Patent Application
20130029928
The invention relates to compositions for treating cancer. In particular there is provided, doses, dosage regimes for the administration of Perfluorooctanoate (PFOA) and in particular, Ammonium Perfluorooctanoate (APFO) in the treatment of cancer.
Ammonium Perfluorooctanoate (APFO) has the molecular formula C8F15O2.H4N
APFO is the ammonium salt of straight chain perfluorooctanoic acid (PFOA). Commercially available ammonium perfluorooctanoate (APFO) is a mixture of approximately 75% straight chain and 25% various branched isomers.
Preliminary experiments to evaluate mode of action were performed using this mixture (APFO). We have previously described (WO 2004/019927 WO 2002/66028) the use of perfluorinated carboxylic acids for the treatment of cancer.
Subsequently, the purified straight chain isomer was obtained, and the results obtained with APFO were verified with this isomer (CXR1002).
CXR1002 is a fatty acid mimetic in that it interacts with fatty acid homeostasis and/or a fatty acid mediated pathway. Both CXR1002 and APFO isomers and also perfluoroalkyls of different chain lengths possess these properties. This has been demonstrated in Vanden Heuvel (1996) where it was shown that different nuclear hormone receptors were activated by PFOA and how this compared to natural fatty acid activation of the same receptors. Wolf (2008) showed a dose response of various chain length perfluoroalkyls against PPAR alpha (FIG. 3 of Wolf (2008)) in a transiently transfected COS-1 cell model to compare the C4 to C9 chain lengths.
It has now been shown that APFO and the CXR1002 isomer has additional mechanisms of action accounting for some of its anti-tumour effects.
APFO has been shown to cause Endoplasmic Reticulum (ER) stress (see Example 8). Endoplasmic reticulum stress induction has been shown to have an anti-tumour effect, including in pancreatic cancer, myeloma and thyroid cancer. For example, sorafenib, bortezomib and Hsp90 cause cell death by induction of ER stress pathways and bortezomib is used clinically to treat multiple myeloma and mantle cell lymphoma. Review articles discussing the association with ER stress and Cancer are Healy (2009), Strasser (2008) and Moenner (2007).
APFO has also been shown to have activity against PIM kinases (see Example 9). PIM kinases are cytoplasmic serine/threonine kinases that are known to be involved in regulation of apoptosis and cellular metabolism. Certain PIM kinases have been shown to be upregulated in cancers and as such their inhibition represents a mechanism of action by which CXR1002 can have an anti-tumour effect in conditions such as leukaemia, lymphoma, prostate cancer, colon cancer and pancreatic cancer. The below studies have shown this link:
PIM kinases are constitutively active and their activity as shown above and in Amaravadi (2005) and Shah (2008) supports in vitro and in vivo human cell growth and survival.
APFO is a perfluorinated carboxylic acid that exerts its anti-tumour effects via multiple mechanisms of action. Previously it had been know that APFO acts by one or more peroxisome proliferator activated receptor (PPAR)-mediated mechanisms. PPARs are members of the nuclear hormone receptor family of transcription factors. They modulate DNA transcription by binding to specific peroxisome proliferator-response elements (PPREs) on target genes.
CXR1002 is a white, odourless solid that is freely soluble in water. CXR1002 and its family of compounds are extremely stable.
The investigational medicinal product being made in the clinical trials described in the examples consists of Size 1 white opaque gelatin capsules containing the active substance, CXR 1002. There is no bulking agent. One strength of capsule has been manufactured with a target strength of 50 mg of CXR1002 per capsule.
Laboratory studies have indicated that CXR1002 can interact with cells in a number of different ways which could be associated with its pharmacological effectiveness as an anti-tumour agent. For example, CXR1002 is an agonist of PPARs and also induces ER-stress in tumour cells. CXR1002 has also been shown to have a range of biological effects probably related to its surfactant properties, including; alteration of cell membrane potential and cytostolic pH (Kleszczynski (2009)); induction of oxidative stress (Fernandez (2008)) that was closely linked to cell cycle arrest; dissipation of mitochondrial membrane potential (Hu (2009)) and dysregulation of gap-junctional intercellular communication (GJIC) and activation of extracellular receptor kinase (ERK) (Upham (2009)). CXR1002 is cytotoxic to tumour cells with an IC50 ranging upwards from 273 μM.
The data presented demonstrate that CXR1002 has anti-tumour activity both in vitro and in xenograft models. The mechanism of action, involving agonism of PPARs α and γ in association with neutral or inhibitory action on PPARδ, is distinct from those of currently available chemotherapeutic agents. In addition CXR1002 induces ER-stress in some cancer cell lines; this may be an effect that is related to its effects on PPARs. Furthermore CXR1002 is an inhibitor of the PIM kinase family of serine/threonine kinases. CXR1002 could provide anticancer activity against a range of tumour types. Humans have already received environmental exposure to CXR1002 and workers involved in the manufacture of APFO have been recorded as having serum concentrations as high as 275 μM without reported adverse effects. Furthermore, patients in the ongoing CXR1002-001 study have exposure in the 200 μM to 800 μM range after a few weeks of dosing with CXR1002. This level of exposure to cells in vitro or to a xenografted tumour would be expected to have a biological effect.
As of February 2011, 43 patients with advanced cancers from one Phase I study have received CXR1002. CXR1002 is not metabolised and dosing is accumulative. It is presumed that CXR1002 will eventually reach a steady state level after a number of doses, in an analogous way to its accumulative exposure in monkeys. The lack of metabolism of CXR1002 provides an advantage over other chemotherapeutic agents such that inter-patient variability in exposure is low as metabolism of the active ingredient at different rates in different patients is not an issue for CXR1002.
Significant occupational exposure to PFOA and its salts, including APFO, has occurred over many years and APFO has been found in the blood of workers exposed in the workplace. The dogma derived from studies such as these is that CXR1002 has a long serum half-life in humans (range=109 to 1308 days). Data from the CXR1002-001 clinical trial, demonstrate that after a single dose of CXR1002, the plasma level of the drug is constant over the 6 week sampling period, indicating that the half life is >6 weeks.
However, patients in the phase I study receiving >100 mg weekly dose have higher exposure after 6 weeks of dosing than the maximal values recorded in occupationally exposed workers.
A large database of experimental studies on the potential health hazards of APFO is available, as are recent toxicology reviews (USEPA (2005)), (Kennedy (2004)). In addition to toxicology studies in laboratory animals, the potential association of APFO exposure with health effects in fluorochemical production workers has been studied since 1976 through medical monitoring and epidemiological investigations (Ubel (1980)), (Olsen (1998)), (Olsen (2000)).
The majority of studies reported in the literature have used APFO itself, although some studies using other salts have also been described. The biological effects of APFO are thought to be due to its dissociation to form perfluorooctanoate (PFOA), the anionic form of perfluorooctanoic acid. Perfluorooctanoic acid and its salts are soluble in water and readily dissociates to the carboxylate anion, perfluorooctanoate (PFOA) (Kennedy (2004)).
The consensus is that, since the active constituent of each of these compounds is the perfluorooctanoate anion, these studies are directly comparable. An extensive toxicology and occupational health database already exists for this compound. Several studies of relevance have been commissioned by commercial companies but the reports are not in the public domain. However, the field has been thoroughly reviewed by Kennedy et al. (2004) and the USEPA (2005). In addition, key studies have been published in the scientific literature or are available through the USEPA public docket.
Most commercial studies on APFO/PFOA have used a commercial material e.g. FC-143 FLUORAD, which comprises 93-97% APFO and the remaining consisting of a mixture of Ammonium perfluoropentanoate, Ammonium perfluoroheptanoate and Ammonium perfluorohexanaote.
Unlike most other anti-tumour agents, PFOA is efficiently absorbed following oral exposure. It is not metabolised and is eliminated intact. PFOA exhibits only moderate acute oral toxicity. Signs and symptoms of toxicity include body weight loss, liver weight increase and liver effects as demonstrated by increased serum transaminase activity and diffuse hepatocellular hypertrophy accompanied, at higher doses, by acidophilic degeneration and/or necrosis of the liver. PFOA exhibits no teratogenic or foetotoxic effects in rats at doses below those causing maternal toxicity and there is no evidence of any adverse effects on reproductive success in a two-generation reproduction study. Two year cancer bioassays in rats resulted in increased incidence of benign tumours (adenomas) of the liver, pancreas (acinar cell) and testes (Leydig cell) at 300 ppm in the diet, but not at 30 ppm. A battery of tests for genotoxicity demonstrated that PFOA does not cause either point mutations or chromosomal aberrations.
None of the toxicology studies give any indication of changes in cardiovascular, central nervous system, respiratory or renal function induced by PFOA. Studies in rats have revealed no clinical signs that suggested adverse pharmacological effects. Furthermore, there was no evidence of such effects in a 26-week toxicity study in male cynomolgus monkeys.
Although no specific studies have been carried out in humans on the potential unwanted pharmacological effects of PFOA, there are no significant toxicities reported in workers with significant occupational exposure.
PFOA is well absorbed following oral exposure. After a single oral dose of 14C-PFOA (11 mg/kg) to male rats at least 93% of total radioactivity was absorbed at 24 hrs (58). Following a single gavage administration to rats (25 mg/kg), peak blood levels were attained 1-2 hours after dosing (Kennedy (2004)). There was a clear sex difference in clearance. Blood levels in female rats showed >95% clearance 24 hrs after dosing, while blood levels in males remained relatively high throughout this period. The sex difference in clearance was even more marked 1 week after treatment, when blood levels in males remained relatively high and those in females had declined to very low levels.
Importantly, PFOA does not appear to accumulate in blood of female rats, since the blood profile of an oral dose of 25 mg/kg following 10 previous similar doses was quite similar to that observed after a single oral dose (Kennedy (2004)).
The amounts of PFOA deposited in the tissues of different species are inversely related to the species-specific rate of urinary excretion. In species which excrete PFOA slowly, the compound distributes primarily to the liver, plasma and the kidney and to a lesser extent other tissues of the body, including testis and ovary. For example, following 28 days gavage administration to male rats the major sites of deposition were the serum, liver and kidney. Little transfer to the brain occurs in adults. In female rats, the pattern of tissue deposition is dose-dependent. At 3 mg/kg more PFOA is deposited in the liver than the kidney whereas this is reversed at higher doses, suggesting the existence of a saturable renal excretory mechanism in the (female) rat (Kennedy (2004)).
There is no evidence that APFO is metabolised in mammals once dissociated to form perfluorooctanoate. However, analysis of five major drug metabolising cytochrome P450 (CYP) isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) indicated that CXR1002 is an inhibitor of CYP2C9, having an IC50 of 0.76 μM under the conditions used (unpublished data). Similar results were obtained using APFO, which had an IC50 of 0.78 μM towards CYP2C9.
The main factor determining the elimination rate of PFOA in different species is the rate of urinary excretion. In female rats, the extent of biliary excretion is <1.0% (Vanden (1991)).
The human renal clearance of PFOA has been evaluated in Japanese volunteers (Hasada (2005)). There were no significant differences in the renal clearance of PFOA with regard to sex, age group, medication, and medical or residential history.
To date, studies of PFOA have primarily related to the effects of the compound as a contaminant and occupational exposure in humans. Little is known regarding its safe and effective use as a therapeutic agent. Safe and efficacious dosages and therapeutic administration regimes have now been identified, specifically in relation to the treatment of cancer.
Furthermore, combinations of PFOA and other chemotherapeutic agents that are unexpectedly advantageous have also been identified as part of the invention.
APFO, and in particular the CXR1002 isomer has a large number of beneficial properties in comparison to existing chemotherapeutic agents. For example, CXR1002 is highly water soluble and as such is highly bioavailable. The high bioavailability is partially explained by CXR1002 possessing a long half life (shown to be greater than 6 weeks of half life in the clinical trials discussed in the examples). CXR1002 is now known not to be a substrate for human metabolism and as such dose and plasma concentration are closely linked and importantly variation between individuals is minimal (as there is no metabolism of CXR1002 there is no variability between individuals in metabolism). The slow clearance of CXR1002 means that a missed dose can be easily compensated for at a later date without an extensive loss of exposure to CXR1002. Due to the low variability of CXR1002 metabolism and clearance between individuals, dose strength and dose frequency required to achieve a desired plasma concentration is readily calculable by a skilled person because circulating plasma concentration can be reliably predicted from each dose taken.
CXR1002 has been shown in the clinical trials described in the examples to be orally bioavailable and this allows for simpler administration than current chemotherapeutic treatments (which are often given by intravenous administration), even to the point of allowing CXR1002 to be taken by patients outside of a hospital setting. In addition the CXR1002 capsule formulation has at least a 57 month shelf life that is commercially useful. The clinical trial work being conducted on CXR1002 has shown that CXR1002 is relatively non-toxic (at the doses examined to date CXR1002 does not cause toxicity commonly associated with anti-cancer drugs (no myelosuppresion, no anaemia, no transfusion requirement, no hair loss, mild or no effect on digestive system (individual variability apparent), no mouth ulcers, no skin problems, no lung effects, no heart effects, no neuropathy or nerve changes).
Although there is some reported nausea and vomiting with CXR1002, study subjects are not receiving concomitant anti-emetics, and these adverse events are of short duration.
Although CXR1002 causes liver enzyme changes in many toxicological test species (such as rats), the frequency of this in study subjects is low, with the predominant side effects being relatively mild including lethargy and mild gastrointestinal disturbance, nausea/vomiting and diarrhoea). The low toxicity of CXR1002 is supported by evaluation of pharmacodynamic markers in the clinical trials as discussed in the examples, which has shown there to be no significant changes.
The low toxicity profile and lack of metabolism allow CXR1002 to be used in combination with other therapeutic regimes with significant side-effects including cytotoxic chemotherapeutics and radiotherapy. Unlike other chemotherapeutics, CXR1002 can be used at the same time or prior to surgery with no wash out period required as CXR1002 would not exhibit the same side-effects as other chemotherapeutics on wound healing and immune response (due to the low toxicity of CXR1002).
Hence CXR1002 has been shown to possess significant advantages over other chemotherapeutics, these advantages allowing the specific compositions, dosage regimes and combination therapies to be identified and optimized as herein described.
In a first aspect of the invention there is provided a composition comprising between 10 mg and 2000 mg of an active ingredient per dosage unit, wherein the active ingredient is perfluorooctanoic acid (PFOA) or a derivative, salt or variant thereof.
By dosage unit we mean the unit of medicament administered to a patient at one time. For example, the dosage unit, or single dose may be administered by a single capsule/tablet, single injection, or single intravenous infusion, a single subcutaneous injection, or by a single procedure using other routes of administration, as discussed below. Alternatively, the single dose may be administered to the patient by two or more capsules/tablets or injections given simultaneously or sequentially to deliver the entire dose to the patient in the continuous, single and defined treatment period; by two or more intravenous infusions given simultaneously or sequentially to deliver the entire dose to the patient in the continuous, single and defined treatment; or by multiple procedures using other routes of administration as discussed below.
Alternatively, the single dose to be administered to the patient can be delivered by a combination of routes to deliver the entire dose to the patient in the continuous, single and defined treatment.
The dosage unit may then be repeated at intervals of time such as a few hours, days, weeks, or months later.
Dosage units can be administered to patients in such a way that the patient receives a loading dose followed by one or more maintenance doses. For example the loading dose may be a high dose in order to quickly reach a desired plasma concentration and then subsequent maintenance doses are a lower dose than the loading dose in order to maintain the required plasma concentration.
By active ingredient we mean the molecule having the desired effect. In this case of this invention we primarily mean PFOA and derivatives, salts or variants thereof.
By variants and derivatives we mean any molecules of substantially identical chemical structure but including minor modifications that do not alter activity but may offer improved or alternative properties for formulation, such as formation into a salt.
In human therapy, the PFOA containing composition, and medicaments of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
For example, the PFOA containing composition, and medicaments of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The PFOA containing composition, and medicaments of the invention may also be administered via intracavernosal injection.
Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the PFOA containing composition, medicaments and pharmaceutical compositions of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
The PFOA containing composition, and medicaments of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The medicaments and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
The PFOA containing composition, and medicaments of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active agent, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a PFOA containing composition, of the invention and a suitable powder base such as lactose or starch.
Aerosol or dry powder formulations are preferably arranged so that each metered dose or “puff” contains an effective amount of an agent or polynucleotide of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
Alternatively, the PFOA containing composition, and medicaments of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, gel, ointment or dusting powder. The PFOA containing composition, and medicaments of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.
For ophthalmic use, the PFOA containing composition, and medicaments of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
For application topically to the skin, the PFOA containing composition, and medicaments of the invention can be formulated as a suitable ointment containing the active agent suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
Generally, in humans, oral or parenteral administration of the PFOA containing composition, medicaments and pharmaceutical compositions of the invention is the preferred route, being the most convenient.
For veterinary use, the PFOA containing composition, and medicaments of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
The PFOA containing composition, as defined herein may be formulated as described in the accompanying Examples.
Preferably the PFOA is ammonium perfluorooctanoic acid (APFO), the ammonium salt.
The composition may comprise any effective amount of active ingredient, this may be between 10 mg and 2000 mg of active ingredient per dosage unit, and preferably is between 50 mg and 1000 mg. Advantageously it is 1000 mg. Conveniently, the dosage unit contains an amount of active ingredient per dosage unit selected from 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 450 mg, 600 mg, 750 mg, 950 mg, 1000 mg and 1200 mg.
Alternatively, the composition may comprise between 10-50 mg, 10-75 mg, 10-100 mg, 10-200 mg, 10-300 mg, 10-400 mg, 10-600 mg, 10-750 mg, 10-950 mg, 10-1000 mg, 10-1200 mg, 50-75 mg, 50-100 mg, 50-200 mg, 50-300 mg, 50-450 mg, 50-600 mg, 50-750 mg, 50-950 mg, 50-1000 mg, 50-1200 mg, 75-100 mg, 75-200 mg, 75-300 mg, 75-450 mg, 75-600 mg, 75-750 mg, 75-950 mg, 75-1000 mg, 75-1200 mg, 100-200 mg, 100-300 mg, 100-450 mg, 100-600 mg, 100-750 mg, 100-950 mg, 100-1000 mg, 100-1200 mg, 200-300 mg, 200-450 mg, 200-600 mg, 200-750 mg, 200-950 mg, 200-1000 mg, 200-1200 mg, 300-450 mg, 300-600 mg, 300-750 mg, 300-950 mg, 300-1000 mg, 300-1200 mg, 400-600 mg, 400-750 mg, 400-950 mg, 400-1000 mg, 400-1200 mg, 450-600 mg, 450-750 mg, 450-950 mg, 450-1000 mg, 450-1200 mg, 600-750 mg, 600-950 mg, 600-1000 mg, 600-1200 mg, 700-950 mg, 700-1000 mg, 700-1200 mg, 950-1000 mg, 950-1200 mg and 1000-1200 mg
Preferably there is 400-600 mg of active ingredient. More preferably there is 400-1200 mg of active ingredient. Most preferably there is 1000 mg of active ingredient.
Conveniently, the composition is pharmaceutically acceptable, and may optionally contain a pharmaceutically acceptable excipient, diluent, carrier or filler.
In a second aspect of the invention there is provided a composition as defined in the first aspect of the invention for use as a medicine.
In a third aspect of the invention there is provided a composition as defined in the first aspect of the invention for use in the treatment of cancer.
By “treatment” we include the meanings that tumour size is reduced and/or further tumour growth is retarded and/or prevented and/or the tumour is killed. We also include the reduction of other symptoms associated with the cancer being treated such as (but not limited to) a reduction in pain, cachexia and metastasis. The treatment may incorporate multiple aspects including chemotherapy, surgery and radiotherapy. The composition of the invention may be used on its own as a chemotherapeutic or with any other treatment for cancer, including before, during and after any other treatment type.
By ‘treatment’ we include both therapeutic and prophylactic treatment of a subject/patient. The term ‘prophylactic’ is used to encompass the use of composition described herein which either prevents or reduces the likelihood of the occurrence or development of cancer in a patient or subject.
A ‘therapeutically effective amount’, or ‘effective amount’, or ‘therapeutically effective’, as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce or prevent a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host.
In a fourth aspect of the invention there is provided a use of a composition as defined in the first aspect of the invention in the manufacture of a medicament for the treatment of cancer.
In a fifth aspect of the invention there is provided a method of treating cancer comprising administering an effective amount of a composition as defined in the first aspect of the invention. Preferably the effective amount is between 10 and 2000 mg per dose, preferably between 50 and 600 mg per dose, and more preferably between 50 and 1200 mg per dose. Alternatively the effective amount is between 1 and 20 mg/kg, preferably between 1 and 7 mg/kg.
As is appreciated by those skilled in the art, the precise amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
In a particularly preferred embodiment, the amount of the active ingredient administered to a patient is approximately between: 0.02 mg/kg to 0.10 mg/kg; or 0.10 mg to 0.20 mg/kg; or 0.20 mg to 0.30 mg/kg; or 0.30 mg to 0.40 mg/kg; or 0.40 mg to 0.50 mg/kg; or 0.50 mg to 0.60 mg/kg; or 0.60 mg to 0.70 mg/kg; or 0.70 mg to 0.80 mg/kg; or 0.80 mg to 0.90 mg/kg; or 0.90 mg to 1.00 mg/kg; or 1.00 mg to 1.10 mg/kg; or 1.10 mg to 1.20 mg/kg; or 1.20 mg to 1.30 mg/kg; or 1.30 mg to 1.40 mg/kg; or 1.40 mg to 1.50 mg/kg; or 1.50 mg to 1.60 mg/kg; or 1.60 mg to 1.70 mg/kg; or 1.70 mg to 1.80 mg/kg; or 1.80 mg to 1.90 mg/kg; or 1.90 mg to 2.00 mg/kg; or 2.00 mg/kg to 2.10 mg/kg; or 2.10 mg to 2.20 mg/kg; or 2.20 mg to 2.30 mg/kg; or 2.30 mg to 2.40 mg/kg; or 2.40 mg to 2.50 mg/kg; or 2.50 mg to 2.60 mg/kg; or 2.60 mg to 2.70 mg/kg; or 2.70 mg to 2.80 mg/kg; or 2.80 mg to 2.90 mg/kg; or 2.90 mg to 3.00 mg/kg; or 3.00 mg to 3.10 mg/kg; or 3.10 mg to 3.20 mg/kg; or 3.20 mg to 3.30 mg/kg; or 3.30 mg to 3.40 mg/kg; or 3.40 mg to 3.50 mg/kg; or 3.50 mg to 3.60 mg/kg; or 3.60 mg to 3.70 mg/kg; or 3.70 mg to 3.80 mg/kg; or 3.80 mg to 3.90 mg/kg; or 3.90 mg to 4.00 mg/kg; or 4.00 mg to 4.10 mg/kg; or 4.10 mg to 4.20 mg/kg; or 4.20 mg to 4.30 mg/kg; or 4.30 mg to 4.40 mg/kg; or 4.40 mg to 4.50 mg/kg; or 4.50 mg to 4.60 mg/kg; or 4.60 mg to 4.70 mg/kg; or 4.70 mg to 4.80 mg/kg; or 4.80 mg to 4.90 mg/kg; or 4.90 mg to 5.00 mg/kg; or 5.00 mg/kg to 6.00 mg/kg; or 6.00 mg to 7.00 mg/kg; or 7.00 mg to 8.00 mg/kg; or 8.00 mg to 9.00 mg/kg; or 9.00 mg to 10.00 mg/kg; or 10.00 mg to 11.00 mg/kg; or 11.00 mg to 12.00 mg/kg; or 12.00 mg to 13.00 mg/kg; or 13.00 mg to 14.00 mg/kg; or 14.00 mg to 15.00 mg/kg; or 15.00 mg to 16.00 mg/kg; or 16.00 mg to 17.00 mg/kg; or 17.00 mg to 18.00 mg/kg; or 18.00 mg to 19.00 mg/kg; or 19.00 mg to 20.00 mg/kg.
A composition, use or method of any of the third to fifth aspects wherein the treatment comprises the step of administering to a patient in need thereof an effective amount of the composition, in a single dosage at a frequency of once or twice per week (weekly or semi-weekly). Conveniently, the single dosage is administered at a frequency of less than once per week, preferably fortnightly or once per six weeks or less.
The dosage may be administered as a higher loading dose followed by one or more lower maintenance doses.
In a sixth aspect of the invention there is provided a therapeutic system for the treatment of cancer comprising administration of a composition as defined in the first aspect in a single dosage of between 10 mg and 2000 mg at a frequency of once per week or less.
By therapeutic system we mean a system of administering compositions to a patient in an effective manner to treat a specific disease. The system may be characterised by the dosages to be administered, the intervals between dosages and the methods of administration, or combinations thereof. The system may also be interchangeably known as a dosage regime.
Preferably, the dosage is between 200 mg and 1200 mg. Conveniently, the dosage is selected from 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 450 mg, 600 mg, 750 mg, 950 mg, 1000 mg and 1200 mg.
Alternatively, the dosage is selected from 1 mg/kg to 7 mg/kg.
Preferably, the dosage frequency is once per six weeks or less.
In the third to sixth aspects of the invention, the cancer may be selected from pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, liver cancer, chondrosarcoma, lung cancer, head and neck cancer, colon cancer, sarcoma, leukaemia, lymphoma, kidney cancer, thyroid cancer and brain cancers such as glioblastoma.
In a seventh aspect of the invention there is provided a composition comprising perfluorooctanoic acid (PFOA) or a salt, derivative or variant thereof; and a further chemotherapeutic agent. Alternatively, there is provided a composition comprising an active ingredient as defined in the first aspect and a further chemotherapeutic agent.
Preferably, the further chemotherapeutic is selected from Doxorubicin, Gemcitabine, Roscovitine, Rapamycin, 5-FU, PARP inhibitors, kinase inhibitors including PIM kinase inhibitors and MAP kinase inhibitors, Hsp90 inhibitors (including Geldanamycin), proteasome inhibitors (including Bortezomib) and HDAC inhibitors (including SAHA); and prodrugs thereof.
Preferably, the further chemotherapeutic is present in an individually effective dose.
By individually effective dose we mean the dose at which the further chemotherapeutic is known to be effective when administered on its own.
Alternatively, the further chemotherapeutic is present in a lower than individually effective dose.
By lower than individually effective dose we mean a dose which is lower than that which is known to be the effective dose when the further chemotherapeutic is administered on its own. In other words, a lower dose than normal is administered because the combination provides a synergistic effect. This has the effect of reducing the administration of chemotherapeutics with unpleasant or dangerous side effects.
In an eighth aspect of the invention there is provided a composition as defined in the seventh aspect for use as a medicine.
In a ninth aspect there is provided a composition as defined in the seventh aspect for use in the treatment of cancer.
In a tenth aspect there is provided a use of a composition as defined in the seventh aspect in the manufacture of a medicament for the treatment of cancer.
In an eleventh aspect there is provided a method of treating cancer comprising administering an effective amount of a composition as defined in the seventh aspect.
In a twelfth aspect there is provided a therapeutic system for the treatment of cancer comprising a combination of component (i) a composition as defined in the first aspect; and (ii) a further chemotherapeutic agent, the components (i) and (ii) being provided for the use in the treatment of cancer and wherein components (i) and (ii) are administered in combination with one another.
By “in combination with one another” regarding the PFOA and chemotherapeutic agent treatments we include the meaning not only that the PFOA and chemotherapeutic agents are administered simultaneously, but also that they are administered separately and sequentially.
In one embodiment, administration of component (i) precedes administration of component (ii). In an alternative embodiment, administration of component (ii) precedes administration of component (i). In a further alternative embodiment, administration of component (i) occurs at the same time as administration of component (ii).
It is envisaged that the components may be administered in any order depending on individual circumstances including, need, drug availability, administration routes used. Preferably the PFOA and chemotherapeutic agents are administered between 0 and 24 hours apart with either the PFOA or the chemotherapeutic being administered first.
Preferably, the further chemotherapeutic of the therapeutic system is selected from Doxorubicin, Gemcitabine, Roscovitine, Rapamycin, 5-FU, PARP inhibitors, kinase inhibitors including PIM kinase inhibitors and MAP kinase inhibitors, Hsp90 inhibitors (including Geldanamycin), proteasome inhibitors (including Bortezomib) and HDAC inhibitors (including SAHA); and prodrugs thereof.
In particular chemotherapeutics that enhance or complement the mechanisms of action of the composition of the invention (CXR1002) are preferred e.g. Hsp90 inhibitors, proteasome inhibitors and HDAC inhibitors.
Hsp90 inhibitors, including geldanamycin, target the chaperone Hsp90 and promote ubiquitin-dependent proteasomal degradation of proteins, leading to ER stress. Bortezomib, a proteasome inhibitor, also promotes the accumulation of aggregated, ubiquitinated proteins in the ER and therefore also cause ER stress. HDAC inhibitors have been shown to act synergistically with bortezomib, indicating that they may be id useful together with agents that induce ER stress (such as CXR1002). PIM kinase inhibition can restore sensitivity to FLT3 and BCR/ABL mutations that confer resistance to tyrosine kinase inhibitors.
In the ninth to twelfth aspects, the cancer may be selected from pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, liver cancer, chondrosarcoma, lung cancer, head and neck cancer, colon cancer, sarcoma, leukaemia, lymphoma, kidney cancer, thyroid cancer and brain cancers such as glioblastoma.
In one embodiment when the cancer is pancreatic cancer, the further chemotherapeutic is selected from Doxorubicin, Gemcitabine, Geldanamycin and Roscovitine.
In an alternative embodiment, when the cancer is chondrosarcoma, the further chemotherapeutic is Gemcitabine.
In a further embodiment, when the cancer is ovarian cancer, the further chemotherapeutic is selected from Doxorubicin, Gemcitabine, Geldanamycin, Roscovitine, Rapamycin and 5-FU or pro-drugs thereof.
In a yet further embodiment, when the cancer is prostate cancer, the further chemotherapeutic is selected from Doxorubicin, Geldanamycin and Roscovitine
In another embodiment, when the cancer is breast cancer, the further chemotherapeutic is 5-FU or pro-drugs thereof.
In an alternative embodiment, when the cancer is liver cancer, the further chemotherapeutic is selected from Gemcitabine, Geldanamycin, Roscovitine and Rapamycin.
In a thirteenth aspect of the invention there is provided a kit of parts comprising:
(i) a composition as defined in the first embodiment; and
(ii) a further chemotherapeutic agent.
The kit may optionally comprise:
(iii) means of administering (i) and (ii) to a patient, wherein the administration may be at the same time or in succession.
Preferably the further chemotherapeutic agent of the kit is selected from Doxorubicin, Gemcitabine, Roscovitine, Rapamycin, 5-FU, PARP inhibitors, kinase inhibitors including PIM kinase inhibitors and MAP kinase inhibitors and Hsp90 inhibitors (including Geldanamycin), proteasome inhibitors (including Bortezomib) and HDAC inhibitors (including SAHA); prodrugs thereof.
The kit may also comprise instructions for use.
Examples embodying certain aspects of the invention will now be described with reference to the following figures in which:
The earliest recognised characteristic of PPARα agonists was their ability to induce peroxisome proliferation in hepatocytes. The PPARα response is reflected in the increased transcription of mitochondrial and peroxisomal lipid metabolism, sterol, and bile acid biosynthesis and retinol metabolism genes (Andersen (2008)). Administration of APFO to rats led to hepatic peroxisome proliferation as measured by the induction of the peroxisomal marker activity cyanide-insensitive palmitoyl CoA oxidation (unpublished data).
Peroxisome proliferation occurs as a result of the interaction of a chemical with PPARα. This leads to an increase in the synthesis of peroxisomal and lipid-metabolising enzymes and, consequently, an increase in size and number of peroxisomes. Cyanide-insensitive palmitoyl CoA oxidation is an accepted marker of peroxisome proliferation, and was used to highlight PPARα activation in vitro and in vivo.
In vivo, APFO exhibits aspects of pharmacology typical of both PPARα and γ agonism. Male Sprague Dawley rats (n=6) were administered APFO (300 ppm) in powdered diet daily while control animals received powdered diet only. Rats were sacrificed at 7, 14, 28 and 84 days. Blood from each study animal was taken by cardiac puncture into lithium/heparin-coated tubes for separation of plasma. Plasma was analysed for glucose, triglycerides, cholesterol, AST and ALT (unpublished data).
Administration of APFO resulted in decreases in the plasma concentrations of triglycerides (PPARα-mediated) and glucose (PPARγ); plasma cholesterol levels were also reduced at all time points (Table 1). No adverse clinical observations were noted even after one year of continuous dietary dosing, although at early time points (1-2 weeks) slight elevations in plasma aspartate and alanine aminotransferase (AST and ALT) levels were observed. At this dietary dose level (300 ppm), plasma concentrations of APFO were 157.00±77.80 μM at week 2 and 256.96±38.93 μM at week 4.
Interaction of CXR1002 with PPARs
Activation of PPARs is a transcriptional signature for PFOA in rats and mice, as well as common carp and zebrafish (Andersen (2008)). The effects of APFO and CXR1002 on the three PPAR isoforms in Cos-1 cells using a GAL4 binding assay and a transactivation assay using full length PPAR reporter gene constructs have been conducted using truncated PPAR constructs. The transactivation assay was performed in both agonist and antagonist mode (unpublished data). In antagonist mode for PPAR the finding from earlier assays suggesting reduced reporter expression, was confirmed by observation of direct antagonism activity for CXR1002. These findings are in keeping with those reported in independent studies by Vanden Heuvel et al., (2006) and Takacs & Abbott (2007), and are summarized together in Table 2.
The effects of CXR1002 are not limited to PPARs. The non-selective pan-activation of numerous nuclear receptors is apparent not only by the transcriptional activation of many genes in PPARα-null mice (Rosen (2008)), but also by the scope of metabolic and regenerative pathways elicited by CXR1002 exposure. In particular, constitutive androstane receptor (CAR) and pregnenolone X receptor (PXR) are activated (Ren (2009)), although this appears to be on a species-specific basis. Further studies are needed, particularly on the human genes, to determine the significance of this in humans. Neither liver X receptor β (LXRβ) nor the common heterodimerization partner retinoid X receptor α (RXRα) are activated by PFOA (14).
To investigate the anti-tumour effects of CXR1002 in a non-biased manner, transcription profiling analysis was performed using the human pancreatic carcinoma cell line PANC-1 cultured in vitro. Gene expression changes observed in the normal pancreas are different from those in the liver, and suggest possible effects on gluconeogenesis and glutamine metabolism (Anderson (2008)). PANC-1 cells were treated with CXR1002 for 24 hrs at a concentration that has been found to cause 15% inhibition of cell growth (IC15) and RNA was subsequently extracted. Analysis of the transcription profiles was made using pathways analysis in the Ingenuity system (unpublished data).
A list of 4996 genes was generated that showed changes in the treated samples compared to the untreated samples. Representation analysis of the in vitro 4996 signature list identified a number of pathways that were over-represented. In particular, genes in the endoplasmic reticulum (ER) stress pathway were over-represented in the signature list,
The endoplasmic reticulum (ER) serves two major functions in the cell. It facilitates the proper folding of newly synthesised proteins destined for secretion and it provides the cell with a calcium reservoir. ER stress occurs in various physiological and pathological conditions where the capacity of the ER to fold proteins becomes saturated. Examples of these situations include calcium flux, glucose starvation, hypoxia or defective protein secretion, modification or degradation.
Cells respond to the accumulation of unfolded proteins in the ER by a rescue process called the unfolded protein response (UPR). However, if the unfolded protein accumulation is persistent and the stress cannot be relieved, UPR signalling switches from prosurvival to proapoptotic (Kim (2006)), (Szegezdi (2006)), usually involving processing of caspases (Chang (2006)). Consistent with this hypothesis, CXR1002-treated PANC-1 cells show reduced proliferation and cleavage of the caspase substrate poly-ADP ribose polymerase (PARP) (
Disruption of the UPR is particularly significant in certain tissues or organs, particularly those dedicated to extracellular protein synthesis e.g. glandular tissues such as the pancreas and thyroid. The pancreatic β-cell is particularly dependent on efficient UPR signalling due to the constantly varying demands for insulin synthesis (Marciniak (2006)).
Chemical toxicants such as tunicamycin and thapsigargin cause an accumulation of unfolded protein aggregates in the ER lumen (Schroder (2008)), (Harding (2002)), (Zhang (2008)). Whilst it is fair to say that many chemicals, drugs and toxicants induce ER stress, not all do. Microarray data from a previous unpublished study requires further analysis, but superficially at least seems to indicate that the ER stress effect may be specific to the pancreatic cancer cell line PANC-1 and not a feature of normal pancreas tissue since the ER stress response is not seen in normal pancreas treated with APFO (28 day study in rat). The phthalate DEHP and the PPARα agonist WY14,643 were also studied. No evidence of ER stress response was detected with either of these compounds.
In studies of primary rat hepatocytes, PFOA concentrations of 30 μM and above caused increased expression of DNA damage-inducible transcript 3 (DDIT3/CHOP/GADD153), suggesting ER stress (Bjork (2009)).
ER stress can be caused by the induction of oxidative enzymes and the CXR1002 PANC-1 microarray signatures showed some mRNA level induction of enzymes involved in redox homeostasis. Altered genes included glutamate-cysteine ligase modifier subunit (GCLM), glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase (HO-1), glutathione reductase (GSR) and thioredoxin reductase (TRXR1) which are reflected by the over-representation of genes in the NRF2 signalling pathway. This is an indication that the PANG-1 cells are undergoing an oxidative stress response. The mechanism of this is unclear, however the induction of DNA damage response genes such as Growth arrest and DNA damage alpha (GADD45α), DDIT3, p21 and p53 suggest that oxidative stress may result in DNA damage. However, in follow-up experiments CXR1002 did not activate transcription of p21WAF1, when examined using β-human chorionic gonadotrophin (hCG) excretion from reporter cell line A2780/p21WAF1 exposed to CXR1002 for 24 hrs (unpublished data).
The above data demonstrate that CXR1002 activates both PPARα and PPARγ at similar concentrations, potentially conferring the benefits of both receptors, including growth inhibition, induction of apoptosis and induction of terminal differentiation. Furthermore, CXR1002 may inhibit PPARδ. Given that PPARδ is able to oppose the effects of PPARα and PPARγ (Vosper (2001)) via repression of transcription mediated by competition for DNA binding (Shi (2002)), there may be a benefit to PPAR α/γ agonist which is inhibitory or neutral at the PPARδ receptor. CXR1002 may have effects on other nuclear receptors, such as CAR and PXR.
Induction of ER stress in tumour cells is a mechanistically important mode of action for a variety of anti-cancer drugs including bortezomib (Velcade) (Healy (2009)). It has also been shown to occur in mechanistic studies of PPAR agonists, such as the dual agonist thiazolidinedione TZD18 (Zang (2009)) and PPARγ ligands such as prostaglandin J2 (Weber 2004)), (Chamber (2007)). A direct correlation between ER stress and PPAR effect remains to be determined for CXR1002.
Overloading the UPR to induce cell death is a possible anticancer strategy (Healy (2009)). Recently, the UPR has been linked to hepatic lipid metabolism (Lee (2009)), and the finding that the transcription factor XBP1, best known as a key regulator of the UPR, is required for de novo fatty acid synthesis in the liver suggests this gene or gene pathway to be a key link (Lee (2008)).
The Sulphorhodamine B (SRB) assay was used to determine the in vitro cytotoxicity of APFO (CXR1001) and CXR1002 towards a panel of human tumour-derived cell lines in a 48 hr assay. The SRB assay was performed according to the method specified by the NIC/NIH. The results for ten cell lines using the SRB assay are summarised in Table 4. The lowest IC50 values (˜160 μM) were seen with HepG2 cells and the highest (˜740 μM) were seen with CaCo-2 cells. In every case the cytotoxic effects of APFO and CXR1002 were similar. In subsequent experiments with CXR1002 an ATP cytotoxicity assay was used on a panel of 18 tumour cell lines. The effects of CXR1002 were assessed after 48 hr treatment. Assay replicates were independent in time and up to 4 replicates were performed per cell line. In this study, some cell lines were resistant to CXR1002, or produced dose response curves which did not allow for IC50 determination. A 48 hr assay may not produce optimal cytotoxicity; recent data shows that a 7 day endpoint gives lower cytotoxicity IC50 values (data not shown).
The mechanism of cytotoxicity of APFO and CXR1002 was evaluated using bromodeoxyuridine (BrdU) incorporation to quantify cell proliferation and Hoechst 3342 staining to identify apoptotic cells. Significant suppression of BrdU incorporation was observed in all but one of the cell lines used in the SRB cytotoxicity assay following treatment with 300 μM APFO or CXR1002 for 48 hrs; in five cell lines, no proliferating cells were detectable at this concentration. No marked effects were observed at 10 μM, whereas the response to 30 μM was variable. The concentration dependence of induction of apoptosis was similar, with marked induction of apoptosis at 300 μM, little effect at 10 μM and variable responses at 30 μM.
CXR1002 has been examined in a small number of xenograft models, using both intra-peritoneal (i.p) and oral dosing (p.o). The effect of PFOA on HT-29 (colon adenocarcinoma) tumours was assessed in nude mouse xenografts, initially using APFO and subsequently using CXR1002. Animals were inoculated with a tumour cell suspension on each flank and the tumours were allowed to grow for 16 days. CXR1002 was administered intra-peritoneally three times per week for 28 days; results were graphed using a curve-fitting programme (
A parallel experiment was carried out using the prostate tumour cell line PC3. Xenograft tumours derived from PC3 cells grew much more slowly than HT-29 xenografts; nevertheless, CXR1002 had a marked anti-tumour effect in this model. The effects of different doses of CXR1002 (5, 15 and 25 mg/kg given by the i.p route) were very similar in this experiment, but for simplification, only data from the 25 mg/kg group is shown (
In both the HT-29 and PC-3 xenograft experiments, slight reductions in plasma glucose and triglyceride levels were detected following CXR1002 treatment of tumour-bearing nude mice, consistent with activation of the PPARγ and PPARα receptors, respectively. Slight increases (up to 3.5 fold) in plasma AST occurred in response to CXR1002 in mice bearing either HT-29 or PC3 cell xenografts. Plasma ALT levels were only slightly increased in PC3-tumour bearing mice (up to 1.8 fold) and were actually decreased in mice bearing HT-29 xenografts. These effects are consistent with a transient effect on the liver associated with mild toxicity and reversible liver enlargement. In rodents, this type of effect is usually due to hepatic PPARα activation associated with peroxisome proliferation.
A further xenograft model was performed using the human pancreatic cell line PANC-1. This tumour is slow growing in vivo. Female nude mice were implanted with PANG-1 cells and once the tumours reached a pre-determined size the animals were dosed with CXR1002 at 25 mg/kg, 3 times per week. For various reasons, animals were lost during the study and the final group sizes were small. Nevertheless, the CXR1002 treated animals showed substantially delayed tumour growth and the weights and rigidity of the tumours were also different between the vehicle treated and untreated animals (
In-life and terminal blood samples taken from the mice were analysed for CXR1002 levels using a validated analytical method. In-life samples averaged 146 μM and terminal blood samples (24 hours post final dose) averaged 474 μM (
CXR1002 was also tested in a xenograft model of liver carcinoma using the cell line HepG2. In this experiment CXR1002 was dosed at 25 mg/kg in two different regimens: 2× per week and 3× per week. Although this tumour cell line is particularly sensitive to CXR1002 in vitro, the xenografted tumours showed a modest response in terms of growth inhibition. There was no obvious difference between the two different dosing regimens. The data in
To summarise, CXR1002 has been tested in four human tumour xenograft models, HT-29 (colon), PC3 (prostate), PANC-1 (pancreatic) and HepG2 (liver). Anti-tumour effects were detected in all models as shown in Table 6. No significant toxicity was observed, although there was evidence for minor changes in liver enzyme function, associated with a liver enlargement effect, which is probably rodent-specific. The exposure to CXR1002 in nude mice was lower than the blood levels achieved in patients at the higher doses in the CXR1002-001 phase I trial.
#Data from Table 5
PPARs play key roles in nutritional homeostasis, the primary effects of PPARα being in the regulation of fatty acid catabolism and those of PPARγ being in adipose differentiation and insulin-mediated regulation of glucose levels (2), (3). The hypolipidaemic effects of PPARα agonists are well characterised, while more recent studies have demonstrated the hypoglycaemic effects of PPARγ agonists (47), (48), (49), (50). While these effects may be peripheral to the anticancer effects of CXR1002, they are relevant as hypotriglyceridaemia and hypoglycaemia may be used as pharmacodynamic markers of PPAR α and γ agonism respectively.
CXR1002 monotherapy has been evaluated in a single Phase I trial in cancer patients with the primary objective of determining the maximum tolerated dose (MTD) of a weekly dosing schedule. A summary of this trial is provided in Table 7.
The bulk active pharmaceutical ingredient will be manufactured under GMP conditions by Chimete Srl, Italy; and the capsules manufactured to cGMP by Penn Pharmaceutical Services LTD, UK.
Storage: All trial medication was held in a dry place at room temperature (15° C. to 25° C.) and protected from light.
The starting dose of CXR1002 was 50 mg administered orally as a single dose. This is approximately 0.24× the Lowest Observed Effect dose level in the monkey which is the most sensitive species that was tested.
CXR1002 was administered to patients, as a capsule by the oral route, orally as a single dose of 50 mg in the morning after an overnight fast in the first cohort of 3 patients. Prophylactic anti-emetics were not administered, and patients fasted for 1 hour after ingestion of CXR1002. PK samples, PD (fasting) samples, blood glucose, and blood triglyceride samples, were taken over a 6-week period.
These patients then underwent repeat dosing schedule with the same dose of CXR1002. The repeat dosing schedule was weekly administration of CXR1002 as a single oral dose in the morning and patients fasted for 1 hour before and after ingestion of CXR1002. Dose limiting toxicity (DLT) will be based on the toxicity assessments over the first 3-week period of the repeat dosing schedule. PK samples (single blood sample) were taken on the following basis:
In all dose cohorts subsequent to the initial dose cohort, all patients will be treated with weekly administration of study drug from the start of dosing. Dose escalation was performed after all patients at the preceding dose level had completed a 3-week repeat dosing period. The dose of CXR1002 was increased in successive dose cohorts until ≧Grade 2 drug-related toxicity was observed, after which dose escalation was in approximately 30% increments.
As of February, 2011, 43 patients with advanced cancers from one Phase I study have received CXR1002. The weekly dose administered ranges from 50 to 1200 mg.
The best response to CXR1002 treatment was stable disease by investigator assessment. One patient with pancreatic cancer had stable disease lasting 7 months.
Pharmacokinetic analysis of CXR1002 was carried out in the Phase I study using a validated assay. After oral administration of a single dose of CXR1002, the plasma concentration reached a Cmax at 1.5 hours in all 3 patients examined. After a single 50 mg dose the exposure in 3 patients varied between 8 and 16 μM and this was maintained at a constant level over the 6 week sampling period following the dose. The data indicates the half-life of elimination of CXR1002 could not be defined but is >6 weeks.
After weekly repeat doses of CXR1002 the plasma level increased in stepped increments. The maximal plasma level recorded to date was from a patient who had received a 1200 mg weekly dose over a 5 week period and had a plasma level of 1530 μM.
There appeared to be no gender difference in CXR1002 exposure following CXR1002 administration. The drug is eliminated extremely slowly and accumulates following a weekly dose.
Study CXR1002-001 is an open label, two centre, phase I study in patients with advanced cancer to assess the tolerability, safety and pharmacokinetics of CXR1002 administered weekly. The study synopsis is shown in Table 8.
Forty three patients were enrolled in the study, as of February 2011. Thirty two patients were enrolled at the Beatson West of Scotland Cancer Centre, Glasgow, and eleven patients were enrolled at Aberdeen Royal Infirmary.
CXR1002 is being given orally as a weekly dose. The starting dose was a 50 mg single dose. The starting weekly repeat dose was 50 mg, with 2 patients continuing to the repeat dose schedule after receiving a single dose. Doses were escalated in groups of three patients. The dose escalation is continuing. A summary of the dose escalation is provided in Table 9.
A validated analytical assay consisting of non-GLP LC-MS/MS was used to quantitate CXR1002 in human plasma. Plasma samples were collected after the single 50 mg dose at the following timepoints: Pre-dose, and then 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 24, 48, and 72 hours after administration and then once weekly at weeks 2, 3, 4, 5, and 6 (days 8, 15, 22, 29, and 36). For patients treated with the weekly repeat dose, plasma samples were collected at the following timepoints: Pre-dose and then 2, 3, 4 and 24 hours after administration for a total of 6 weeks. Thereafter a single sample was collected every 6 weeks for monitoring of exposure during long term treatment. Plasma samples were processed at site and stored at −80° C. prior to batch shipment to the analytical laboratory.
Of the 43 patients enrolled in study CXR1002-001, 24 were males and 19 were females. The majority of patients had received 2 prior therapies. Two patients had received 5 prior therapies. The tumour types of the patients are shown in Table 10.
Pharmacodynamic samples were also collected from patients for the measurement of pharmacodynamic markers. Samples were collected using the same time schedule as that used for the pharmacokinetic samples.
The following data shows for each patient the plasma levels over time. The particular weekly dose is shown, as is the gender and age of each patient. Graphical plots of the data for each patient are shown in
The half life of CXR1002 is extremely long and could not be defined during the period of evaluation (6 weeks) (
The best response to CXR1002 treatment was stable disease lasting 7 months. Four patients had stable disease ≧4 months (range 20 to 35 weeks) (Table 12). Of these, 1 patient diagnosed with pancreatic cancer had radiographic evidence of tumour shrinkage which did not meet the criteria of partial response.
The aim of this study was to combine CXR1002 with other agents to ascertain whether an enhanced response to the combination of drugs was observed.
The results as presented are from a single assay in which the cell lines listed in Table 13 below were exposed to CXR1002 or the test items listed in Table 14 or the test items in combination with CXR1002.
The cell lines were purchased from The American Type Culture Collection (ATCC) via LGC Promochem (London, UK), the European Collection of Cell Cultures (ECACC) via Sigma-Aldrich, UK, or the Health Science Research Resources Bank of the Japan Health Science Foundation (JHSF): (Refer to Table 13). Cell line H was supplied by the Biomedical Research Centre, Ninewells Hospital, Dundee.
Test compounds were dissolved in DMSO to make stock solutions of an appropriate concentration. The stock solutions were further diluted in DMSO to produce additional stock solutions as necessary. The amount of DMSO added to the medium was 1% of the final volume.
Cells were plated at the optimal plating density for that cell line in 96-well plates and allowed to attach overnight. The next day, the medium was removed and replaced with fresh medium containing the dose ranges of test items. The cells were exposed to 5-FU, cisplatin, docetaxel, doxorubicin, geldanamycin, gemcitabine, rapamycin or roscovitine in Roswell Park Memorial Institute (RPMI) medium containing 10% Foetal Calf Serum (FCS) and 2 mM Glutamine at 37° C. and 5% CO2 for 48 hours. The concentrations of CXR1002 or other agents to which the cells were exposed were as previously determined or as suggested by relevant literature (see Table 14 below). There were 3 replicates for each test item concentration.
From the results of the single compound assays, appropriate doses ranges were determined for use in the combinatorial assays with CXR 1002. These are shown in Table 16:
Following exposure to Test Items, the CellTitre-Glo Luminescent Cell Viability Assay to measure ATP content was performed according to the manufacturer's detailed instructions (Promega Corporation, Technical Bulletin No. 288, and Cell notes, Issue 10, 2004).
The results of the ATP depletion assay were corrected for background luminescence and expressed as a percentage of the vehicle control value using Microsoft Excel software. Point-to-point spline analysis was performed and the results graphed in GraphPad Prism as cell viability (percentage of vehicle control) versus Test Item concentration.
Doxorubicin, gemcitabine, geldanamycin and roscovitine were shown to increase the sensitivity of a number of the cell lines. Rapamycin increased sensitivity in the four ovarian lines tested and in HepG2 cells. Interestingly from the clinical perspective, when MDA-MB-157 (breast) cells were treated with the combination of 5-FU, a drug used in the treatment of breast cancer, and CXR1002, there was an apparent increase in sensitivity.
The objective of this study is to combine CXR1002 with known anti-cancer agents, both investigational and marketed drugs, in an effort to achieve enhanced tumour cell killing ie. to potentiate mode of action.
In a 48 hour cytotoxicity assay the following compounds were tested at fixed concentrations, derived from a review of the literature, in the presence of CXR1002 (0-1 mM):
1. MAP kinase inhibitor (MEK1/2) (compound name, UO126)
2. AKT/PI3K inhibitor (compound name, LY294002)
3. PARP inhibitor (compound name, DPQ)
The cell lines were purchased from The American Type Culture Collection (ATCC) via LGC Promochem (London, UK), the European Collection of Cell Cultures (ECACC) via Sigma-Aldrich, UK, or the Health Science Research Resources Bank of the Japan Health Science Foundation (JHSF): (Refer to Table 19). Sarcoma cell line H was supplied by the Biomedical Research Centre, Ninewells Hospital, Dundee.
Test compounds were dissolved in DMSO to make stock solutions of an appropriate concentration. The stock solutions were further diluted in DMSO to produce additional stock solutions as necessary. The concentrations of the original stock solutions and the additional stock solutions will be recorded in the appropriate CXR Study folder and in the Study Report. The final amount of DMSO added to the medium was 1% of the final volume.
Cells were plated at the optimal plating density for that cell line in 96-well plates and allowed to attach overnight. The next day, cells were pre-treated with the inhibitors UO126 or LY294002 (see Tables 18 & 20) for 2 hrs, the medium was removed and replaced with fresh medium containing the appropriate dose of the test item. After 2 hrs, CXR1002 (concentration range 0-1 mM) together with the appropriate inhibitor was then added. Cells that were to be treated with DPQ received no pre-treatment. Cells were exposed to these compounds in Roswell Park Memorial Institute (RPMI) medium containing 10% Foetal Calf Serum (FCS) and 2 mM Glutamine at 37° C. and 5% CO2 for 48 hours. There were 3 replicates for each test item concentration.
Following exposure to Test Items, the CellTitre-Glo Luminescent Cell Viability Assay to measure ATP content will be performed according to the manufacturer's detailed instructions (Promega Corporation, Technical Bulletin No. 288, and Cell notes, Issue 10, 2004).
The results of the ATP depletion assay were corrected for background luminescence and expressed as a percentage of the vehicle control value using Microsoft Excel software. The results were graphed as ATP content (percentage of appropriate control) versus Test Item concentration (CXR1002).
Use of the CXR1002/UO126 combination compared to CXR1002 alone revealed increased sensitivity in the following cell lines:
It is clear from this data that concomitant inhibition of MEK1/2 may enhance the efficacy of CXR1002 and that the effect may be selective to certain cell lines and therefore tumour types.
LY294002 is a potent inhibitor of phosphoinositide 3-kinases. When used in conjunction with CXR1002 increased efficacy was noted in a select number of cell lines most notably the sarcoma cell line H.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair, replication and cell cycle. However, its overactivation leads to nicotinamide adenine dinucleotide and ATP depletion and cell death. Use of the PARP inhibitor, DPQ, in conjunction with CXR1002 led to increased sensitivity in the following cell lines: HPAFII and Capan2 (pancreas) and SW1353 (sarcoma). Again, the synergistic response with the two drugs was selective.
It is clear from this preliminary data that certain combinations of CXR1002 and the inhibitors UO126 or LY294002 or DPQ result in an increased level of cytotoxicity when compared to the use of CXR1002 alone. The exact mechanisms underpinning these observations remain to be elucidated. However, with regard to kinase pathways, the pathways (MAPK/PI3K/Akt) are known to form the core intracellular signalling routers in the stimulation of growth factors. Their expression, particularly that of PI3K/Akt, or that of their phosphorylated (activated) forms has been reported as a significant prognosis marker in sarcoma (Tomita, Y (2006)), gastric cancer (Cinti, C (2008)), pancreatic cancer (Chada, K S (2006)) and breast cancer (Park, S S (2007)). Therefore, inactivation of the PI3K/Akt or MAPK pathways should be effective as a specific chemotherapy against malignant tumours because of lower expression of activated forms in the surrounding tissues. In addition, these pathways play an essential role as survival signal pathways when cancer cells are exposed to a cellular stress. CXR1002 may cause cellular stress e.g. oxidative stress and therefore may activate certain stress-related responses. Therefore, use of inhibitors of these pathways might be expected to enhance the degree of cytotoxicity in cancer cell lines exposed to CXR 1002 and perhaps even in vivo.
Poly(ADP-ribose)polymerase (PARP) or poly(ADP-ribose)synthase (PARS) has an essential role in facilitating DNA repair, controlling RNA transcription, mediating cell death, and regulating immune response. In various cancer models, PARP inhibitors have been shown to potentiate radiation and chemotherapy by increasing apoptosis of cancer cells, limiting tumour growth, decreasing metastasis, and prolonging the survival of tumour-bearing animals. Again, it appears that use of PARP inhibitors in conjunction with CXR1002 potentiates cytotoxicity.
Investigation into the ER stress effects of CXR1007 were conducted by looking at whether CXR1002 induces expression of ER stress-regulated proteins and then splicing of XBPI MRNA upon CXR1002 induced ER stress.
Panc-1 (pancreatic tumour) cells were treated with vehicle control (lane 1), with 500 μM of CXR1002 for 4 h (lane 2), with 500 μM of CXR1002 for 1 day (lane 3), with 500 μM of CXR1002 for 2 days (lane 4), with 500 μM of CXR1002 for 3 days and with 500 μM of CXR1002 for 4 days.
Western blots were performed on the protein extracts (equivalent protein concentrations were loaded onto the gels). The antibodies used were for known ER stress regulated protein Bip/GRP78, CHOP/DDIT3, IRE1alpha, TRB3 (Tribbles3), cleaved PARP (marker of apoptosis), and tubulin (loading control) (see
This showed that CXR1002 altered expression of ER stress-regulated proteins.
Splicing of XBP1 mRNA as an Indicator of ER Stress Inclusion
Panel (A) of
Panel (B) of
Tunicamycin, 10 mg/mL. This is a control compound known to induce ER stress and XBP-1 splicing.
The RT-PCR analysis shows that XBP-1 splicing varies from predominately unspliced to spliced after treatment with CXR1002. XBP-1 is known to be spliced when ER stress is induced.
PIM kinase activity after CXR1002 exposure. PIM kinase inhibition has been investigated for each of PIM-1, PIM-2 and PIM-3 kinase molecules.
The PIM-1 assay is performed using the Upstate IC50 Profiler Express™ service. In a final reaction volume of 25 μl, human recombinant PIM-1 (5-10mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 100 μM KKRNRTLTV, 10 mM MgAcetate and [γ-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μl of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
The PIM2 assay is performed using the Upstate IC50 Profiler Express™ service. In a final reaction volume of 25 μl, human recombinant PIM-2 (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 300 μM RSRHSSYPAGT, 10 mM MgAcetate and [?-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μl of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in menthanol prior to drying and scintillation counting.
The PIM-3 assay is performed using the Upstate IC50 Profiler Express™ service. In a final reaction volume of 25 μl, human recombinant PIM-3 (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 300 μM RSRHSSYPAGT, 10 mM MgAcetate and [?-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μl of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in menthanol prior to drying and scintillation counting.
In each of the three PIM kinase assays, CXR 1002 shows inhibition of the kinase molecules.
CXR1002 pharmacokinetics. PK sampling (repeat dose) in CXR1002 clinical trial
The methodology of this clinical trial is detailed in Example 5.
Plasma was collected at regular intervals (2, 3, 4 and 24 hrs post dose) every week from fasted patients (fasted minimum of 1 hour pre and post dose) administered weekly doses of CXR1002
(*pt. 031 was not dosed)
Urine was collected over a 24 hour duration post each weekly dose and CXR1002 levels were measured in the total sample.
As shown in
For detailed methodology, see Example 5.
Patients 004-036 were included in the analysis (*patients 34, 35 & 38 were not available for analysis. CXR1002 was administered (at dose increments; n=3-6) daily for a 6-week period. Plasma was collected and analysed for PK & PD effects. Data was initially grouped by dose and then re-grouped by PK (peak plasma exposure on wk 6). PD data: Plasma samples=baseline vs. wk 6 (peak plasma). Comparable graphs were plotted whether grouped by dose or drug exposure.
Individual patient raw data was captured and represented in graphs as % change from baseline (screening). Individual patient data (% baseline) was plotted in Prism and data was grouped according to PEAK plasma [CXR1002] at wk 6. Data represents either mean±SEM values or median, range+individual data points.
The data suggests an effect of CXR1002 on LDL (i.e. lowering effect) but not HDL (i.e. CXR1002 lowers ‘bad’ cholesterol but ‘good’ cholesterol remains unchanged). This effect is entirely predicted from the animal data and suggests a possible use in patients with conditions such as high cholesterol and hyperlipidemia.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1002861.1 | Feb 2010 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2011/000232 | 2/18/2011 | WO | 00 | 10/17/2012 |
