy (given as a single agent)

[0081] Figure 1. Photographs of canine with a large mast cell tumour on the dorsum (back) of the animal: before (Figure 1A) and after 12 weeks (Figure 2B) of oral rimcazole treatment at a total daily dose of 10 mg / kg body weight. This demonstrates a circa 22% reduction in the longest diameter of the tumour from baseline (90 mm to 70 mm) after 12 weeks of daily rimcazole treatment. This qualifies as stabilisation of tumour growth by RECIST criteria. In addition, the tumour margins became more circumscribed (better defined) after rimcazole treatment.

[0082] Figure 2. Photographs of canine with mast cell tumour located on the forelimb: before (Figure 2A) and after 4 weeks of oral rimcazole at 5 mg / kg / day (Figure 2B). Despite administration of rimcazole at the lowest dose (5mg / kg / day), tumour stabilisation was achieved by 4 weeks with further signs of improvement after 8 weeks of treatment.

[0083] Figure 3. Photographs of canine with a large fibrosarcoma tumour on the underbelly: before (Figure 3A) and after 8 weeks (Figure 3B) of oral rimcazole at 10 mg / kg daily for 4 weeks and 20 mg / kg daily for a further 4 weeks.

[0084] Figure 4. Photographs of canine with aggressive squamous cell carcinoma of the forelimb before (Figure 4A) and after (Figure 4B) 8 weeks of oral rimcazole treatment at a total daily dose of 10 mg / kg body weight. This demonstrates clear evidence of tumour slowing after 8 weeks of treatment.

[0085] Figure 5. Photographs of canine with aggressive, recurrent conjunctival melanoma at the site of enucleation - surgical removal of the eye - before (Figure 5A) and after 2 months of daily treatment with a topical formulation of rimcazole applied to the skin (Figure 5B). Topically applied rimcazole resulted in symptomatic benefit (reduction in bleeding and exudation and improved quality of life). Disease coincides with the onset of inflammation:

[0086] Figure 6 demonstrates the loss of disease control (emergence of disease progression) beyond 8 weeks. This coincides with evidence of a peri-tumour inflammatory response. Refer also to Table 3 for a summary of efficacy outcomes at time points through to 12 weeks.

[0087] Photographs of canine with aggressive squamous cell carcinoma of the forelimb before (Figure 6A), after 8 weeks (Figure 6B) and 12 weeks (Figure 6C) of oral rimcazole treatment at a total daily dose of 10 mg / kg body weight. Whereas there was clear evidence of disease control (stabilisation) through to 8 weeks, disease progression was observed beyond this time point which coincided with evidence of an inflammatory response (redness and swelling of the surrounding tissue - Figure 6C).

[0088] Combination regimen (rimcazole plus a COX-2 inhibitor) enhances magnitude and duration of anti-tumour efficacy:

[0089] Figures 7 onwards demonstrate the enhancement and duration of anti-tumour efficacy - such that partial and complete remissions of target lesions are now observed - when rimcazole is combined with a COX-2 inhibitor (firocoxib)

[0090] Figure 7 Photographs of canine with aggressive squamous cell carcinoma before (Figure 7A), after 4 weeks (Figure 7B) and 8 weeks (Figure 7C) treatment with oral rimcazole at a total daily dose of 10 mg / kg body weight in combination with firocoxib at a standard antiinflammatory dose for canines (5 mg / kg body weight). A clear objective, partial response was observed at 4 weeks, with evidence of further reduction in tumour size at 8 weeks.

[0091] Figure 8 Further photographs of canine with aggressive squamous cell carcinoma before (Panel A), after 8 weeks (Panel B) and 16 weeks (Panel C) treatment with oral rimcazole at a total daily dose of 10 mg / kg body weight in combination with firocoxib at a standard antiinflammatory dose for canines (5 mg / kg body weight). A clear and sustained partial response of the target lesion through to 16 weeks of treatment is observed, with concomitant signs suggestive of a tumour healing response (scab formation) at 16 weeks.

[0092] Tumour growth is measured by the gold standard RECIST criteria - response evaluation criteria in solid tumours; a partial response is concluded (by RECIST criteria) from the observed greater than 30% reduction in the longest diameter of the principal target lesion.

[0093] Figure 9 Photographs of a canine with malignant melanoma of the oral cavity (Figure 9A) that has spread (metastasised) to the lungs (Figures B - D, serial pulmonary radiographs - chest X- rays - lateral view). The baseline, pre-treatment radiograph (Figure 9B) illustrates the presence of multiple pulmonary (lung) metastases - opaque, globular shadows on the radiograph indicative of secondaries from the oral melanoma. Figures 9C and 9D demonstrate sustained, complete resolution of the lung metastases after 4 weeks (9C) and 8 weeks (9D) treatment with rimcazole at a total daily dose of 10 mg / kg body weight in combination with firocoxib at 5 mg / kg body weight.

[0094] Rimcazole monotherapy is effective in the adjuvant treatment of metastatic disease following treatment of the primary tumour

[0095] Figure 10 Photographs of a canine with metastatic mammary carcinoma that has spread (metastasised) to the lungs (Figures 10A and 10B, pulmonary radiographs - chest X-rays - lateral view). The baseline, pre-treatment radiograph (Figure 10A) illustrates a discrete metastatic - secondary - tumour deposit, marked up within a ring. After 4 weeks treatment with rimcazole (total daily dose 10 mg / kg body weight) there has been a complete resolution of lung metastases (Figure 10B).

[0096] Detailed Description of the Invention

[0097] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0098] Dopamine modulation

[0099] As described herein, it has surprisingly been found that the dopamine-modulatory action of rimcazole enables it to substantially inhibit tumour growth at concentrations in the submicromolar (below 1 micromolar) range, thus avoiding the risk of toxicity. Inhibition of cellular uptake of dopamine by rimcazole leads to a rise in extracellular dopamine that exerts a dopaminergic effect at cell surface dopamine receptors.

[0100] Dopamine is classically considered to have a role as a neurotransmitter in the central nervous system but is increasingly recognized to have important roles outside the central nervous system - including as an immunomodulator in the peripheral immune system with diverse immunostimulatory actions including the promotion of cytotoxic T lymphocyte mediated cell death, an important safeguard against tumour development that is also harnessed in the treatment of tumours via immunotherapy. Moreover, there is an increasing body of evidence that dopamine signalling in peripheral tissues is perturbed in cancer, leading to efforts to identify ways to modulate dopamine (DA) pathways as a novel route to cancer treatment (reviewed in Grant et al, 2022). Five cell surface dopamine receptors are known to exist (DRD1 - DRD5) that fall into either D1-like or D2-like receptor types, both of which have been investigated as potential targets for cancer treatment. In common with other tumour growth modulatory pathways, the interplay between different receptor types is likely to be complex, with agonism or antagonism at different DA receptor types leading variously to inhibition of tumour cell proliferation, induction of cell death, inhibition of angiogenesis and effects on cancer stem cells. The dopamine transporter itself has not thus far been specifically studied as a target for antitumour treatment. The dopamine transporter (DAT) is responsible for the uptake (sequestration) of dopamine inside the cell; inhibition of DAT leads to a rise in extracellular dopamine that is free to act on cell surface dopamine receptors. Rimcazole is a known high affinity inhibitor of the dopamine transporter (Gilmore et al, (2004)) and therefore has a dopaminergic effect, leading to a rise in dopamine-mediated cell signalling.

[0101] To date, the clinical applicability of rimcazole as an inhibitor of the dopamine transporter has been investigated for its potential to mediate favourable neurological effects such as effects in psychiatric patients on mood and in the management of cocaine dependence (Katz et al, (2003)). However, as noted above, its action as an inhibitor of the dopamine transporter (DAT) has been widely considered in the art to be an undesirable side-activity in the context of its potential as a treatment for cancer.

[0102] There is accumulating evidence that agonistic (stimulatory) signalling at the D1 dopamine receptor - as would be achieved by a rise in extracellular dopamine - is linked to the potential for significant tumour growth suppression, prevention of metastasis, as well as the promotion of tumour microvasculature maturation (Pawel et al (2020)). Synergistic effects when dopamine receptor agonists are combined with other anti-cancer agents have also been reported.

[0103] Without wishing to be bound by theory, the present inventors hypothesise that the dopamine transporter has potential to exert a more general tumour-promoting effect, by reducing extracellular dopamine. Studies specifically of the dopamine transporter in tumour tissue are beginning to yield useful information - in particular that the dopamine transporter may be expressed by non-neural tumours where it confers a growth advantage to the tumour that is associated with reduced survival of the patient. Given that the action of the dopamine transporter is to reduce extracellular dopamine levels by promoting dopamine transport (sequestration) inside the cell, the ectopic expression of the dopamine transporter by peripheral tumours could be anticipated to confer a growth advantage. Consistent with this, in humans with renal cell carcinoma, the dopamine transporter has been identified as a biomarker of reduced survival following surgery (Schrodter et al, 2016). It follows from this that a rise in extracellular dopamine by inhibition of the dopamine transporter may have general potential as an approach to anti-tumour treatment.

[0104] DAT Inhibitors:

[0105] Consistent with the findings described herein, the present invention provides for the use of inhibitors of the dopamine transporter (DAT), also known as dopamine reuptake inhibitors, in the treatment of spontaneous cancer in authentic patients, in particular human and canine patients. Examples of dopamine reuptake inhibitors include rimcazole, amineptine, methylphenidate (Ritalin), nomifensine, cocaine, methylenedioxypyrovalerone (MDPV), cocaine, altropane, amfonelic acid, benocyclidine, DBL-58, difluoropine, fluorenol, GBR-12935, GYKI-52895, modafinil, RTI-229, lometopane, vanoxerine, medifoxamine, ketamine, phencyclidine, sertraline, hyperforin and adhyperforin.

[0106] In preferred embodiments of the present invention, the inhibitor of the dopamine transporter is the compound rimcazole.

[0107] Dual DAT inhibition and sigma-1 antagonism

[0108] Consistent with the findings described herein, the present invention provides for the combined use of DAT inhibitors together with sigma-1 antagonists - either as separate compounds or as a single compound, such as rimcazole, that combines both activities.

[0109] The invention exemplifies how a cooperative allosteric interaction between DAT and the sigma- 1 receptor can, in particular situations such as spontaneous cancer in a patient, enhance the binding affinity of sigma-1 antagonists such that sigma-1 mediated properties, hitherto considered to reguire micromolar plasma (extracellular) concentrations, can in fact be revealed at low, submicromolar concentrations. Sigma-1 antagonists that were previously considered too unsafe to be of use as effective anti-tumour agents now become viable clinical candidates.

[0110] Exemplary sigma-1 site antagonists include but are not limited to rimcazole, haloperidol, reduced haloperidol, I PAG (I -(4-iodophenyl)-3-(2-adamantyl)guanidine), BD- 1047 (N(-)[2-(3,4- dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine), BD-1063 (I (-)[2-(3,4- dichlorophenyl)ethyl]-4-methylpiperazine), (+)-SKF- 10047 ((+)-N-allyl normetazocine), (+)- benzomorphans including (+)-pentazocine and (+)-ethylketocyclazocine, (+)-morphinans including dextrallorphan, cis isomers of LI50488 and analogues, arylcyclohexamines including PCP, N,N'-diryl-substituted guanidines including DTG (1 ,3-di(2-tolyl)guanidine), phenylpiperidines including (+)-3-PPP and OHBQs, steroids including progesterone and desoxycorticosterone, butryophenones, BD614,(+ / -)-cis-N-methyl-N-[2-(3,4- dichlorophenyl)ethyl]-2-(l-pyrrolodinyl)cyclohexylamine, perphenazine, fluphenazine, (-)- butaclamol, acetophenazine, trifluroperazine, molindone, pimozide, thioridazine, chlorpromazine, triflupromazine, BMY 14802, BMY 13980, remoxipride, tiospirone, cinpuperone WY47384, and antidepressants including amitriptyline and imipramine and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, derivatives or metabolites thereof.

[0111] Rimcazole

[0112] Rimcazole (c / s-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]carbazole) is known in the art and routes for its manufacture are well established. Rimcazole has two stereogenic centres at the 3 and 5 positions of the piperazinyl ring and, as used herein, the term "rimcazole" also includes stereoisomers of rimcazole. In one embodiment, the stereoisomer of rimcazole is the (3S,5R) isomer, i.e. 9-[3-[(3S,5R)-3,5-dimethylpiperazTn-l- yl]propyl]carbazole, having the structural formula shown below.

[0113] Rimcazole may be administered as rimcazole base with the chemical formula shown below, or as a pharmaceutically acceptable salt, hydrate, solvate, ester, prodrug, or derivative thereof. The term rimcazole is used to include all pharmaceutically acceptable forms of rimcazole.

[0114] In other words, as used herein, the term ‘rimcazole’ is understood to include all pharmaceutically acceptable salts, esters, hydrates, solvates, prodrugs, or derivatives thereof that have in common the same active principle - rimcazole base - that is available to the systemic circulation and through which its biological effects are mediated. These forms of rimcazole may be referred to as rimcazole base and rimcazole equivalents, where distinction is helpful.

[0115] Salts

[0116] Pharmaceutically acceptable salts are known in the art. Examples may be found in Berge et al (J. Pharm. Sci, 1977).

[0117] Suitable pharmaceutically acceptable salts include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

[0118] Suitable salts of rimcazole which are known in the art include rimcazole dihydrochloride and rimcazole hemifumarate.

[0119] The term "prodrug" means a pharmaceutically acceptable form of a functional derivative of rimcazole (or a salt thereof), wherein the prodrug may be: 1) a relatively active precursor which converts in vivo to an active prodrug component; 2) a relatively inactive precursor which converts in vivo to an active prodrug component; or 3) a relatively less active component of the compound that contributes to therapeutic biological activity after becoming available in vivo. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in, for example, EP 0 012 208 which is herein incorporated by reference in its entirety).

[0120] Metabolites

[0121] In humans, rimcazole is known to be metabolized by CYP2D6 (IMB_Report_RimcazoleTrial_031011.Pdf), an enzyme subject to genetic variation that influences clearance of the drug from the bloodstream. An orthologue (functional equivalent) of CYP2D6 has been identified in canines: CYP2D15 (Jimenez et al. (2023).

[0122] Whereas metabolism by CYP2D6, and its orthologues, generates complexity through interspecies pharmacokinetic differences, CYP2D6 metabolism also affords a therapeutic opportunity through the formation of bioactive CYP2D6-generated metabolites (including those generated by a CYP2D6 orthologue). It is well understood in the art that CYP2D6 metabolism can lead to the generation of bioactive molecules and in some cases, CYP 2D6 metabolism is required for drug activity: for example, the pharmacological activity of codeine is dependent on its conversion to morphine via CYP2D6. (Dean L, Kane M. in: Pratt VM et al, 2012)

[0123] It follows from this that bioactive metabolites of rimcazole generated by the enzyme CYP2D6, or a canine CYP2D6 orthologue, have potential to act as dopamine reuptake inhibitors, and therefore as safe and effective anti-tumour agents, in their own right. In a further embodiment therefore, the invention is understood to include bioactive metabolites of rimcazole, generated by the enzyme CYP2D6 or a canine orthologue.

[0124] Rimcazole or other agents of the invention used in combination with NSAIDs, in particular selective COX-2 inhibitors (“coxibs”)

[0125] Consistent with the findings described herein, the present invention provides for the combined use of compounds of the invention, rimcazole being the exemplar, with anti-inflammatory drugs including non-steroidal agents (NSAIDs) and in particular, selective inhibitors of the cyclooxygenase-2 (COX-2) pathway in order to enhance anti-tumour efficacy.

[0126] The scope of the invention also encompasses combination with broader spectrum agents used to treat inflammation such as corticosteroids including, but not limited to, prednisone and dexamethasone.

[0127] Such agents are preferably administered at non-immunosuppressive doses so that the cytotoxic immunotherapy actions of rimcazole are not impeded. Those skilled in the art - clinical practitioners responsible for prescribing the medicines to cancer patients - are well able to determine what an inflammatory dose versus an immunosuppressive dose of a corticosteroid would be, for the relevant patient population.

[0128] The rationale for the choice of this as a combination regimen is explained in more detail below. cancer - 1 of animal models

[0129] The action of rimcazole as an inhibitor of the dopamine transporter has not been uncovered in any of the experimental model systems used in the art - leading to a false presumption of a need for micromolar blood (plasma) concentrations for it to mediate anti-tumour action in patients with cancer. It is hypothesised that rimcazole’s ability to mediate anti-tumour activity via inhibition of the dopamine transporter was not capable of being deployed in the prior experimental model systems studied owing to fundamental differences in the biology of the experimental neoplasms (engineered in the laboratory) and spontaneous cancer in an authentic patient. This is further explained below.

[0130] The experimental model systems studied to date during the investigation of rimcazole as a potential treatment for cancer are flawed to teach safe and effective doses of rimcazole for the treatment of spontaneous cancer in patients. Deficiencies in the model systems led to a false assumption that doses of rimcazole leading to micromolar plasma concentrations would be required for effective treatment of cancer and that these doses would in turn be well tolerated. As explained above, human trials in 2010 demonstrated clearly that such doses were severely toxic.

[0131] A number of deficiencies exist in the experimental model systems:

[0132] 1. Mice are less susceptible to induction of seizures by rimcazole (in contrast to humans and canines)

[0133] Rimcazole appeared to be safe and effective as an anti-tumour agent in the mouse xenograft models but this was misleading as it is now understood that mice have a higher seizure threshold (viz. they are less susceptible to seizures) when exposed to rimcazole, meaning they withstand micromolar plasma concentrations without ill effect. Concentrations of 2-3 micromolar rimcazole were reached in the blood of the host mice after a single dose of 40 mg / kg. It can be deduced from this that repeat daily dosing at 40 mg / kg for a period of two weeks would have led to blood concentrations in excess of 2 - 3 microM at steady state. Despite this, there was no evidence of seizure induction.

[0134] 2. The model systems in the art do not recapitulate the selective pressures faced by a naturally occurring spontaneous tumour

[0135] The in vitro models and experimental animal models described in the art to investigate rimcazole as a potential therapeutic, employed tumour cell lines grown for extended periods in the laboratory under growth factor-rich conditions. These are very different from the conditions faced by a cancer that arises spontaneously in a patient where the cancer self-selects to survive in a nutrient and growth factor deprived environment.

[0136] Mouse xenograft models are well recognised to have many limitations and to be poor overall predictors of clinical benefit (B. W. Simons and C. Braytonin Patient Derived Tumor Xenograft Models (2017)). In contrast to mouse xenograft models, spontaneous cancer that arises in a patient is therefore inherently different from an experimental model of cancer and in therapeutic terms can be considered an inherently different clinical condition.

[0137] 3. Experimental xenograft models used immunodeficient mice as hosts

[0138] Immunodeficient mice are unsuitable to evaluate potential immunotherapies for cancer treatment, as their defective immune system means they are incapable of mounting an immune response to a foreign (tumour) antigen. It is taught herein that rimcazole has the potential to act as an immunotherapy agent through its modulation of dopamine pathways. In particular, modification of the dopamine transporter that in turn enhances the binding affinity of rimcazole to the sigma-1 receptor uncovers an immune checkpoint inhibition mechanism, and other immunomodulatory actions, at non-toxic submicromolar concentrations of rimcazole. This could never be predicted from models using immunodeficient hosts.

[0139] Indeed, in a published world-wide patent (Patel et al, 2010) rimcazole as monotherapy failed to produce any anti-cancer activity at any dose level (30, 22.5, 10, 3 mg / kg by daily oral administration) against advanced, aggressive breast cancer (MDA MB 231) xenografts in an immunodeficient mouse host. Combination with a taxane was required to produce anti-tumour effects and even then, a dose of rimcazole (22.5 mg / kg) was required that, as explained elsewhere in this document, is associated with plasma concentrations that are safe in mice (do not provoke epileptic seizures) but are unsafe in dogs and humans. It is further explained herein that a mg / kg dose in mice cannot be empirically extrapolated to a mg / kg dose in dogs or humans for two reasons: the fundamental principle of allometric scaling between species that adjusts for body surface area when calculating drug dose; secondly, the well-known pharmacokinetic differences between animal species. This prior art supports the inadequacy of immunodeficient mouse hosts to demonstrate anti-cancer effectiveness of rimcazole at safe (submicromolar) plasma concentrations.

[0140] Deficiencies in the model systems used thus far are reasonably hypothesised to have arisen because the model systems cannot accurately reflect the evolution and selective pressures upon tumour growth during the development of spontaneous cancer in a patient. The model systems have comprised cultured tumour cells grown in the laboratory or mice engineered to have a defective immune system so the host animals can support the growth of engrafted human tumours. Both are highly artificial systems.

[0141] The inventors therefore assert that spontaneous cancer is a fundamentally different clinical condition from the highly artificial model systems used to date to explore the potential of rimcazole as a treatment for cancer. This step change in rationale has enabled the new invention - use of rimcazole for the safe and effective treatment of spontaneous cancer in authentic patients, which the prior art taught was implausible. cancer

[0142] In one aspect of the invention, dopamine transporter inhibitors that may optionally and advantageously be combined with sigma-1 antagonists, including the exemplar rimcazole, are of use in the treatment of spontaneous cancer.

[0143] The term “spontaneous” cancer will be understood to refer to malignant neoplasia which has arisen naturally, in an authentic patient, in particular a human or canine patient. It therefore expressly excludes engineered tumours, such as would arise from an animal model developed in a laboratory.

[0144] The term “treatment” as used herein includes one or more of: controlling the growth of the disease; controlling the spread of the disease; extending the life of the patient; maintaining or improving the quality of life; inducing partial or complete remission; producing curative benefit.

[0145] The present invention provides new understanding, previously unknown in the art, that rimcazole’s action at the dopamine transporter causes a marked enhancement of its anti-tumour activity such that it is now effective at submicromolar plasma concentrations which are nontoxic. Indeed, the effectiveness of rimcazole at submicromolar concentrations in the blood could not have been predicted from the prior art which consistently taught that rimcazole’s antitumour actions require micromolar concentrations to be achieved in the bloodstream (in the case of in vivo xenograft studies) and tissue culture media (in the case of in vitro studies) - concentrations considered to be consistent with rimcazole’s known low affinity for sigma-1 sites - in the micromolar range (Gilmore et al (2004)).

[0146] The enhancement of rimcazole’s anti-tumour activity, such that it is effective at non-toxic, submicromolar plasma concentrations, is reasoned to be achieved in two ways: firstly, by the action of rimcazole as an inhibitor of the dopamine transporter, in its own right; secondly, by a cooperative allosteric interaction between the dopamine transporter and the sigma-1 receptor, such that the binding affinity of rimcazole for sigma-1 sites is enhanced - enabling sigma-1 antagonist effects at submicromolar plasma concentrations.

[0147] Patient populations - the relevance of immune competence

[0148] The present invention provides generally for the treatment of patients with spontaneous cancer, in particular human and canine patients.

[0149] Cancer patients may have a compromised immune system - this may be as a result of chemotherapy or radiotherapy, or it may be due to the disease itself. Moreover, immunosuppressive drugs are commonly prescribed to cancer patients for concomitant illnesses or conditions from which the patient may also be suffering; such medications would include corticosteroids at immunosuppressive doses and broad spectrum immunosuppressants for the treatment of inflammatory conditions such as rheumatoid arthritis. Without wishing to be bound by theory, cancer patients with defective immune systems may be less suitable to receive treatment with rimcazole, as the full spectrum of rimcazole’s anti-tumour actions, mediated through immunomodulatory dopaminergic effects, cannot be harnessed in such patients.

[0150] New patient populations are therefore taught from those that were disclosed previously. These new patient populations are those with a functioning immune system.

[0151] In a particular aspect of the invention, therefore, inhibitors of the dopamine transporter and rimcazole in particular can mediate anti-tumour action by an immunotherapy action, in patients that are not immunocompromised.

[0152] In particular embodiments, rimcazole is used in the treatment of spontaneous cancer in canine or human patients where the patient has a competent (functioning) immune system, i.e. wherein the human or canine patient is an immunocompetent cancer patient.

[0153] Although the dopamine transporter is more usually studied in the context of neural tissue, it is increasingly recognized to be expressed by non-neural tissues - notably in cells of the immune system - where dopamine is considered to have an important immunomodulatory role, with dopamine having the ability to stimulate the cytotoxic activity of T lymphocytes (Broome et al, 2020). It is further hypothesized therefore that rimcazole-mediated inhibition of the dopamine transporter in cytotoxic T lymphocytes recruited to a tumour site will lead to a dopamine- mediated enhancement of cytotoxic T lymphocyte-mediated anti-tumour action. As well as direct actions on the tumour and its microenvironment, submicromolar plasma concentrations of rimcazole are consistent with an immunotherapy action against tumours, mediated through dopamine pathways.

[0154] It is further taught for the first time herein, that rimcazole may mediate an immunotherapy action as an immune checkpoint inhibitor at safe, non-toxic, submicromolar plasma concentrations through a cooperative allosteric interaction between the dopamine transporter and sigma-1 receptor. This is explained in more detail below.

[0155] The present invention therefore teaches, for the first time, a new clinical population of immunocompetent patients for the use of rimcazole as an immunotherapy in the safe and effective treatment of spontaneous cancer, mediated through the modulation of dopamine acting in its own right, as well as through enhanced binding affinity of rimcazole to the sigma-1 receptor resulting from a cooperative allosteric interaction with the dopamine transporter.

[0156] Therefore, preferred clinical populations in the present invention are immunocompetent canine or human patients with spontaneous cancer. Immunocompetence can be established in the patient by white blood cell (WBC) counts - total and differential - measured in peripheral blood samples that should be within the laboratory reference range for the species and the age of the patient. The differential WBC count measures the percentage of each of the five types of white blood cell. Correspondingly, patients receiving concomitant broad spectrum immunosuppressant drugs for other illnesses (such as corticosteroids at immunosuppressive doses) are less suitable to receive rimcazole as cancer treatment as the immunosuppressant drugs they are taking will antagonise a cytotoxic immune response elicited by rimcazole.

[0157] Tumour types I stages

[0158] Advanced, aggressive cancers

[0159] In an aspect of the invention, rimcazole is used at safe and effective doses in the treatment of advanced and / or aggressive spontaneous tumours. This is demonstrated herein by way of clinical examples in canine patients with cancer. This exploits the multi-pronged action of the molecule that exerts direct anti-tumour action, anti-angiogenic activity and, as disclosed herein for the first time, immunomodulatory actions at safe, submicromolar concentrations.

[0160] The combination of these actions renders advanced, aggressive cancers suitable for treatment with rimcazole. The invention discloses for the first time that rimcazole is effective in patients with advanced cancer at safe, non-toxic plasma concentrations; the invention elucidates that this is due to the involvement of the dopamine transporter (DAT) and a secondary enhancement by the DAT of sigma-1 site binding affinity so that anti-tumour efficacy is achieved at safe plasma concentrations.

[0161] As discussed by Gilmore et al (supra) rimcazole is well known to have a substantially higher affinity for the dopamine transporter, with affinity constants in the nanomolar range, compared to the sigma-1 receptor (micromolar affinity). Rimcazole binding affinities (Kj) and IC50 values for the dopamine transporter are in the range 58 to 423 nanomolar (0.058 to 0.423 microM). It is also reported in Gilmore 2004 that rimcazole can bind to cell surface dopamine receptors D1 and D2; however, this is with lower affinity (range from 5 microM to greater than 10 microM) compared to its binding at the dopamine transporter. In tissues that express the dopamine transporter, rimcazole at submicromolar concentrations will therefore increase levels of free, extracellular dopamine that mediates a so-called dopaminergic effect.

[0162] It follows from this that at submicromolar plasma concentrations (which correspond to submicromolar concentrations in the extracellular fluid - the interstitial fluid that bathes tumour cells), rimcazole would be expected from the art to act through dopamine pathways, but not via the sigma- 1 receptor.

[0163] Notwithstanding the clear evidence of low affinity binding of rimcazole for sigma-1 sites, an in vivo molecular association between the sigma-1 receptor and dopamine transporter can increase the affinity of ligands for a sigma-1 receptor I dopamine transporter complex, through a cooperative allosteric interaction (Hong et al 2017) in some situations.

[0164] The inventors therefore hypothesised that it may be possible for rimcazole to mediate its multipronged anti-tumour action via dual DAT inhibition and sigma-1 antagonism at safe, submicromolar concentrations - made possible through a cooperative physical interaction between the sigma- 1 receptor and dopamine transporter that occurs in the particular situation of spontaneous cancer in a patient.

[0165] At doses of the drug associated with submicromolar plasma concentrations, rimcazole may not be capable of mediating an anti-tumour effect via the sigma- 1 receptor. Over time, it is possible there may be sufficient tumour-selective accumulation to reach micromolar levels within the tumour tissue (a known property of the drug, as described in Spruce et al, 2004, supra) which might then permit rimcazole to mediate anti-tumour action via sigma-1 receptor antagonism at a later stage of treatment. However, the inventors have reasonably hypothesised that in the early stages of treatment - recognised to be crucial to a successful treatment outcome - submicromolar plasma concentrations of rimcazole will mediate anti-tumour effects through its action as an inhibitor of the dopamine transporter, in its own right or through a cooperative allosteric interaction with the sigma-1 receptor. This will be of particular importance in aggressive, rapidly growing tumours.

[0166] In an aspect of the invention, therefore, rimcazole may be of particular use in the treatment of advanced and / or aggressive, rapidly growing tumours.

[0167] Aggressive tumours are particularly prone to spread (metastasise) to distant sites even at an early stage of disease. Metastatic disease is ultimately what most often kills the patient. Therefore, even if surgery or another method of treatment is successful to control the primary tumour, so-called adjuvant treatment may be required to treat or prevent metastatic spread.

[0168] The invention demonstrates that rimcazole, including as monotherapy, is effective as adjuvant treatment to treat metastatic disease following surgical excision of a primary tumour.

[0169] In an aspect of the invention, therefore, rimcazole may be of particular use in adjuvant treatment, following surgery or another method of treatment such as radiotherapy, for the primary tumour. Adjuvant treatment has the aim to treat metastatic (secondary) tumours that have spread from the primary tumour to distant sites. Adjuvant treatment may be used to treat “gross” (clinically evident) metastatic disease. It can also be used to prevent the emergence of clinically apparent metastatic disease by treating so-called micrometastases that may not be clinically evident. This is particularly useful where a tumour is aggressive, with a high propensity to spread.

[0170] In a further aspect, rimcazole may be useful as neo-adjuvant treatment where it is administered prior to surgery or another method of treatment for the primary tumour. Neo-adjuvant treatment can be followed by adjuvant treatment, subsequent to the surgery or other method of treatment.

[0171] In an aspect of the invention, rimcazole is of particular use to treat or prevent pulmonary metastatic disease where the tumour has spread to the lungs. Particular tumour types are further taught, based on a prediction of enhanced anti-tumour response arising from dopamine modulation that may in one embodiment, act in concert with sigma-1 antagonism:

[0172] Mast cell tumours:

[0173] In one embodiment the spontaneous cancer is a malignant mast cell tumour in a canine or human patient.

[0174] Mast cells are derived from the myeloid lineage and are involved in the immunoinflammatory response. Mast cells can give rise to malignant tumours - typically in the skin but they can also arise in other areas of the body including the spleen, liver, intestine and bone marrow. Mast cell tumours are very common in dogs - less so in humans - and are the commonest type of skin cancer in dogs. Mast cell tumours are challenging to treat due in part to significant variability in their biological behaviour.

[0175] Mast cells contain intracellular granules that are rich in dopamine, histamine and other biogenic amines (Ronnberg et al, (2012)). The publication by Ronnberg describes expression of the dopamine transporter (DAT) in skin mast cells which permits the uptake of extracellular dopamine for storage in intracellular granules. It follows from this that inhibition of the dopamine transporter by rimcazole, or another agent, will lead to high levels of extracellular dopamine derived from the rich intracellular stores. The inventors hypothesised therefore that mast cell tumours will be susceptible to rimcazole- mediated inhibition of DAT, leading to growth inhibitory effects mediated through elevated extracellular dopamine.

[0176] It is shown herein by way of clinical example that rimcazole inhibits the growth of canine mast cell tumours, leading to stabilisation of the disease even at an advanced stage, in the absence of toxicity. This occurs when rimcazole is administered at doses clearly associated with submicromolar concentrations of rimcazole in the blood, consistent with its high affinity binding and inhibitory action at the dopamine transporter.

[0177] Squamous cell carcinoma:

[0178] In one embodiment the spontaneous cancer is a squamous cell carcinoma in a canine or human patient.

[0179] Dopamine has been reported to prevent the development of squamous cell carcinoma in ultraviolet B-induced premalignant cutaneous lesions (Lu et al (2021)). The inventors hypothesised that squamous cell carcinoma would be susceptible to inhibition of DAT by rimcazole, or another DAT inhibitor compound, given the importance of dopamine in preventing malignant progression in this cell type. Moreover, immunotherapies have demonstrated effectiveness in squamous cell carcinoma thought to be due in part to the large number of mutations present in squamous cell carcinomas; the heavy mutational burden increases the immunogenicity of the tumour (the capacity of the immune system to recognize the tumour as “foreign”) - causing the immune system to go on the attack.

[0180] It is shown herein by way of clinical example that rimcazole causes marked slowing of tumour growth (disease stabilisation by RECIST criteria - Eisenhauer et al 2009) in a canine patient with an aggressive squamous cell carcinoma at a daily dose of rimcazole (10 mg / kg) that is associated with blood (plasma) concentrations clearly in the submicromolar (non-toxic) range. In a second patient with extensive squamous cell carcinoma, disease stabilisation has been demonstrated with rimcazole as monotherapy (refer to Table 3 for a summary of clinical outcomes in a pilot efficacy and safety evaluation of rimcazole administered as monotherapy to ten canines with advanced cancers, of whom 2 were squamous cell carcinoma cases).

[0181] Refer to Clinical examples Section 2. - Rimcazole in combination with a COX-2 inhibitor (firocoxib) for exemplification of a marked enhancement in the magnitude and duration of rimcazole-mediated anti-tumour response when rimcazole was combined with firocoxib, a licensed COX-2 inhibitor. Using this regimen, a patient with advanced squamous cell carcinoma demonstrated a clear, partial response (by RECIST criteria) of more than 80% shrinkage of the target lesion (Figures 7 and 8). A partial response has been sustained through to at least 16 weeks, in contrast to squamous cell carcinoma patients that received rimcazole as monotherapy where disease stabilization was observed up to 8 weeks. It should be borne in mind that the aforementioned cases had advanced, aggressive carcinoma.

[0182] The ability of dopamine to prevent the progression of premalignant skin lesions to squamous cell carcinoma further teaches the use of dopamine transporter inhibitors in the treatment of premalignant skin lesions.

[0183] Squamous cell carcinoma (SCC) comprises any type of cancer that arises from squamous cells - a type of epithelial cell. Squamous cell carcinoma is diagnosed histologically by those skilled in the art such as pathologists trained in the microscopic investigation of tissue specimens. Many of the common cancers arise from squamous cells. For example, approximately 90% of head and neck cancers - that include mouth and throat cancer - are SCCs. SCC is the second most prevalent type of non-small cell lung cancer (NSCLC).

[0184] The invention therefore includes, but is not limited to, a range of different types of cancer where a histological diagnosis of squamous cell carcinoma has been made.

[0185] In some types of cancer, squamous cell carcinomas comprise a high or significant proportion of cases. In other types of cancer, a diagnosis of squamous cell carcinoma (SCC) is rare but carries a worse prognosis than non-SCC histological types. Cancers where a SCC diagnosis is common: non-melanoma skin cancer; head and neck cancer including mouth and throat cancer; oesophageal cancer; non-small cell lung cancer (NSCLC); cancer of the cervix; vulval cancer; vaginal cancer; penile cancer; scrotal cancer; anal cancer; non-transitional bladder cancer; peritoneal (serous) cancer; ocular (conjunctival) cancer.

[0186] Cancers where SCC comprises a rare subset (worse prognosis): cancer of the stomach, breast, prostate, ovary; testis; liver, gall bladder and biliary tract, kidney; pancreas, rectum, thyroid.

[0187] The ability of dopamine to prevent the progression of premalignant skin lesions to squamous cell carcinoma further teaches the use of dopamine transporter inhibitors in the treatment of premalignant lesions. Premalignant lesions of squamous cell type can occur at a number of locations - in particular the skin but at other sites also e.g. the oesophagus, cervix and bladder.

[0188] In a further embodiment, therefore rimcazole or other compound(s) of the invention can be used in the treatment of a premalignant lesion of squamous cell type in a canine or human patient.

[0189] Malignant melanoma:

[0190] Dopamine is well known as an intermediate in the biosynthetic pathway for melanin within melanocytes. Melanocytes and melanoma tumours are rich in dopamine and can be deduced to require expression of the dopamine transporter to sustain high intracellular dopamine levels.

[0191] Dopamine, and its precursor DOPA, have been demonstrated to have growth inhibitory effects on melanoma cells, suggesting the potential of dopamine pathway modulation as a treatment for malignant melanoma (Doepner et al (2022)). Clinical circumstantial evidence also points to the importance of normal dopamine regulation in prevention of melanoma given the cooccurrence of Parkinson’s disease (associated with dopamine deficiency) and melanoma at significantly higher than expected rates. (Grant et al, supra).

[0192] The inventors hypothesised that malignant melanoma would be suitable for treatment with rimcazole due to inhibition of the dopamine transporter - causing movement of dopamine from inside to the outside of the cell where it then becomes free to act on cell surface dopamine receptors. The inventors further reasoned that the immunotherapy action of rimcazole would render malignant melanoma suitable for treatment.

[0193] It is shown herein by way of clinical proof of principle example that malignant melanoma in a canine patient responds to rimcazole. It is further shown herein that when rimcazole is combined with a COX-2 inhibitor, there is a complete and sustained resolution of extensive pulmonary (lung) metastases in a patient with Stage 4 oral melanoma (advanced disease with distant metastatic spread). Refer to clinical examples section 2 - rimcazole in combination with a COX-2 inhibitor. Mammary carcinoma

[0194] Triple negative (hormone insensitive) mammary carcinoma is known to be responsive to rimcazole in laboratory models. However, the prior art teaches that micromolar concentrations of rimcazole are required for anti-tumour efficacy in mammary carcinoma - concentrations that cannot be achieved safely in either humans or canines.

[0195] Triple negative breast cancer in humans and canine mammary carcinoma have a similar genetic signature and also share other important, clinically relevant similarities (Namagerdi et al, 2020) - demonstrating the power of spontaneous cancer in dogs to inform treatment of the human disease.

[0196] It is shown herein by way of clinical proof of principle example that metastatic mammary carcinoma in a canine patient responds to rimcazole. It is further shown herein that rimcazole as monotherapy is effective as adjuvant treatment, resulting in complete resolution of pulmonary (lung) metastases in a patient whose primary mammary tumour had been surgically excised. Refer to clinical examples section 3 - rimcazole is effective as monotherapy in the adjuvant treatment of metastatic disease.

[0197] Tumours responsive to immunotherapies:

[0198] In a further embodiment, the spontaneous cancers are tumours that are known to respond to immunotherapy. In some case these overlap with tumours susceptible to more direct antitumour effects of rimcazole-mediated dopamine modulation.

[0199] Dopamine has wide-ranging immunomodulatory effects in the periphery that include the promotion of T lymphocyte cytotoxic activity (Broome et al, 2020). It follows from this that the modulation of dopamine pathways and of the dopamine transporter specifically, has potential as an immunotherapy approach to treat cancer.

[0200] It is further taught for the first time herein, that rimcazole may mediate an immunotherapy action as an immune checkpoint inhibitor at safe, non-toxic, submicromolar plasma concentrations through a cooperative allosteric interaction between the dopamine transporter and sigma-1 receptor. The sigma-1 receptor is known in the art to promote accumulation of the immune checkpoint protein, PD-L1 on tumour cells, a mechanism that assists tumours to evade the body’s immune defences. Moreover, inhibition of the sigma-1 receptor, such as with a small molecule sigma-1 antagonist, inhibits the immune checkpoint and activates a T lymphocyte response (Maher et al, 2018). It is therefore known in the art that sigma-1 antagonists have potential as immune checkpoint inhibitors to treat cancer. However, the teaching in the art is also consistent that rimcazole mediates sigma-1 antagonism at micromolar concentrations; therefore, the art teaches that while it is theoretically possible for rimcazole to mediate immune checkpoint inhibition, this could not be achieved at safe, submicromolar concentrations. Contrary to the expectation in the art, in a further aspect of the present invention, rimcazole can in fact mediate immune checkpoint inhibition in patients with spontaneous cancer, at safe, submicromolar plasma concentrations; the new invention teaches that this arises from enhanced affinity of rimcazole for sigma- 1 sites due to a cooperative allosteric interaction between the dopamine transporter and sigma-1 receptor in the clinical situation of patients with spontaneous cancer.

[0201] It is described herein by way of clinical example that rimcazole leads to effects in canine patients that are consistent with engagement of an anti-tumour immune response. These effects are observed at plasma concentrations of rimcazole in the submicromolar range - supporting action of rimcazole as an immunotherapy agent through its inhibition of the dopamine transporter.

[0202] A number of tumour types respond to immunotherapy. In some case these overlap with tumours susceptible to more direct anti-tumour effects of rimcazole-mediated dopamine modulation.

[0203] Tumours that are likely to be responsive to dopamine-mediated immunomodulatory actions of rimcazole include sarcoma of bone or soft tissue origin or melanoma (a tumour of melanocytes). Examples of soft tissue sarcoma are: fibrosarcoma, histiocytic sarcoma, liposarcoma and rhabdomyosarcoma.

[0204] The poor therapeutic outcomes for sarcomas in canines and humans are common to both species but the impact is greater in the veterinary arena owing to the much higher prevalence of sarcomas in canines compared to humans. There is therefore a particular need for effective treatments for canine sarcomas.

[0205] It is shown herein by way of two clinical examples - fibrosarcoma and histiocytic sarcoma in canine patients - that rimcazole is effective and safe for the treatment of soft tissue sarcoma.

[0206] It is further shown, by way of clinical example, that an advanced soft tissue sarcoma in a canine patient - a fibrosarcoma - demonstrated clinically relevant disease stabilisation, with improved definition of the tumour margins, in response to rimcazole at a daily dose associated with submicromolar, non-toxic plasma concentrations. This is clinically important as fibrosarcoma tumours in canines are difficult to surgically excise due to invasiveness at the tumour margins, leading to a high risk of recurrence. Rimcazole treatment prior to, alongside, or after surgery will therefore reduce the risk of recurrence.

[0207] Osteosarcoma (a sarcoma of bone) is a very aggressive tumour in human and canine patients but is substantially more prevalent in the canine compared to the human population, with some canines (generally the larger and giant breeds) at particularly high risk. Osteosarcomas are among the tumour types responsive to immunotherapies.

[0208] Sarcomas of bone are, like their soft tissue counterparts, aggressive and therapy resistant. Osteosarcomas (usually affecting the limbs) are particularly prevalent in dogs. Surgical amputation extends life by around 6 months owing to a high incidence of metastatic spread to the lungs.

[0209] In some cases of canine osteosarcoma rimcazole may be used instead of amputation to inhibit the growth of the primary tumour and prevent metastatic spread. In other cases, rimcazole may be administered as neoadjuvant and adjuvant treatment, prior to and following surgical limb amputation, to prevent metastatic spread to major organs and hence to extend life. It is shown herein by way of clinical example that rimcazole leads to a complete and sustained resolution of lung metastases in a canine melanoma patient and in a canine mammary carcinoma patient - supporting its potential to treat metastatic disease.

[0210] Other tumours known to be susceptible to immunotherapies include non-melanoma skin cancer including squamous cell carcinoma, stomach cancer, colon cancer, anorectal cancer, bladder cancer, mammary cancer in particular triple negative mammary carcinoma, kidney cancer, lung cancer, liver cancer, oral cancer, oesophageal cancer, B and T cell lymphoma, blood cancer (leukaemia).

[0211] In another embodiment, therefore, the spontaneous cancer is selected from the group consisting of sarcoma of bone or soft tissue origin, or melanoma (a tumour of melanocytes). Examples of soft tissue sarcoma are haemangiosarcoma (a tumour of blood vessels), fibrosarcoma, histiocytic sarcoma, liposarcoma and rhabdomyosarcoma.

[0212] It is shown herein by way of clinical example that an advanced soft tissue sarcoma in a canine patient - a fibrosarcoma - demonstrated disease stabilization, with improved definition of the tumour margins, in response to rimcazole.

[0213] Therefore, in a further embodiment rimcazole is administered prior to, alongside or following surgical excision to improve the chance of curative removal of locally invasive tumours such as canine fibrosarcomas.

[0214] In a further embodiment, also taught by responsiveness of the tumour to agents that stimulate an anti-tumour immune response, the spontaneous cancer is selected from the group of nonmelanoma skin cancer, stomach cancer, colon cancer, anorectal cancer, bladder cancer, mammary cancer in particular, triple negative mammary cancer, kidney cancer, lung cancer, liver cancer, oral cancer, oesophageal cancer, B and T cell lymphoma, blood cancer (leukaemia).

[0215] Splenic haemangiosarcoma

[0216] Dopamine promotes tumour microvasculature maturation (Pawel et al (2020)). This will lead to reduced leakiness of tumour-associated vasculature.

[0217] Haemangiosarcoma is a common tumour in dogs - much less so in humans and other animal species. It is a highly aggressive tumour arising from the cells that line the walls of blood vessels (endothelial cells). A major risk of the tumour is rupture leading to catastrophic blood loss. The tumour most often appears in the spleen, heart or liver although it can occur at other locations. The inventors reasoned that dopamine-mediated stabilisation of tumour-associated vasculature by administration of a dopamine transporter inhibitor such as rimcazole would reduce the risk of tumour rupture and may extend survival.

[0218] It is shown herein by way of clinical example that rimcazole substantially improves the patient’s quality of life in canine splenic haemangiosarcoma - even when treatment is initiated in very advanced disease, post-splenectomy and with documented progressive disease following epirubicin chemotherapy. In the specific clinical situation of canine patients with advanced haemangiosarcoma, rimcazole can extend life by delaying euthanasia due to improved quality of life.

[0219] In a specific embodiment, rimcazole or another dopamine transporter inhibitor can therefore be used to extend life by delaying euthanasia, due to improved quality of life, in canine patients with advanced haemangiosarcoma.

[0220] Combination with NSAIDs / COX-2 inhibitors: enhancement of anti-tumour

[0221] In a further embodiment of the invention, rimcazole or other compounds of the invention are combined with an anti-inflammatory drug such as a non-steroidal anti-inflammatory drug (NSAID) preferably of the COX-2 inhibitor class (known as “coxibs”) or with a corticosteroid such as prednisone or dexamethasone administered at anti-inflammatory, non-immunosuppressive doses.

[0222] As a general principle, inflammation may either promote or suppress the development of cancer and it may correspondingly help or hinder an anti-tumour response to cancer treatment, depending on the inflammatory pathways involved. The relationship of inflammation to cancer is complex therefore and not fully understood in the art.

[0223] The aim of this embodiment of the invention is to increase the anti-tumour efficacy of rimcazole, or other compound(s) of the invention, by combining the principal agent(s) of the invention, such as rimcazole, with an anti-inflammatory drug in order to inhibit an undesirable pro-tumour inflammatory drive. An inflammatory drive may be intrinsic to the tumour, causing it to offset an anti-tumour response from the start of treatment; an inflammatory response may also occur if cancer treatment results in a high level of tumour cell death that outstrips the body’s disposal mechanisms - allowing noxious cell contents to be released. Undesirable inflammation that opposes an anti-tumour response may also arise following treatment with immune checkpoint inhibitors.

[0224] It is shown herein by way of clinical example that rimcazole administered by mouth as a single agent (monotherapy) slowed tumour growth (resulting in disease stabilisation) for a period of 8 weeks in 80% of canine patients with advanced, therapy-resistant tumours. However, all patients relapsed between 8 and 12 weeks with clinical evidence of disease progression using RECIST criteria. The emergence of disease progression coincided with clear, clinical evidence of a peri-tumour inflammatory response (refer to Figure 6C).

[0225] In addition to gross (macroscopic) evidence of inflammation by clinical observation, there was histological evidence of an intense peri-tumour inflammatory response at necropsy (postmortem) in a patient that died from an unrelated cardiogenic cause (Table 3, patient#008). This patient had stable disease through to 8 weeks but died at 10 weeks. Post-mortem findings described the presence of marked reactive follicular hyperplasia in the lymph nodes draining the tumour with no evidence of metastasis - reflecting engagement of an anti-tumour immune response (including immune checkpoint inhibition). An inflammatory response was evident from intense fibroplasia (increase in the number of inflammatory fibroblasts) in the tumour and surrounding tissue as well as a marked ingress of inflammatory (non-neoplastic) mast cells. These findings are consistent with an inflammatory drive in the tumour microenvironment. Another finding of note was the absence of mitotic figures in the residual tumour (as well as in an incidental haemangioma) suggestive of a neoplasia-selective cell cycle arrest given there were mitotic figures in the bone marrow and elsewhere.

[0226] The inventors reasoned that the uniformity of the trend to initial disease stabilisation through to 8 weeks with subsequent relapse (disease progression) accompanied by clinical and histological evidence of a peri-tumour inflammatory response, was consistent with a pro-tumour inflammatory response. The inventors therefore hypothesised that the pre-emption of an inflammatory drive by combining rimcazole with an anti-inflammatory drug would enhance and prolong an efficacy response.

[0227] The induction of signs of an intense peri-tumour inflammatory drive in response to rimcazole as monotherapy was surprising when the teaching in the prior art is consistent that rimcazole has an anti-inflammatory role; for example, prior studies had demonstrated the induction of a pro- apoptotic, anti-inflammatory switch to hypoxia inducible factor and the p65 RelA subunit of NF- kappaB.( Achison et al 2007 and Spruce et al W02009 / 074809.).

[0228] The use of rimcazole as an anti-inflammatory agent was therefore taught in the prior art and it is surprising therefore that rimcazole induces an inflammatory response when administered to authentic patients with spontaneous cancer - another example of how the models used in the laboratory setting do not authentically recapitulate the clinical situation.

[0229] Indeed, tumour xenograft studies in mice had demonstrated sustained tumour inhibition in response to rimcazole; the absence of an inflammatory drive can be explained by the mice being comprehensively immunocompromised (outbred nude and athymic mice) which is not reflective of spontaneous cancer in a patient.

[0230] There is particular interest in the role of COX-2 (cyclo-oxygenase-2) upregulation in opposing the anti-tumour effect of a range of anti-cancer treatments including traditional, cytotoxic therapies as well as immunotherapies including immune checkpoint inhibitors (Zelenay et al (2015); Pelly et al (2021); Bell et al., (2022)). As explained above, the relationship of inflammation to tumour growth is complex with evidence that inflammation may help or hinder an anti-tumour response depending on the pathways involved. The goal is therefore to switch off the type of inflammation that reduces an anti-tumour response.

[0231] The inventors reasoned that by combining rimcazole, or other principal compounds of the invention, with anti-inflammatory agents, in particular those that inhibit the COX-2 pathway, a more effective anti-tumour response would be achieved. It is shown herein by way of clinical example that this is indeed the case; when rimcazole is combined with firocoxib, a COX-2 inhibitor, there is clear evidence of tumour shrinkage sufficient to qualify as an objective response by RECIST criteria (Figure 7, Figure 8) as well as complete and sustained resolution of metastases (secondary tumours) in patients with advanced disease that had spread to distant sites (Figure 9).

[0232] Exemplary anti-inflammatorv aqents that are of use in combination with rimcazole or other principal compounds of the invention to treat cancer

[0233] Examples of non-steroidal anti-inflammatory drugs for human use include, but are not limited to, ibuprofen, naproxen, diclofenac, indomethacin, meloxicam, piroxicam, ketorolac, sulindac, ketoprofen, diflunisal, flurbiprofen, fenoprofen, oxaprozin, tolmetin, nabumetone, etodolac, aspirin, acetaminophen, tolfenamic acid,

[0234] Examples of selective COX-2 inhibitors (coxibs) that have been used in humans include, but are not limited to, celecoxib, etoricoxib, parecoxib, valdecoxib, rofecoxib, cimicoxib, and lumiracoxib. In some embodiments, the selective COX-2 inhibitor is celecoxib. Although in some countries only celecoxib is available, due to cardiovascular safety concerns over the long term, in the context of short-term use to enhance cancer treatment efficacy the benefits of coxibs in humans may outweigh the risks.

[0235] Examples of selective COX-2 inhibitors for use in dogs include, but are not limited to, firocoxib, deracoxib, robenacoxib, mavacoxib, enflicoxib, carprofen, meloxicam. In some embodiments, the selective COX-2 inhibitor is firocoxib. Less selective agents (that inhibit COX-2 and COX-1) may also be of use.

[0236] Examples of less selective NSAIDs for use in dogs include, but are not limited to, piroxicam, etodolac, vedaprofen, dipyrone.

[0237] The NSAID grapiprant that inhibits PGE2 is also of use in dogs in combination with rimcazole or other principal compounds of the invention.

[0238] Corticosteroids, including but not limited to prednisone / prednisolone, dexamethasone and hydrocortisone, at non-immunosuppressive doses are also of use in both dogs and humans in combination with rimcazole or other principal compounds of the invention. Modes of administration

[0239] Formulations and dosage:

[0240] The understanding now brought about by the new invention teaches that in fact rimcazole is effective at inducing an anti-tumour response at submicromolar blood levels in humans and canines, provided it is administered in the clinical situation of spontaneous cancer in an authentic patient.

[0241] In particular embodiments, rimcazole may be used in dosing regimens that are disclosed herein to be non-toxic but are demonstrated to be surprisingly effective. Exemplary dose regimens for use of immediate release oral formulations of rimcazole in canines and humans are provided herein - both of which are associated with maintenance of rimcazole blood levels below a threshold of 1 micromolar. It is well established, and reported in the literature, that the induction of seizures in humans is associated with plasma concentrations of rimcazole in excess of 1 micromolar (discussed in more detail below).

[0242] Furthermore, it is well known, and reported, that canines and humans have a similar disposition to epilepsy with similar prevalence rates for idiopathic epilepsy in humans and canines. Indeed, the similarity is such that dogs are proposed as a natural animal model of human epilepsy (Loscher (2022)). Therefore, it can be readily deduced from the art that dogs will not experience seizures if rimcazole plasma concentrations are maintained at a safe level - below 1 micromolar. This contrasts with other animal species such as murines (mice) that have a lower susceptibility to seizures and as a result can withstand micromolar plasma concentrations of rimcazole without ill effect.

[0243] Therefore, while it could arguably have been deduced from the art that rimcazole blood concentrations in humans and canines needed to be maintained below 1 micromolar in order to avoid toxicity (principally, epileptic seizures), the art also clearly taught that such doses would be ineffective, as micromolar concentrations of rimcazole are required to achieve anti-tumour efficacy.

[0244] In accordance with the invention described herein, the action of rimcazole as a high affinity inhibitor of the dopamine transporter means it can be administered at a dose that ensures that the peak blood plasma level of rimcazole is maintained below micromolar concentrations, thus avoiding toxicity. Therefore, in an aspect of the present invention, the compound is administered such that the peak blood (plasma) level of rimcazole is consistently below a concentration of 1 micromolar - known in the art as the threshold above which there is a risk of seizures in humans, and correspondingly in canines which are well known to have a similar susceptibility to epilepsy as humans and may be treated with similar medicines.

[0245] As used herein, reference to a ‘peak blood (plasma) level below a concentration of 1 micromolar’ means that measured plasma concentrations for the patient over the treatment duration are consistently (i.e. in a majority of measurements over the treatment duration) less than 1 micromolar. Such a requirement does not exclude brief, but clinically inconsequential, excursions into the micromolar range which would not cause toxicity.

[0246] Dosing regimens for canine patients and human patients that are safe (in that the peak blood (plasma) level of rimcazole is maintained at a safe - submicromolar - level) whilst being effective to treat the cancer are therefore provided herein.

[0247] In an embodiment, the compound is administered in an oral formulation to a canine patient with spontaneous cancer in a dose from 5 mg / kg body weight daily up to a maximum of 30 mg / kg body weight daily. For example, the daily dose may be at least 5 mg / kg, at least 10 mg / kg, or at least 15 mg / kg; and / or may be up to 30 mg / kg, up to 25 mg / kg, or up to 20 mg / kg. Exemplary dosage ranges hence include 5-30 mg / kg daily, 10-30 mg / kg daily, 15-30 mg / kg daily, 5-25 mg / kg daily, 10-25 mg / kg daily, and 5-20 mg / kg daily.

[0248] In an embodiment, the compound is administered in an oral formulation to a human patient with spontaneous cancer in a dose from 1 mg / kg body weight daily up to a maximum of 5 mg / kg body weight daily. For example, the daily dose may be at least 1 mg / kg, at least 2 mg / kg, or at least 2.5 mg / kg; and / or may be up to 5 mg / kg, up to 4.5 mg / kg, or up to 4 mg / kg. Exemplary dosage ranges hence include 1-5 mg / kg daily, 2-5 mg / kg daily, 2.5-5 mg / kg daily, 1-4.5 mg / kg daily, 2-4.5 mg / kg daily, and 1-4 mg / kg daily.

[0249] The daily dose may be administered as a single dose or divided into two or more doses.

[0250] The prior art taught that such regimens (leading to submicromolar concentrations of rimcazole in the blood and extracellular space) would be ineffective for the treatment of cancer. This is because the prior art was based exclusively upon flawed experimental model systems of cancer in the laboratory (not spontaneous cancer in authentic patients) that were insufficient to inform suitable doses of rimcazole and, even worse, the indicative doses to achieve efficacy were found to be severely toxic. It can be understood now that a fundamental flaw of these experimental models is that they did not recapitulate the perturbations in dopamine signalling, recognised to be important in the genesis and progression of spontaneous tumours. Therefore, the pathways enabling rimcazole to be effective at submicromolar concentrations through its inhibitory action on the dopamine transporter were not available in these models - leading to a false conclusion that rimcazole needs to reach micromolar concentrations in the extracellular milieu in order to mediate anti-tumour activity. Based upon the teaching in the prior art, the notion of rimcazole as a safe and effective cancer treatment was therefore implausible.

[0251] The invention further assists the safe and effective administration of rimcazole by teaching specific dose ranges of oral formulations of rimcazole for canine and human patients in the treatment of spontaneous cancer. Dose extrapolations between animal species based upon the allometric scaling calculations understood by those skilled in the art of pharmaceutical drug development (dose adjustment based on body surface area: body mass ratio (FDA guidance) are not sufficient for drugs such as rimcazole that exhibit inter-species differences in the way the body handles the drug (so- called pharmacokinetic differences). Therefore, a rimcazole dosing regimen should be defined for each species, based upon pharmacokinetic and safety data in the species concerned.

[0252] Pharmacokinetic differences commonly arise due to differences in drug metabolizing enzymes between, and even within, animal species (Michael H. Court, (2013)). In humans, rimcazole is known to be metabolized by CYP2D6 (Irish Medicines Board Report), an enzyme subject to genetic variation that influences clearance of the drug from the bloodstream.

[0253] An orthologue (functional equivalent) of CYP2D6 has been identified in canines: CYP2D15 (Jimenez et al (2023)) As with human CYP2D6, genetic variants of the canine orthologue have been identified that are of uncertain functional significance. Simple dose extrapolations based on cross-species allometric scaling are therefore not sufficient to predict an equivalent therapeutic (effective and safe) window.

[0254] In humans, further complexity arises from the fact that many drugs known as substrates for (metabolised by) the human enzyme CYP2D6, are also, paradoxically, auto-inhibitors of 2D6 - via 2D6 generated metabolites or potentially through the parent drug (M. Ingelman-Sundberg, (2005)). In humans therefore drugs metabolised by CYP2D6 come with the risk of time dependent accumulation, due to autoinhibition of their own metabolism, that can lead to marked rises in plasma concentrations (Irish Medicines Board Report). The inventors reason therefore that rimcazole dosing regimens in humans need to be adjusted downwards to account for plasma accumulation arising from auto-inhibition of CYP2D6 metabolism, until steady state is achieved.

[0255] Whereas metabolism by CYP2D6, and its orthologues, generates complexity through interspecies pharmacokinetic differences, CYP2D6 metabolism also affords a therapeutic opportunity through the formation of bioactive CYP2D6-generated metabolites (including those generated by a CYP2D6 orthologue). It is well understood in the art that CYP2D6 metabolism can lead to the generation of bioactive molecules and in some cases, CYP 2D6 metabolism is required for drug activity: for example, the pharmacological activity of codeine is dependent on its conversion to morphine via CYP2D6. (Dean L, Kane M. In: Pratt VM et al 2012)

[0256] It follows from this that, as earlier indicated above, bioactive metabolites of rimcazole generated by the enzyme CYP2D6, or a canine CYP2D6 orthologue, have potential to act as dopamine reuptake inhibitors, and therefore as safe and effective anti-tumour agents, in their own right.

[0257] Alternative formulations of oral rimcazole - such as modified release formulations - are also included within the scope of the present invention as it is well understood by those skilled in the art how to investigate and derive bioequivalent or therapeutically equivalent doses of modified release formulations to those described for immediate release oral formulations, for which clinical exemplification of anti-tumour efficacy is provided herein.

[0258] Pharmaceutical formulations for administration by alternative routes (other than the oral route), that are intended to act systemically are also included within the scope of the present invention. Examples include transdermal formulations (formulations applied to the skin, intended for systemic action), buccal and rectal formulations, intramuscular, intradermal and subcutaneous formulations. The means whereby safe and effective dosing regimens for such formulations can be derived from the safe and effective blood level window for oral, immediate release formulations of rimcazole in canines and humans (namely, comparative pharmacokinetic studies) are well understood by those skilled in the art of pharmaceutical drug development. The invention therefore teaches the person skilled in the art how to derive dose ranges for systemically available formulations of rimcazole that are therapeutically equivalent to those defined for oral immediate release formulations. These findings create new clinical situations for the use of rimcazole in treatment of cancer.

[0259] In a particular aspect, the present invention provides a topical formulation comprising a compound that is an inhibitor of the dopamine transporter. The compound may in one example be rimcazole or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof, for use in a method of treating spontaneous cancer in a human or canine patient. The topical formulations described herein are intended for local application and to act locally on a tumour.

[0260] Examples of locally applied, locally acting formulations include, but are not limited to, cutaneous, rectal, vaginal, and inhalation products.

[0261] This aspect of the invention provides another means whereby rimcazole, or another inhibitor of the dopamine transporter, can be administered safely and effectively to treat a locally invasive spontaneous cancer. Rimcazole has a sufficiently low molecular weight (321 kDa) that permits its passage through the layers of the skin to reach the deeper subcutaneous tissue meaning it is suitable in principle to treat locally invasive tumours.

[0262] In a particular embodiment, topical formulations of rimcazole intended for local cutaneous action may be applied to tumours on the body surface. It is demonstrated herein by way of clinical example that rimcazole, applied in a topical preparation to an invasive melanoma, results in clinical benefit consistent with anti-tumour efficacy. This supports clinical feasibility of using topical preparations of rimcazole, or another inhibitor of the dopamine transporter, applied to the skin in the treatment of locally invasive tumours such as melanoma and squamous cell carcinoma. It is shown herein by way of clinical example that topical (cutaneous) formulations of rimcazole in strengths of 2% w / w and 5% w / w are effective and safe in the local treatment of tumours. Topical formulations of rimcazole may be administered once or twice daily or less often, as required, for periods up to 6 months or longer. The dosing regimen can be titrated to clinical effect (frequency and duration of dosing adjusted according to clinical response).

[0263] By way of example, a preferred salt form of rimcazole for topical cutaneous application (intended for local action) is the hemifumarate salt (rimcazole hemifumarate) - a highly stable form of rimcazole with low aqueous solubility. As shown herein by way of clinical example, this can be readily formulated as a semi-solid suspension of the active substance (rimcazole hemifumarate) in a simple, non-aqueous carrier such as paraffin wax. Formulation of rimcazole as an insoluble, particulate suspension in a waxy base has the advantage that it helps to maintain the stability of the active substance - thereby extending the shelf life of the product - without compromising cutaneous bioavailability and therefore clinical efficacy of the active substance.

[0264] In a further embodiment, the particle size of the active substance (rimcazole hemifumarate) for suspension in the non-aqueous base could be reduced in order to enhance clinical efficacy by enhancing cutaneous penetration, and therefore local bioavailability, of the active substance. A reduction in particle size could be achieved for example by the process of micronisation which uses a range of techniques that are well established in the art of pharmaceutical formulation. This could in turn allow lower strength formulations to be administered that are therapeutically equivalent to higher strength non-micronised formulations.

[0265] Treatment duration:

[0266] In an embodiment, the duration of treatment with rimcazole may be short. The inventors have observed tumour responses and improvement in quality of life with short treatment durations. Accordingly, in some cases the treatment duration is 16 weeks or less, 12 weeks or less, 8 weeks or less, 7 weeks or less, 6 weeks or less, 5 weeks or less, 4 weeks or less, 3 weeks or less, or even 2 weeks or less.

[0267] In an embodiment, rimcazole is administered until tumour shrinkage or regression is observed. In an embodiment, rimcazole is administered until tumour stabilisation is observed.

[0268] In an embodiment, rimcazole is administered to end of life (death from cancer or other causes). In other words, rimcazole may be prescribed for duration of life.

[0269] In an embodiment, rimcazole is given as palliative treatment without curative intent in order to maintain or improve quality of life in terminal disease. In an embodiment, rimcazole is given with curative intent to eradicate the disease in a canine or human patient.

[0270] In some cases, rimcazole is administered to the canine or human patient in a dose-escalating regimen. The dose may be escalated if a tumour is observed to worsen, or in a predetermined escalating regimen. For example, treatment of canine patients may begin at 5 mg / kg / day and increase up to 30 mg / kg / day. It will also be appreciated that the dose may be reduced if regression is observed. Treatment of human patients may begin at 1 mg / kg / day and increase to a maximum of 5 mg / kg / day.

[0271] In a further embodiment, rimcazole is administered in a topical (cutaneous) formulation applied to the skin or mucous membranes of the alimentary or genital tracts for the local treatment of spontaneous cancer.. In particular embodiments, topical preparations of rimcazole applied to the skin may be used for the local treatment of melanoma or non-melanoma skin cancer in human and canine patients and for the treatment of premalignant skin lesions that are destined to become cancerous, if left untreated.

[0272] Adjuvant therapy and combinations:

[0273] As mentioned above, the canine or human patient may have advanced or aggressive spontaneous cancer. Aggressive cancer has a higher chance of recurrence after treatment, and of spread to other sites in the body. In this clinical situation, rimcazole may be administered as first line, or later line, treatment (following any one, or more, of surgery, radiation therapy or chemotherapy). Advanced or aggressive cancer may be present at a single site or at multiple sites resulting from metastatic spread.

[0274] Adjuvant therapy - given in addition to another method of treatment - has the aim to reduce the chance of cancer recurring at the site of the primary tumour and to reduce the risk of secondary spread to other sites in the body. Adjuvant treatment may also be given to treat metastatic disease, where secondary spread has already occurred. Rimcazole may be of use as adjuvant treatment in any of these clinical situations.

[0275] In an embodiment, rimcazole is administered as adjuvant treatment before, alongside or following another method of treatment such as surgery, radiotherapy, chemotherapy or any other physical, pharmaceutical or biological treatment for cancer. In a particular embodiment, rimcazole is administered as adjuvant therapy to treat or prevent metastatic disease following control of the primary tumour by surgery or another method of treatment.

[0276] In a further embodiment the canine or human patient has an advanced or aggressive spontaneous cancer that is resistant to chemotherapeutic drugs (“chemoresistant”). In this clinical situation, rimcazole may be administered alone or as neoadjuvant (before) or adjuvant (alongside or following) treatment with the chemotherapy drug in order to reduce the risk of relapse.

[0277] In a further embodiment the canine or human patient has a radioresistant spontaneous cancer where rimcazole’s potential to act as an immunotherapy via dopamine in its own right and / or in concert with sigma-1 antagonism, will be of particular benefit (Yifan Wang et al (2019)). In this clinical situation rimcazole may be administered to tumours that have acquired radioresistance, in order to sensitise the tumour prior to further cycles of radiotherapy treatment.

[0278] In a further embodiment rimcazole is administered as a neoadjuvant radiosensitizer prior to radiotherapy treatment of an advanced or aggressive spontaneous cancer in a canine or human patient.

[0279] It is further hypothesised herein that in the specific clinical situation of spontaneous cancer in an authentic patient, rimcazole binding to the dopamine transporter at submicromolar concentrations secondarily mediate a favourable steric interaction, that enhances rimcazole’s binding affinity to sigma sites. The result is that rimcazole acts in concert at both the dopamine transporter and sigma- 1 receptor at submicromolar concentrations but this is only revealed in the clinical situation of spontaneous cancer which is why it has not been uncovered in the art thus far. In a further embodiment, rimcazole can therefore be considered to represent a combination therapy that mediates synergistic anti-tumour effects via the dopamine transporter and sigma-1 receptor acting in concert. The result is unexpected effectiveness at submicromolar concentrations.

[0280] It follows from this that in a further aspect of the invention, agents that inhibit the sigma-1 receptor (sigma-1 antagonists) may be used in combination with dopamine reuptake inhibitors in the treatment of spontaneous cancer in canines and humans. These may be administered either as separate agents or as one agent that combines both properties, such as with the molecule rimcazole.

[0281] In a further embodiment, as described above, rimcazole is administered prior to, alongside or following surgical excision to improve the chance of curative removal of locally invasive tumours such as canine fibrosarcomas. The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0282] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0283] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0284] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0285] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0286] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0287] Examples

[0288] It is demonstrated herein by way of clinical example - in canine patients with advanced, aggressive spontaneous tumours - that rimcazole is a safe and effective treatment for cancer. This is achieved at doses of rimcazole associated with plasma concentrations that are consistently in the non-toxic, submicromolar range - against the expectations of all teaching in the art. The invention explains why new understanding in relation to the importance of dopamine transporter modulation, in its own right and in concert with sigma-1 antagonism, results in rimcazole being a highly efficacious treatment for cancer, without signs of toxicity.

[0289] Systemic administration of rimcazole as monotherapy (a single agent) at doses that lead to submicromolar concentrations of rimcazole in the bloodstream produces clinically meaningful slowing of tumour progression, with maintained or improved quality of life without signs of toxicity. This is against all prior expectations in the art.

[0290] Anti-tumour efficacy is demonstrated at low (submicromolar) blood (plasma) concentrations, consistent with the high affinity of rimcazole for the dopamine transporter. Efficacy at submicromolar blood levels is at the same time inconsistent with anti-tumour action mediated via sigma-1 antagonism. Sigma-1 antagonism was regarded as the mechanism whereby rimcazole had potential as a treatment for cancer but this was later dismissed as unachievable owing to the risk of toxicity at the micromolar blood levels considered to be needed for it to be an effective drug.

[0291] It is shown herein by way of clinical example that rimcazole administered by mouth as a single agent (monotherapy) slowed tumour growth (resulting in disease stabilisation) for a period of 8 weeks in 80% of canine patients with advanced, therapy-resistant tumours (Table 3). This was accompanied by maintained or improved quality of life. However, all patients relapsed between 8 and 12 weeks with clinical evidence of disease progression using RECIST criteria. The emergence of disease progression coincided with clear, clinical evidence of a peri-tumour inflammatory response (refer to Figure 6C).

[0292] In addition to gross (macroscopic) evidence of inflammation by clinical observation, there was also histological evidence of an intense peri-tumour inflammatory response at necropsy (postmortem) in a patient that died from an unrelated cardiogenic cause (Table 3, patient#008). This patient had stable disease through to 8 weeks but died at 10 weeks. Post-mortem findings described the presence of marked reactive follicular hyperplasia in the lymph nodes draining the tumour with no evidence of metastasis - reflecting engagement of an anti-tumour immune response (including immune checkpoint inhibition). An inflammatory response was evident from intense fibroplasia (increase in the number of inflammatory fibroblasts) in the tumour and surrounding tissue as well as a marked ingress of inflammatory (non-neoplastic) mast cells; these findings are consistent with an inflammatory drive in the tumour microenvironment. Another finding of note was the absence of mitotic figures in the residual tumour (as well as in an incidental haemangioma) suggestive of a neoplasia-selective cell cycle arrest given there were mitotic figures in the bone marrow and elsewhere.

[0293] The inventors reasoned that the uniformity of the trend to initial disease stabilisation through to 8 weeks with subsequent relapse (disease progression) accompanied by clinical and histological evidence of a peri-tumour inflammatory response, was consistent with a pro-tumour inflammatory response. The inventors therefore hypothesized that the pre-emption of an inflammatory drive by combining rimcazole with an anti-inflammatory drug would enhance and prolong an efficacy response.

[0294] The induction of signs of an intense peri-tumour inflammatory drive in response to rimcazole as monotherapy was surprising when the teaching in the prior art is consistent that rimcazole has an anti-inflammatory role; for example, prior studies had demonstrated the induction of a pro- apoptotic, anti-inflammatory switch to hypoxia inducible factor and the p65 RelA subunit of NF- kappaB.( Achison et al 2007 and Spruce et al W02009 / 074809.).

[0295] The use of rimcazole as an anti-inflammatory agent was therefore taught in the prior art and it is surprising therefore rimcazole induces an inflammatory response when administered to authentic patients with spontaneous cancer - another example of how the models used in the laboratory setting do not authentically recapitulate the clinical situation.

[0296] Indeed, tumour xenograft studies in mice had also demonstrated sustained tumour inhibition in response to rimcazole; however, the absence of an inflammatory drive can be explained by the mice being comprehensively immunocompromised (outbred nude and athymic mice).

[0297] There is particular interest in the role of COX-2 (cyclo-oxygenase-2) upregulation in opposing the anti-tumour effect of a range of anti-cancer treatments including traditional, cytotoxic therapies as well as immunotherapies including immune checkpoint inhibitors (Zelenay et al (2015); Pelly et al (2021); Bell et al., (2022)). The relationship of inflammation to tumour growth is complex with evidence that inflammation may help or hinder an anti-tumour response depending on the pathways involved. The goal is therefore to switch off the type of inflammation that hinders an anti-tumour response.

[0298] The inventors reasoned that by combining rimcazole, or other principal compounds of the invention, with anti-inflammatory agents, in particular those that inhibit the COX-2 pathway, a more effective anti-tumour response would be achieved. It is shown herein by way of clinical example (refer for more detail to the specific examples set out below) that this is indeed the case. When rimcazole is combined with firocoxib, a COX-2 inhibitor, there is clear evidence of tumour shrinkage sufficient to qualify as an objective response by RECIST criteria (Figure 7, Figure 8) in a canine patient with squamous cell carcinoma. This was sustained for at least 16 weeks - twice as long as the period of disease stabilization with monotherapy. A further canine patient with metastatic oral melanoma (an aggressive tumour type) demonstrated complete, sustained resolution of pulmonary metastases (secondary tumours in the lungs) following treatment with rimcazole plus firocoxib (Figure 9).

[0299] Notwithstanding the evidence that firocoxib, or other anti-inflammatory drugs, will enhance the anti-tumour action of rimcazole, it is demonstrated herein that rimcazole as monotherapy can result in complete tumour resolution in some clinical situations. In particular, such situations may include situations where rimcazole is used as an adjuvant treatment to treat metastatic disease where another method of treatment such as surgery is used to treat the primary tumour.

[0300] CLINICAL EXAMPLES

[0301] 1. Rimcazole administered as monotherapy (single agent) - evaluation of primary tumour

[0302] A. Initial tolerability evaluation in canine cancer patients of oral rimcazole administered at a 5 mg / kg and 10 mg / kg daily.

[0303] In the first phase of clinical testing, canine patients with advanced, aggressive disease at end of treatment line were treated with a low dose of oral rimcazole to provide an initial evaluation of tolerability. The doses of oral rimcazole used in this phase - 5 mg / kg and 10 mg / kg daily - is are consistently associated with peak blood (plasma) levels of rimcazole ranging from less than 100 to c.200 nanomolar more than five to ten-fold below a conservative plasma concentration toxicity threshold of 1 micromolar (Table 2).

[0304] The drug was well tolerated with some promising early signs consistent with potential for clinical benefit.

[0305] Three examples are described below.

[0306] Diagnosis: metastatic splenic haemangiosarcoma (soft tissue sarcoma arising from blood vessels). End-stage, aggressive disease. Life expectancy at commencement of rimcazole: 3 - 4 weeks.

[0307] Treatment: oral rimcazole (daily dose 5 mg / kg body weight).

[0308] Oral rimcazole commenced at 4 months post-splenectomy and after 3 cycles of doxorubicin chemotherapy.

[0309] Clinical response:

[0310] In the second week of rimcazole treatment, the patient developed pyrexia (fever), chills, lethargy and anorexia. These symptoms are consistent with those reported for an immunotherapy in canines with spontaneous cancer and signify engagement of an immune response to the tumour antigen (Masaya Igase et al (2020)). The patient was successfully managed with temporary dose interruption and symptomatic treatment.

[0311] A growing body of literature indicates that the onset of immune related adverse events in patients may be predictive ultimately of meaningful clinical anti-tumour response to immunotherapies. Studies report that patients experiencing immune related adverse events go on to demonstrate improvements in progression-free survival, overall survival and overall response rate compared to those patients lacking an immune related adverse event profile (Satya Das and Douglas B. Johnson, (2019)). The onset of immune related adverse events can therefore be considered a surrogate marker that in some cases is predictive of ultimate clinical (anti-tumour) benefit.

[0312] The absence of signs of engagement of an immune response in healthy (tumour-free) beagle dogs administered with oral rimcazole is readily explained by the absence of an immunogenic stimulus in the healthy dogs due to the absence of tumour antigen. Whereas, in canine cancer patients, the tumour may provide an immunogenic stimulus responsive to rimcazole-mediated immunotherapy action.

[0313] Significantly, there was no evidence of vascular disruption or bleeding while on rimcazole - consistent with dopamine-mediated preservation of blood vessel integrity.

[0314] The patient was euthanised due to extensive disease. Rimcazole provided palliative symptomatic benefit at this end of life stage.

[0315] Diagnosis: Soft tissue (suspected histiocytic) sarcoma of the thigh with metastasis to the popliteal and sublumbar nodes. Expected survival 3 weeks at start of oral rimcazole treatment.

[0316] Treatment: oral rimcazole (daily dose 5 mg / kg body weight).

[0317] Clinical response:

[0318] Two weeks after commencement of rimcazole the tumour mass had reduced in size by 15%, indicating disease stabilisation.

[0319] Four weeks after commencement of rimcazole, abdominal ultrasound revealed progressive enlargement of the ipsilateral medial iliac lymph node (which drains the tumour site). This is consistent with rimcazole-mediated immunotherapy action (activation of a local immune response to the tumour antigen).

[0320] There is growing recognition that regional lymph nodes draining a tumour may be pivotal to the engagement of an immune response to the tumour (Marieke F. Fransen et al (2018)). Notably, lymph node enlargement may be confined to the tumour-draining nodes and the resection of these may prevent the action of an immunotherapy.

[0321] This case is considered to demonstrate promising evidence of disease stabilisation with evidence of engagement of an immune response at a very low dose of oral rimcazole - 5 mg / kg daily - that is associated with plasma concentrations of rimcazole below 0.1 microM (100 nanomolar): more than ten-fold below the conservative ceiling for maximum plasma concentration set out in the invention. 3 - Female. French

[0322] Diagnosis: metastatic splenic haemangiosarcoma (soft tissue sarcoma arising from blood vessels). End-stage, aggressive disease. Oral rimcazole commenced at 4 months postsplenectomy, after 4 cycles of epirubicin chemotherapy, and in the presence of documented progressive disease (hepatic metastases). Epirubicin was discontinued due to adverse events.

[0323] Treatment: oral rimcazole (daily dose 10 mg / kg body weight).

[0324] Clinical response:

[0325] The patient’s quality of life quickly improved upon commencement of rimcazole, and this was sustained during treatment. A short interruption in rimcazole treatment led to a deterioration in quality of life. This improved when treatment was resumed.

[0326] The patient survived with good quality of life until 7 months post-splenectomy - longer than the median survival of 4 - 6 months for such patients.

[0327] B. Pilot clinical efficacy and safety evaluation of oral rimcazole in canine cancer patients at a dose level of 10 mq / kq / day, rising to a maximum of 20 mq / kq / day

[0328] In the next phase of clinical evaluation, ten canine patients with advanced and / or aggressive tumour types recognised to have high unmet need were enrolled to receive oral rimcazole at a daily dose of 10 mg / kg, rising to 20 mg / kg daily depending on clinical response. Patients received daily oral rimcazole for a period up to 4 months, depending on tolerability and response.

[0329] Tumour types chosen for the initial evaluation were informed by scientific and clinical rationale to support the use of rimcazole. These included mast cell tumour (fast-growing, round cell tumours of the myeloid lineage), squamous cell carcinoma (highly mutated cutaneous tumours) and fibrosarcoma (connective tissue tumours that are treatment resistant).

[0330] The two dose levels of rimcazole used in this phase - 10 mg / kg and 20 mg / kg daily - are associated with peak plasma concentrations up to circa. 200 nM (0.2 microM) and 500 nM (0.5 microM) respectively for the two dose levels (Table 2). These values are substantially below the conservative plasma concentration toxicity threshold of 1 micromolar defined herein.

[0331] Patients received treatment for up to 16 weeks with 4 weekly clinical assessments that included physical examination, haematology, biochemistry and validated quality of life questionnaire, with 8 weekly chest Xray and abdominal ultrasound examination.

[0332] Tumour growth was measured at 4 weekly intervals using calipers and evaluated using internationally recognised RECIST criteria (as in human oncology). Patients were graded at each time point as having progressive disease (PD), stable disease (SD), partial response (PR) or complete response (CR). Table 3 summarises the tumour growth data for each patient at time points through to week 12 (refer to table legend for evaluation of tumour growth by RECIST criteria).

[0333] Treatment was discontinued upon disease progression or escalated to the ceiling dose of 20mg / kg depending on clinical judgement.

[0334] TABLE 3 - anonymized clinical efficacy outcome data

[0335] PD: Progressive disease (RECIST criteria). Sum of longest diameters of target lesion(s) greater than 20% compared to baseline (pre-treatment). SD: Stable disease (RECIST criteria). Qualifies as neither PD nor partial response viz. Increase in sum of longest diameter(s) less than 20% compared to baseline; or, less than 30% reduction in sum of longest diameter(s) compared to baseline.

[0336] Summary of response to rimcazole as monotherapy - delay in tumour progression, good quality of life, no serious adverse events:

[0337] In the pilot evaluation of 10 canine patients with advanced, aggressive disease, the rate of tumour growth was slowed such that the disease was controlled (stabilised) after 8 weeks of treatment in 80% (8 out of 10) patients. Moreover, quality of life was maintained or improved while on treatment. Significantly, 9 out of 10 patients were considered to be in a geriatric age band for canine patients. Rimcazole can therefore be given safely and improves quality of life in elderly canine patients with cancer - an important benefit in this patient population.

[0338] These benefits are clinically meaningful.

[0339] At the 12 week time point, all patients with the exception of one, had developed disease progression (increase in tumour growth rate).

[0340] Rimcazole was well tolerated at doses up to 20 mg / kg / day throughout the study, with no seizures or other serious adverse events reported. Three deaths occurred that were unrelated to rimcazole.

[0341] These results demonstrate clear evidence of efficacy benefit for rimcazole administered as monotherapy in a canine patient population with advanced cancer, many of whom were in a geriatric age range. The benefits included slowing of disease progression (disease stabilisation) for a period of 2 months in the majority (80%) of patients, despite the disease being at an advanced stage. This was accompanied by maintained or improved quality of life - a significant benefit in its own right in terminally ill, elderly cancer patients. This would hold true for both canine and human cancer patients.

[0342] It is demonstrated in the examples herein that rimcazole at dose levels (10 mg / kg and 20 mg / kg) resulting in plasma concentrations in the submicromolar range) has been well tolerated. Despite the advanced stage of the tumours, there is clear evidence of important clinical benefit indicative of anti-tumour efficacy. In cases treated with rimcazole for up to 2 months so far (with treatment ongoing) there has been significant tumour shrinkage, tumour stabilisation and improvement in quality of life. The clinical evaluation is ongoing.

[0343] Specific clinical examples from the series of 10 canine patients (table 3, above) are described in more detail below:

[0344] Example 4 - Female. Pitbull terrier Dog (patient #002, Table 3). Age 8yrs

[0345] Photographs of this patient before (Figure 1A) and after 12 weeks (Figure 1B) rimcazole treatment are displayed. Diagnosis: Disseminated squamous cell carcinoma (SCC) and concomitant mast cell tumour located on the dorsum.

[0346] Treatment: oral rimcazole (daily dose 10 mg / kg body weight).

[0347] Duration of treatment 12 weeks. Patient died from an incidental, unrelated condition (gastric volvulus) at 14 weeks.

[0348] Clinical response:

[0349] SCC:

[0350] Despite the extensive nature of the patient’s squamous cell carcinoma, the lesions demonstrated evidence of stabilisation.

[0351] Mast cell tumour:

[0352] As depicted in Figure 1 , the large mast cell tumour on the dorsum measured 90 mm in the longest diameter at the commencement of treatment (Figure 1 A); this steadily reduced in size to 70 mm after 12 weeks of daily rimcazole treatment at a dose of 10 mg / kg body weight (Figure 1 B). There was also an improved definition of the tumour margins.

[0353] Tumour response was evaluated in accordance with the widely accepted RECIST 1.1 criteria used in both veterinary and human oncology drug evaluation (Eisenhauer et al (2009)).

[0354] According to RECIST 1. 1, the tumour response (reduction in longest diameter of circa 22%) in this patient can be considered to qualify as stable disease. The patient died at 14 weeks from an unrelated condition (gastric volvulus - a surgical emergency due to twisting of the stomach) Rimcazole was well tolerated throughout, with no adverse events due to the drug.

[0355] Photographs of this patient before (Figure 2A) and after 4 weeks of rimcazole treatment at 5 mg / kg daily (Figure 2B) rimcazole treatment are displayed.

[0356] Diagnosis: Mast cell tumour located on the forelimb.

[0357] Treatment: oral rimcazole 5 mg / kg / day. To note, in part owing to the advanced age of the dog (16 years) a lower dose of rimcazole (5 mg / kg / day) was used from the second week of treatment.

[0358] Clinical response:

[0359] Despite the low dose of 5 mg / kg / day (associated with plasma concentrations less than c. 100 nanomolar range), tumour growth stabilised after 4 weeks of treatment (no change in the longest diameter). After 8 weeks of treatment, one diameter had reduced in length by circa 50% (62 mm at baseline, reduced to 33 mm at 8 weeks), accompanied by reduction in the other diameter).

[0360] Despite the low dose (10-fold below the known toxicity threshold for oral rimcazole in beagle dogs) the patient has demonstrated clear evidence of disease stabilisation after 8 weeks of treatment, with no adverse events.

[0361] Example 6- Male. Golden retriever Dog (patient #001, Table 3). Age 8yrs

[0362] Photographs of this patient before (Figure 3A) and after 8 weeks of rimcazole treatment (Figure 3B) are displayed.

[0363] Diagnosis: Advanced fibrosarcoma located on the underbelly

[0364] Treatment: oral rimcazole (daily dose 10 mg / kg body weight up to 4 weeks; escalated to 20 mg / kg beyond 4 weeks).

[0365] Clinical response:

[0366] Treatment well tolerated including at the 20 mg / kg dose level.

[0367] Despite the very large size of the tumour at commencement of treatment, there was no growth of the tumour through to 8 weeks’ treatment.

[0368] According to RECIST 1. 1, the tumour response in this patient through to week 8 qualified as stable disease.

[0369] Example 7 - Female. Pug Dog. Age 10yrs (patient #008, Table 3). Disease stabilisation throuc to 8 weeks.

[0370] Photographs of this patient before (Figure 4A) and after 8 weeks (Figure 4B) of rimcazole treatment are displayed.

[0371] Diagnosis: Aggressive squamous cell carcinoma located on the forelimb

[0372] Treatment: oral rimcazole (daily dose 10 mg / kg body weight)

[0373] Duration of treatment so far - 12 weeks.

[0374] Clinical response:

[0375] Despite the very large size and aggressive nature of the tumour, early improvement was observed.

[0376] After 8 weeks of treatment, clear slowing of tumour growth had been maintained - sufficient (Figure 4B), to qualify as stable disease by RECIST criteria. Although there was a reduction in tumour size, this did not meet the requirement for a 30% or greater reduction in the longest diameter to qualify as a partial response by RECIST. It nonetheless qualified as stable disease which is indicative of disease control - a clinically relevant response in advanced disease.

[0377] Beyond the 8 week time point, this patient demonstrated disease progression, accompanied by evidence of a peri-tumour inflammatory response (Figure 6C). Refer to rationale (supra) for introduction of the combination regimen - rimcazole with firocoxib (supra) - in the next phase of evaluation.

[0378] Example 8 - Male. Border collie Dog. Age 12 yrs

[0379] Photographs of this patient before (Figure 5A) and after 2 months (Figure 5B) of topical (cutaneous) rimcazole treatment are displayed.

[0380] Diagnosis: Aggressive conjunctival amelanotic melanoma. Recurrence at site of surgery postenucleation (surgical removal of the eye). Received a standard course of therapeutic melanoma vaccine (Oncept) post-surgery, subsequent to which relapse occurred at the surgical site. Further systemic treatment was declined by the owners.

[0381] Treatment: topical rimcazole (ointment formulation for application to the skin).

[0382] Topical rimcazole commenced ~ 12 months following the initial surgery. Rimcazole applied daily as a topical (ointment) formulation - initially 2% w / w then 5% w / w. Very well tolerated.

[0383] Clinical response:

[0384] The patient obtained clear symptomatic benefit (including reduced bleeding and weeping of the lesion) with resultant improvement in quality of life during treatment with rimcazole. Skin regrowth over the surface of the tumour in response to rimcazole is consistent with anti-tumour efficacy.

[0385] The patient went on to survive with good quality of life for several months - beyond expectation. There was no evidence of regional lymph node spread, consistent with anti-tumour efficacy. Photographs provided in Figures 5A and 5B, wherein 5A (Prior to treatment) and 5B (After two months of daily treatment.

[0386] 2. Rimcazole administered in combination with an anti-inflammatory drug (a selective COX-2 inhibitor, firocoxib)

[0387] In the next phase of clinical evaluation, canine patients with advanced cancer received rimcazole in combination with firocoxib (a selective COX-2 inhibitor), administered at a standard anti-inflammatory dose for canines. The rationale for this - to enhance anti-tumour efficacy response by suppressing a pro-tumour inflammatory drive - is explained above.

[0388] A small number of patients have been treated thus far with the combination regimen. The evidence clearly supports that combining rimcazole with an anti-inflammatory drug of the coxib class, enhances anti-tumour efficacy such that an objective anti-tumour response (shrinkage of the tumour sufficient to qualify as a response by RECIST criteria) that is sustained through to at least 16 weeks is now demonstrated (Figure 8). In addition, resolution (sustained regression) of secondary tumours (lung metastases) has also been demonstrated in a patient with aggressive, oral melanoma (Figure 9). This demonstrates the potential for rimcazole to produce curative benefit even in patients with advanced, aggressive disease that has spread to distant sites in the body.

[0389] Photographs illustrate the patient’s tumour before commencement of treatment (Figure 7A) and after 4 weeks (Figure 7B) and 8 weeks (Figure 7C) of oral rimcazole treatment (10 mg / kg / day) in combination with firocoxib (5 mg / kg / day). Further photographs illustrate the patient’s tumour before commencement of treatment (Figure 8A) and after 8 weeks (Figure 8B) and 16 weeks (Figure 8C) of oral rimcazole treatment (10 mg / kg / day) in combination with firocoxib (5 mg / kg / day).

[0390] Diagnosis: Advanced cutaneous squamous cell carcinoma

[0391] Treatment: oral rimcazole (daily dose 10 mg / kg body weight) in combination with oral firocoxib 5 mg / kg / day.

[0392] Clinical response:

[0393] Prior to treatment, the patient’s tumour measured 55 mm in its longest diameter (Figure 7A I 8A). After 4 weeks of treatment, the longest diameter had reduced to 27 mm - an approximate 50% reduction in tumour size, consistent with a partial response by RECIST criteria. After 8 and 16 weeks of treatment, the tumour had further reduced in size.

[0394] Of note, the patient had evidence of enlargement of the left inguinal lymph node draining the tumour site during treatment. As explained above, this is consistent with activation of an immune response to the tumour - consistent with immune checkpoint inhibition. Also of note, the magnitude of lymph node enlargement declined between the 8 and 16 week time points - consistent with slowing of the immune response, coincident with the decline in the size of the target lesion.

[0395] This clinical example clearly demonstrates an enhanced clinical efficacy (anti-tumour) response when rimcazole is combined with a COX-2 inhibitor (firocoxib), with clinical evidence consistent with an anti-tumour immune response.

[0396] Figure 9 illustrates photographs of the patient’s primary tumour (an oral melanoma - Figure 9A) alongside chest radiographs (9B- 9D) prior to commencement of treatment (Figure 9B) and after 4 weeks (Figure 9C) and 8 weeks (Figure 9D) treatment with oral rimcazole (10 mg / kg / day) in combination with firocoxib (5mg / kg / day)

[0397] Diagnosis: Metastatic (stage 4) oral melanoma

[0398] Treatment: oral rimcazole (daily dose 10 mg / kg body weight) in combination with oral firocoxib 5 mg / kg / day.

[0399] Clinical response:

[0400] The patient’s primary (oral melanoma) tumour could not be measured easily given its posterior location in the mouth. Canine oral melanoma is an aggressive disease that commonly spreads to the lungs, as was the case in this patient. Pulmonary (lung) metastases were therefore selected as target lesions for evaluation of tumour growth using serial radiographs.

[0401] Figure 9B - lateral chest radiograph prior to commencement of study treatment shows numerous metastases (secondary tumours) distributed throughout the lungs. After 4 weeks of treatment with rimcazole plus firocoxib, lung metastases were no longer visible on the radiographs (Figure 9C)- suggesting complete resolution. Complete resolution of lung metastases was confirmed after 8 weeks of treatment (Figure 9D).

[0402] This clinical example demonstrates a clear efficacy response in a patient with extensive metastatic disease when rimcazole is combined with a COX-2 inhibitor (firocoxib) This supports use of rimcazole as an adjuvant therapy to treat or prevent metastatic disease.

[0403] 3. Rimcazole monotherapy administered as adjuvant treatment - evaluation of metastatic disease

[0404] Notwithstanding the clear, objective response to rimcazole in combination with an antiinflammatory drug in clinical examples of an advanced primary tumour (squamous cell carcinoma) and metastatic oral melanoma, the potential of rimcazole as monotherapy in the adjuvant treatment of metastatic disease was evaluated.

[0405] It was reasoned by the inventors that - particularly, though not exclusively - in clinical circumstances where the primary tumour had been successfully treated with another method of treatment such as surgery, rimcazole may have potential as a monotherapy adjuvant treatment - to treat or prevent metastatic disease.

[0406] Figure 10A and B illustrate chest radiographs prior to commencement of treatment (Figure 10A) and after 4 weeks (Figure 10B) treatment with oral rimcazole (10 mg / kg / day)

[0407] Diagnosis: Metastatic (stage 5) mammary carcinoma following mastectomy (surgical removal of the primary tumour)

[0408] Treatment: oral rimcazole (total daily dose 10 mg / kg body weight)

[0409] Clinical response:

[0410] Prior to rimcazole treatment, the baseline pulmonary radiographs (in 3 orientations) were reported, by an independent radiology expert, to reveal 3 discrete metastatic lesions in the lung tissue. A representative lesion is marked up with a ring in Figure 10A, in the lateral orientation.

[0411] After 4 weeks of treatment with rimcazole 10 mg / kg total daily dose, the expert recorded the absence (resolution) of all pulmonary lesions - indicating a complete response (Figure 10B).

[0412] This clinical example supports the use of rimcazole as a single agent (monotherapy) adjuvant treatment to treat or prevent metastatic disease, following another method of treatment for the primary tumour.

[0413] References

[0414] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these citations is incorporated herein by reference.

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Claims

Claims:

1. A compound which is rimcazole, or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof, for use in a method of treating spontaneous cancer in a human or canine patient by increasing the levels of extracellular dopamine in the cancer tissue.

2. The compound for use according to claim 1 wherein the method comprises increasing the level of extracellular dopamine in the cancer tissue, optionally in combination with antagonism of the sigma- 1 receptor.

3. The compound for use according to claim 1 or claim 2 wherein the compound is administered such that the peak plasma level of rimcazole is maintained below a concentration of 1 micromolar.

4. The compound for use according to any one of the preceding claims, wherein the method comprises administration of the compound in combination with an anti-inflammatory agent.

5. The compound for use according to claim 4, wherein the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent (NSAID).

6. The compound for use according to claim 5, wherein the anti-inflammatory agent is an NSAID selected from ibuprofen, naproxen, diclofenac, indomethacin, meloxicam, piroxicam, ketorolac, sulindac, ketoprofen, diflunisal, flurbiprofen, fenoprofen, oxaprozin, tolmetin, nabumetone, etodolac, aspirin, acetaminophen, and tolfenamic acid.

7. The compound for use according to claim 4 or claim 5, wherein the anti-inflammatory agent is a COX-2 inhibitor, optionally selected from celecoxib, etoricoxib, parecoxib, valdecoxib, rofecoxib, cimicoxib, lumiracoxib, firocoxib, deracoxib, robenacoxib, mavacoxib, enflicoxib, carprofen, and meloxicam.

8. The compound for use according to claim 4, wherein the anti-inflammatory agent is a corticosteroid, administered at a non-immunosuppressive dose.

9. The compound for use according to any one of claims 4 to 8, wherein the compound and the anti-inflammatory agent are administered separately, simultaneously, or sequentially.

10. The compound for use according to any one of the preceding claims, wherein the compound is administered in an oral formulation.11 . The compound for use according to any of the preceding claims, wherein the compound is administered to a canine patient with spontaneous cancer at a dosage in the range from 5 mg / kg daily to 30 mg / kg daily.

12. The compound for use according to any of the claims 1 to 10, wherein the compound is administered to a human patient with spontaneous cancer at a dosage in the range from 1 mg / kg daily to 5 mg / kg daily.

13. The compound for use according to any one of claims 1 to 9, wherein the compound is administered in a topical formulation.

14. The compound for use according to claim 13, wherein the topical formulation is for application to the skin and wherein the treatment is local treatment of melanoma (melanocyte) cancer in a human or canine patient.

15. The compound for use according to any one of the preceding claims, wherein the cancer is at an advanced (late) stage.

16. The compound for use according to any one of claims 1 to 14, wherein the cancer is aggressive and at an early stage.

17. The compound for use according to any one of the preceding claims, wherein the human or canine patient is a therapy resistant spontaneous cancer patient, optionally wherein the human or canine patient is a patient with treatment-refractory or relapsed disease.

18. The compound for use according to any one of the preceding claims, wherein the human or canine patient is an immunocompetent patient.

19. The compound for use according to any one of the preceding claims, wherein the spontaneous cancer is a mast cell tumour.

20. The compound for use according to any one of claims 1 to 18, wherein the spontaneous cancer is a squamous cell carcinoma, optionally selected from the group of non-melanoma skin cancer; head and neck cancer including mouth and throat cancer; oesophageal cancer; non-small cell lung cancer (NSCLC); cancer of the cervix; vulval cancer; vaginal cancer; penile cancer;scrotal cancer; anal cancer; non-transitional bladder cancer; mammary cancer, peritoneal (serous) cancer; ocular (conjunctival) cancer.

21. The compound for use according to any one of claims 1 to 18, wherein the spontaneous cancer is a malignant melanoma.

22. The compound for use according to any one of claims 1 to 18, wherein the spontaneous cancer is a fibrosarcoma.

23. The compound for use according to any one of claims 1 to 18, wherein the spontaneous cancer is selected from the group consisting of: sarcoma of bone or soft tissue origin, melanoma or haemangiosarcoma.

24. The compound for use according to any one of claims 1 to 18, wherein the spontaneous cancer is selected from the group of non-melanoma skin cancer, stomach cancer, colon cancer, anorectal cancer, bladder cancer, mammary cancer, kidney cancer, lung cancer, liver cancer, oral cancer, oesophageal cancer, lymphoma, blood cancer (leukaemia).

25. The compound for use according to any one of the preceding claims, wherein the compound is administered as adjuvant treatment prior to, alongside, or following, another method of treatment to reduce the risk of relapse.

26. The compound for use according to any one of the preceding claims, wherein the compound is the hemifumarate or dihydrochloride salt of rimcazole.

27. Use of rimcazole, or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof, in the manufacture of a medicament for use in treating spontaneous cancer in a human or canine patient by increasing the levels of extracellular dopamine in the cancer tissue.

28. The use according to claim 27, wherein the medicament is for use in treating spontaneous cancer by increasing the level of extracellular dopamine in the cancer tissue in combination with antagonism of the sigma-1 receptor.

29. The use according to claim 27 or claim 28 wherein the medicament is for administration such that the peak plasma level of rimcazole is maintained below a concentration of 1 micromolar.

30. The use according to any one of claims 27 to 29, wherein the medicament is for administration of the compound in combination with an anti-inflammatory agent.

31. The use according to claim 30, wherein the anti-inflammatory agent is a non-steroidal antiinflammatory agent (NSAID).

32. The use according to claim 31, wherein the anti-inflammatory agent is an NSAID selected from ibuprofen, naproxen, diclofenac, indomethacin, meloxicam, piroxicam, ketorolac, sulindac, ketoprofen, diflunisal, flurbiprofen, fenoprofen, oxaprozin, tolmetin, nabumetone, etodolac, aspirin, acetaminophen, and tolfenamic acid.

33. The use according to claim 30 or claim 31 , wherein the anti-inflammatory agent is a COX- 2 inhibitor, optionally selected from celecoxib, etoricoxib, parecoxib, valdecoxib, rofecoxib, cimicoxib, lumiracoxib, firocoxib, deracoxib, robenacoxib, mavacoxib, enflicoxib, carprofen, and meloxicam.

34. The use according to claim 30, wherein the anti-inflammatory agent is a corticosteroid, administered at a non-immunosuppressive dose.

35. The use according to any one of claims 30 to 34, wherein the compound and the antiinflammatory agent are administered separately, simultaneously, or sequentially.

36. The use according to any one of claims 27 to 35, wherein the medicament is for administration as an oral formulation.

37. The use according to any one of claims 27 to 35, wherein the medicament is for administration as a topical formulation.

38. The use according to any one of claims 27 to 37, wherein the cancer is at an advanced (late) stage.

39. The use according to any one of claims 27 to 37, wherein the cancer is aggressive and at an early stage.

40. The use according to any one of claims 27 to 39, wherein the human or canine patient is a therapy resistant spontaneous cancer patient, optionally wherein the human or canine patient is a patient with treatment-refractory or relapsed disease.41 . The use according to any one of claims 27 to 40, wherein the human or canine patient is an immunocompetent patient.

42. The use according to any one of claims 27 to 41 , wherein the spontaneous cancer is a mast cell tumour; or a squamous cell carcinoma, optionally selected from the group of nonmelanoma skin cancer; head and neck cancer including mouth and throat cancer; oesophageal cancer; non-small cell lung cancer (NSCLC); cancer of the cervix; vulval cancer; vaginal cancer; penile cancer; scrotal cancer; anal cancer; non-transitional bladder cancer; mammary cancer, peritoneal (serous) cancer; ocular (conjunctival) cancer; or a malignant melanoma; or a fibrosarcoma.

43. The use according to any one of claims 27 to 41 , wherein the spontaneous cancer is selected from the group consisting of: sarcoma of bone or soft tissue origin, melanoma or haemangiosarcoma; or from the group of non-melanoma skin cancer, stomach cancer, colon cancer, anorectal cancer, bladder cancer, mammary cancer, kidney cancer, lung cancer, liver cancer, oral cancer, oesophageal cancer, lymphoma, blood cancer (leukaemia).

44. The use according to any one of claims 27 to 43, wherein the medicament is for administration as adjuvant treatment prior to, alongside, or following, another method of treatment to reduce the risk of relapse, wherein said reduction comprises (i) prevention of primary tumour recurrence and / or (ii) treatment or prevention of secondary spread (metastases).

45. The use according to any one of claims 27 to 44, wherein the compound is the hemifumarate or dihydrochloride salt of rimcazole.

46. A method of treating spontaneous cancer in a human or canine patient by increasing the levels of extracellular dopamine in the cancer tissue, wherein the method comprises administering an effective amount of a compound which is rimcazole or a pharmaceutically acceptable salt, ester, hydrate, solvate, prodrug, or derivative thereof, to the patient.

47. The method of claim 46, wherein the method comprises increasing the level of extracellular dopamine in the cancer tissue in combination with antagonism of the sigma-1 receptor.

48. The method of claim 46 or claim 47 wherein the compound is administered such that the peak plasma level of rimcazole is maintained below a concentration of 1 micromolar.

49. The method of any one of claims 46 to 48, wherein the method comprises administration of the compound in combination with an anti-inflammatory agent.

50. The method of claim 49, wherein the anti-inflammatory agent is a non-steroidal antiinflammatory agent (NSAID).

51. The method of claim 50, wherein the anti-inflammatory agent is an NSAID selected from ibuprofen, naproxen, diclofenac, indomethacin, meloxicam, piroxicam, ketorolac, sulindac, ketoprofen, diflunisal, flurbiprofen, fenoprofen, oxaprozin, tolmetin, nabumetone, etodolac, aspirin, acetaminophen, and tolfenamic acid.

52. The method of claim 49 or claim 50, wherein the anti-inflammatory agent is a COX-2 inhibitor, optionally selected from celecoxib, etoricoxib, parecoxib, valdecoxib, rofecoxib, cimicoxib, lumiracoxib, firocoxib, deracoxib, robenacoxib, mavacoxib, enflicoxib, carprofen, and meloxicam.

53. The method of claim 49, wherein the anti-inflammatory agent is a corticosteroid, administered at a non-immunosuppressive dose.

54. The method of any one of claims 49 to 53, wherein the compound and the antiinflammatory agent are administered separately, simultaneously, or sequentially.

55. The method of any one of claims 46 to 54, wherein the compound is administered in an oral formulation.

56. The method of any of claims 46 to 55, wherein the compound is administered to a canine patient with spontaneous cancer at a dosage in the range from 5 mg / kg daily to 30 mg / kg daily.

57. The method of any of claims 46 to 55, wherein the compound is administered to a human patient with spontaneous cancer at a dosage in the range from 1 mg / kg daily to 5 mg / kg daily.

58. The method of any one of claims 46 to 54, wherein the compound is administered in a topical formulation.

59. The method of claim 58, wherein the topical formulation is for application to the skin and wherein the treatment is local treatment of melanoma (melanocyte) cancer in a human or canine patient.

60. The method of any one of claims 46 to 59, wherein the compound is the hemifumarate or dihydrochloride salt of rimcazole,61 . The method of any one of claims 46 to 60, wherein the cancer is at an advanced (late) stage.

62. The method of any one of claims 46 to 60, wherein the cancer is aggressive and at an early stage.

63. The method of any one of claims 46 to 62, wherein the human or canine patient is a therapy resistant spontaneous cancer patient, optionally wherein the human or canine patient is a patient with treatment-refractory or relapsed disease.

64. The method of any one of claims 46 to 63, wherein the human or canine patient is an immunocompetent patient.

65. The method of any one of claims 46 to 64, wherein the spontaneous cancer is selected from: a mast cell tumour; a squamous cell carcinoma, optionally selected from the group of nonmelanoma skin cancer; head and neck cancer including mouth and throat cancer; oesophageal cancer; non-small cell lung cancer (NSCLC); cancer of the cervix; vulval cancer; vaginal cancer; penile cancer; scrotal cancer; anal cancer; non-transitional bladder cancer; mammary cancer, peritoneal (serous) cancer; ocular (conjunctival) cancer; a malignant melanoma; or a fibrosarcoma.

66. The method of any one of claims 46 to 64, wherein the spontaneous cancer is selected from the group consisting of: sarcoma of bone or soft tissue origin, melanoma or haemangiosarcoma; or from the group of non-melanoma skin cancer, stomach cancer, colon cancer, anorectal cancer, bladder cancer, mammary cancer, kidney cancer, lung cancer, liver cancer, oral cancer, oesophageal cancer, lymphoma, blood cancer (leukaemia).

67. The method of any one of claims 46 to 66, wherein the compound is administered as adjuvant treatment prior to, alongside, or following, another method of treatment to reduce the risk of relapse, wherein said reduction comprises (i) prevention of primary tumour recurrence and / or (ii) treatment or prevention of secondary spread (metastases).

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