WO1997035569A1 - Use of heme oxygenase inhibitors to treat cancer - Google Patents

Use of heme oxygenase inhibitors to treat cancer Download PDF

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Publication number
WO1997035569A1
WO1997035569A1 PCT/GB1997/000844 GB9700844W WO9735569A1 WO 1997035569 A1 WO1997035569 A1 WO 1997035569A1 GB 9700844 W GB9700844 W GB 9700844W WO 9735569 A1 WO9735569 A1 WO 9735569A1
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tumour
heme oxygenase
agent
inhibitor
nitric oxide
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PCT/GB1997/000844
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French (fr)
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Adrian Richard Moore
Dean Willis
Derek Albert Willoughby
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William Harvey Research Limited
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Priority to AU21689/97A priority Critical patent/AU2168997A/en
Publication of WO1997035569A1 publication Critical patent/WO1997035569A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/223—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of alpha-aminoacids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/295—Iron group metal compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol

Definitions

  • This invention relates to the treatment of cancers and other tumours, and to pharmaceutical compositions and therapies for such treatment.
  • Tumours are usually recognizable as a mass of abnormal cells. If these grow in one location and do not spread to other tissues or areas they are usually classified as benign.
  • An in-situ tumour is one that develops in the epithelium, and contains typically small cells showing abnormalities, but which does not invade other tissues. Cancers are fully developed, malignant tumours that invade and destroy adjacent tissues.
  • cancer is one of the commonest causes of death in western populations and in certain groups of individuals is indeed the commonest. Even non- fatal cancers often have drastic effects on sufferers; as an example, mastectomy is frequently required to prevent spread of cancer from a diseased breast.
  • Heme ferrulferri-protoporphyrin-IX plays a vital role in cellular metabolism, functioning as the prosthetic group of hemeproteins (eg. hemoglobin and cytochromes). Its catabolism is a two-step process. The first, and rate limiting reaction, is the production of biliverdin and carbon monoxide by the microsomal enzyme heme- oxygenase. The second step is the production of bilirubi ⁇ from biiiverdin by the cytosolic enzyme, biliverdin reductase.
  • the first, and rate limiting reaction is the production of biliverdin and carbon monoxide by the microsomal enzyme heme- oxygenase.
  • the second step is the production of bilirubi ⁇ from biiiverdin by the cytosolic enzyme, biliverdin reductase.
  • Heme-oxygenase is found in liver, kidney, spleen and skin, and has also been localised to specific cell types, notably fibroblasts and macrophages.
  • the enzyme exists in at least two isoforms, one constitutive and the other inducible.
  • Heme, heavy metal ions eg. tin, gold, platinum and mercury
  • transition metal ions eg. iron, cobalt, chromium and nickel
  • heme- oxygenase is induced as part of a generalised stress response to stimuli such as thermal shock (hence the alternative name heat-shock protein 32: hsp32), oxidative stress and cytokines such as interleukin-1 (IL— 1), tumour necrosis factor and 1L-6.
  • stimuli such as thermal shock (hence the alternative name heat-shock protein 32: hsp32), oxidative stress and cytokines such as interleukin-1 (IL— 1), tumour necrosis factor and 1L-6.
  • Known compounds that inhibit heme-oxygenase are structural analogues of ferriprotoporphyrin (FePP), which itself induces the enzyme, and some examples include tin protoporphyrin (SnPP) and tin mesoporphyrin (SnMP).
  • FePP ferriprotoporphyrin
  • SnPP tin protoporphyrin
  • SnMP tin mesoporphyrin
  • U.S. Patent No. 4657902 relates to the use of tin mesoporphyrin and compositions containing it to inhibit heme metabolism in mammals to control the rate of tryptophan metabolism in mammals, and to increase the rate at which heme is excreted by mammals.
  • U.S. Patent No. 5010073 relates to liposomal metalloporphyrin preparations for targeting the spleen for inhibition of heme-oxygenase activity in the spleen.
  • the present invention is based on the observation of reduction in tumour growth and tumour dry mass following administration of a heme oxygenase inhibitor.
  • a first aspect of the invention provides the use of an inhibitor of heme oxygenase in the manufacture of a medicament for the treatment of a tumour.
  • an inhibitor of heme oxygenase it is hereby intended to include compounds that directly inhibit heme oxygenase and those whose inhibitory action is indirect but nevertheless induce a reduction in the amount of heme oxygenase activity - the reduction can be due to a reduced amount of heme oxygenase, a reduced amount of active heme oxygenase or to heme oxygenase of reduced efficiency.
  • the inhibitor inhibits an inducible form of HO-1.
  • Agents suitable for inhibiting heme-oxygenase are typically structural analogues of FePP (nb. which induces heme-oxygenase) in which the Fe ion is replaced by another metal ion, or the PP is replaced.
  • FePP structural analogues of FePP (nb. which induces heme-oxygenase) in which the Fe ion is replaced by another metal ion, or the PP is replaced.
  • Suitable dosage amounts of these agents range from 0.1 to 50 ⁇ moles/kg of body weight of, for example, a human.
  • suitable dosage amounts of SnPP would be from 0.07mg/kg to 46mg/kg of body weight of a human.
  • Route of administration of a heme oxygenase inhibitor according to the invention is optionally via any conventional pharmaceutical route, these including but being not limited to oral, rectal, subcutaneous, parenteral, intravenous, intraperitoneal and topical. It is further explained below how certain specific formulations also represent further aspects of the invention; though, generally, the preparation of pharmaceutical compositions comprising a heme oxygenase inhibitor for oral use is described in US patents numbers 4657902 and 5010073, and to which the skilled person is referred for further guidance on the formulation and preparation of such compositions.
  • the FePP analogue tinprotoporphyrin (SnPP) was used to treat a tumour of colon-26 cells. SnPP was administered subcutaneously and over a period of 7 days, at the end of which period significant reduction in tumour was observed.
  • Antagonists of prostaglandin A receptors are further agents suitable for decreasing heme-oxygenase (HO) activity.
  • agents for reducing HO activity are agents that increase nitric oxide (NO) levels in a patient.
  • Such agents are, optionally, a substrate for nitric oxide synthase (NOS) or a stimulator of this enzyme or a NO donor.
  • a known substrate for NOS is L-arginine and a known NO donor is sodium nitroprusside.
  • Nitric oxide (NO) formed from L-arginine and molecular oxygen by isoforms of the enzyme nitric oxide synthase (NOS EC 1.14.13.39), is involved in a variety of physiological and pathophysiological processes. The reactivity of this molecule, and its capacity to complex with metalloproteins, underlies many of its biological actions.
  • the medicament is for treatment of cancer.
  • a heme oxygenase inhibitor is for use in combination with a cytotoxic agent.
  • the inhibitor is for use at such concentrations that both inhibit heme oxygenase and are cytotoxic.
  • the inhibitor is for use with a separate agent that is cytotoxic.
  • the inhibitor is for use in combination with a cytotoxic therapy such as radiotherapy.
  • the heme oxygenase inhibitor is for use in combination with a cytotoxic agent and elevated levels of nitric oxide in the patient, optionally achieved using a nitric oxide donor or a substrate for NOS.
  • nitric oxide is used as an adjunct to tumour treatment by a heme oxygenase inhibitor in combination with a cytotoxic agent.
  • the invention thus provides the use of known pharmaceuticals in the manufacture of a medicament for the treatment of tumours, such as cancerous tumours.
  • tumours such as cancerous tumours.
  • the invention offers an alternative therapy for tumours, which therapy has been neither described nor suggested in any prior art.
  • the inventors have tested the treatment of tumours according to the invention and have observed useful anti-tumour results.
  • a pharmaceutical composition comprising a compound that inhibits heme oxygenase and an anti-tumour pharmaceutical that is not an inhibitor of heme oxygenase.
  • the pharmaceutical composition of the second aspect is thus a combination typically of a heme oxygenase inhibitor with a known anti-tumour agent.
  • the pharmaceutical compositions of the invention can be prepared using any suitable pharmaceutically acceptable carrier.
  • the inhibitor is tin protoporphyrin and this is present in a pharmaceutical in an amount of between 0.1 and 50 ⁇ mols/kg.
  • a suitable anti-tumour agent is one that inhibits cell division, for example an anti- mitotic agent.
  • a pharmaceutical composition comprises an inhibitor of heme oxygenase with an anti-cancer drug selected from alkylating agents, antimetalbolites, alkaloids, cytotoxic antibodies, nitrosoureas and synthetic anti-neoplastic drugs selected from amacrine, carboplatin, cisplatin, crisantaspase, dacarbazine, hydroxyurea, paclitaxei, pentostatin, procarbazine, mitotane, dibromomannitol and razoxane.
  • an anti-cancer drug selected from alkylating agents, antimetalbolites, alkaloids, cytotoxic antibodies, nitrosoureas and synthetic anti-neoplastic drugs selected from amacrine, carboplatin, cisplatin, crisantaspase, dacarbazine, hydroxyurea, paclitaxei,
  • a patient with a tumour is administered a combination of two pharmaceutically active agents; the first being an inhibitor of heme oxygenase and the second a cytotoxic drug.
  • An anti-tumour effect is obtained through the combined actions of the two active ingredients.
  • Another suitable anti-tumour agent for combination with a heme oxygenase inhibitor, is a latent agent which is activated by radiation either to become a toxic agent or to release a toxic agent.
  • a latent agent which when activated by ultra violet radiation produces free radicals, the free radicals being damaging to the tumour.
  • the use of such a latent agent can be of particular use as the radiation is targetted to the tumour area, thus producing activation of the latent agent in areas where only healthy tissue is present.
  • a problem with known anti-tumour agents is that of tachyphylaxis, that is to say the observation that after a period of administration of such an agent it is observed that the anti-tumour agent loses part or all of its effectiveness and must be administered in ever increasing doses.
  • the invention provides a pharmaceutical composition that combines an inhibitor of heme oxygenase with a known anti-tumour agent and offers the possibility of administering a reduced amount of the known agent.
  • a common visible side effect of chemotherapy for the treatment of cancers is both hair loss and weight loss. The opportunity to use reduced amounts of such anti-cancer agents opens the possibilities for amelioration or reduction in these known side effects.
  • the dose of an anti-cancer agent is often limited to the maximum dose the patient can withstand without risk to life.
  • the invention also offers the possibility that a similar dose of anti-cancer agent, used in combination with a heme oxygenase inhibitor, may give an enhanced anti-cancer effect without increasing the risk to the patient's life.
  • the invention provides a topical pharmaceutical composition
  • a topical pharmaceutical composition comprising an inhibitor of heme oxygenase and a topical, pharmaceutically acceptable carrier.
  • a suitable topical carrier can comprise an oil, a wax, an emulsion of an oil, an emulsion of a wax, a gel or a cream.
  • this topical composition may be applied directly to a tumour, such as one found on the skin of a patient.
  • the pharmaceutical composition is a suppository.
  • a pharmaceutical composition comprises a compound that inhibits heme oxygenase in a solution for infusion into a patient.
  • This composition can be administered to a patient with cancer by intravenous drip; this route is convenient for a sleeping or anaesthetized patient, or one not physically capable of taking medicaments orally.
  • a fifth aspect of the invention provides a composition that inhibits heme oxygenase and has formula: -
  • X represents an analogue of FePP and A represents a moiety capable of releasing nitric oxide or of inducing release of nitric oxide.
  • A is optionally at least one arginine molecule; this composition is thus the arginate form of a structured analogue of FePP.
  • the composition is tin protoporphyrin arginate or zinc protoporphyrin arginate.
  • Nitric oxide can be cytoxic at high levels, and this composition thus combines inhibition of heme oxygenase with formation of cytotoxic nitric oxide.
  • A is further optionally a nitric oxide donor, a substrate for NOS or a moiety capable of releasing NO, such as a -N0 2 group.
  • the invention provides a pharmaceutical composition comprising a composition according to the fifth aspect and a pharmaceutically acceptable carrier.
  • a method of treatment of a tumour in a patient comprising administering to that patient a compound that inhibits heme oxygenase.
  • a structural analogue of FePP which inhibits heme oxygenase is administered in an amount of from 0.1 to 50 ⁇ r ⁇ ols per kilogram of body weight of the patient per day.
  • a structural analogue of FePP, such as SnPP is administered orally to a patient is an amount sufficient to exert a therapeutic effect.
  • the amount administered can be in the range of 0.1 -50 ⁇ mols/kg body weight of the patient. More specifically, the amount can range from 1.0-30 ⁇ mols/kg body weight. In a specific embodiment of the invention described in an example hereafter, the dosing rate is about 14 ⁇ mois/kg.
  • the method further comprises administering at least two pharmaceutically active agents, one being an inhibitor of heme oxygenase and one being an anti-tumour agent that is not an inhibitor of heme oxygenase.
  • the two agents are administered simultaneously or separately.
  • the timing of administration is such that a cytotoxic Ievel of the anti-tumour agent is achieved whilst heme oxygenase activity is depressed by the heme oxygenase inhibitor.
  • a HO inhibitor is administered in combination with an anti-tumour agent selected from alkylating agents, antimetabolites, alkaloids, cytotoxic antibodies, nitrosoureas, and synthetic anti-neoplastic drugs selected from amacrine, carboplatin, cisplatin, crisantaspose, decarbazine, hydroxyurea, paclitaxel, pentostatin, procarbazine, dibromomannitol and razoxane.
  • an anti-tumour agent selected from alkylating agents, antimetabolites, alkaloids, cytotoxic antibodies, nitrosoureas, and synthetic anti-neoplastic drugs selected from amacrine, carboplatin, cisplatin, crisantaspose, decarbazine, hydroxyurea, paclitaxel, pentostatin, procarbazine, dibromomannitol and razoxane.
  • the method further comprises providing an elevated Ievel of nitric oxide in the patient, such as by administration of a nitric oxide donor or a substrate for NOS.
  • an elevated Ievel of nitric oxide in the patient such as by administration of a nitric oxide donor or a substrate for NOS.
  • the brains and spleens of male Wistar rats (160 ⁇ 20g, Tuck Co. UK) were homogenised using a glass homogenizer, in protease inhibitory buffer; phenylmethylsulfonyl fluoride 1 mM, pepstatin A 1.5mM and leupeptin 0.2mM, in 10mM phosphate buffered saline pH 7.3. Protein determinations were carried by the Bradford method, bovine serum albumin was used as protein standard.
  • Nitric oxide synthase activity was determined by the citrulline assay and the data calculated as pmol citrulline/mg protein/30min (Vane, J.R. et al (1993) Proc. Natl. Acad. Sci. USA 91 , 2046-2050).
  • Heme-oxygenase activity was assayed as previously described (Sierra, E.E. et al, (1992) Analytical Biochem. 200, 27-30).
  • the 15 ⁇ l reaction mixture consisted of 11.2 ⁇ M [ C] heme (specific activity 54 Ci/mol), 1 mM NADPH, 2mM glucose-6-phosphate, 0.1 units of glucose-6-phosphate dehydrogenase, 3mg/ml liver cytosolic protein, 100-50 ⁇ g of sample protein and the relevant concentration of test drugs; L-arginine, D-arginine, NG-nitro-L-arginine methyl ester (L-NAME) or sodium nitroprusside.
  • the reaction mixture containing the test drugs was allowed to equilibrate at 37°C for 15 mins before the reaction was started by the addition of the heme.
  • the reaction was terminated after 30 mins by the addition of excess cold heme and bilirubin and placed on ice.
  • the reaction mixture was spotted on to the silica gel thin-layer chromatography sheet by 2, 2 ⁇ l applications. All samples were run in duplicate.
  • the chromatogram was developed using a 20:1 dilution of chloroform: acetic acid. Spots corresponding to heme and bilirubin were excised and placed in 10ml of scintillation fluid to be counted. The data was calculated as pmois bilirubin formed/mg protein/hour.
  • Statistical analysis of the raw data was carried using Student's unpaired t test. Results expressed as mean ⁇ s.e.mean with P ⁇ 0.05 considered as significant.
  • Homogenates of rat brain contained both HO and NOS activity as determined by our assay systems.
  • rat spleen homogenates had double the HO activity, but lacked NOS activity.
  • Addition of the NOS inhibitor, L-NAME resulted in a dose dependent increase in brain HO activity, with 5mM L-NAME significantly increasing activity by 80%.
  • addition of L-arginine, the NOS substrate, to brain homogenates resulted in a dose dependent decrease in HO activity.
  • the highest concentration of L-arginine used, 10mM reduced HO activity by 75%.
  • the enantiomer of L-arginine, D-arginine which cannot be utilised as a substrate by NOS, had no significant effect on brain HO activity.
  • Spleen HO activity unlike brain activity was not modified by the addition of L-NAME, L-arginine or D-arginine.
  • the addition of the NO donor sodium nitroprusside resulted in a dose dependent decrease in HO activity in both spleen and brain homogenates.
  • the addition of 10mM sodium nitroprusside resulted in a 75% and 80% decrease in the HO activity of brain and spleen homogenates respectively.
  • Coion-26 cells were cultured in DMEM supplemented with 10% foetal calf serum, 1000U/ml penicillin, 1000U/ml streptomycin and 100 ⁇ g/ml gentamycin. These cells have been shown to produce mRNA for HO-1 by Northern blot and to express HO-1 protein by Western blot and immunocytochemistry.
  • Mouse spleen RNA was isolated using the Clontech RNA extraction kit.
  • First strand cDNA synthesis was performed using the Ready-to-Go kit from Pharmacia and PCR carried out using the following parameters: 1x3' at 94.0°C, (45" at 95.0°C, 45" at 59.1 °C, 1 ' at 72.0°C)x30, 1x10' at 72.0°C.
  • Forward primer 5' gCCTgA ATCgAggAgAACCA3'
  • reverse primer 5' CTTTTggTgAgggAACTgTgTCA3'
  • expected size of the product was 1 kb.
  • the fragment was gel-isolated (using the Geneclene BIO 01 system, Anachem), and ligated into PCR 2 cloning vector from Clontech. Screening was carried out in E. coli. XLI-blue (Stratagene). The fragment was checked for orientation using Hind III and the antisense construct was digested with Hind III and Spe I for ligation into the expression vector pRC/RSV (Clontech) which had been digested with the same enzymes. 400ml cultures were processed using the Quiagen maxi-prep kit, providing sufficient DNA for the transfection experiment.
  • Transfection of the colon26 cells was carried out for the antisense construct and the vector alone. Transfection was carried out using Lipofectin (Life Technologies). 5ug of plasmid were used per transfection. Transfection was carried out in a T75 cell culture flask, and the cell were left overnight with the Lipofecti ⁇ -plasmid mix in serum- free medium. The medium was replaced with 10% FCS-DMEM the following day. 24hrs later, the cells were removed from the flask, and re-seeded in a 96-well plate in 10% FCS-DMEM containing 1 mg/ml G418 (Geneticin). Medium was changed once a week.
  • the screening procedure involved observation for medium colour-change (indicating cell proliferation), and occurred from about one week following transfection.
  • the cells were transferred to a 6-well plate, and allowed to proliferate until enough cells were available for characterisation and freezing down. All medium contained G418. Diminished HO-1 expression was confirmed by isolating protein and RNA for Western blot and Northern blot respectively.
  • the protein isolation was carried out using the method routinely used in the Department and Westerns were carried out using the Amersham ECL-kit, and the antibody used was SP-895 from stressgen.
  • RNA extraction was carried out using the Trizol method (Life Technologies), and RNA was transferred to a nylon support following electrophoresis in a denaturing formaldehyde gel. Northern hybridisation was performed using the Amersham Rapidhybe solution and protocol provided with it for use with radionuclides. The HO-1 cDNA fragment was used as a probe.
  • Sterile polyether polyurethane sponge discs (8mm x 4mm, 5mg) were implanted subcutaneously in the dorsum of anaesthetized BALB/c mice (20-23g). Three days later the sponges were seeded by injection of 10 coion-26 cells in a volume of 50 ⁇ l sterile saline. Ten days after seeding, animals were killed in line with Home Office guidelines for the use of experimental animals.
  • tumours were excised at 10 days. In comparison to tumours carrying the control vector, there was a significant (p ⁇ 0.05) rise in tumour dry mass when the tumour cells carried antisense HO-1. These tumours were also of greater mass than control colon26 tumours but this difference did not reach statistical significance.
  • Table 1 The effects of control vector and antisense HO-1 on the development of colon-26 tumours in mice. Tumours were excised 10 days after seeding. * p ⁇ 0.05 comparison with control vector.
  • the data from the experiment are shown in Table 2.
  • the wet and dry data are inclusive of the 5mg sponge.
  • Daily dosing with 40 ⁇ mole/kg tin protoporphyrin from day 4 reduced tumour growth by 48% compared to vehicle treated controls (p ⁇ 0.05). Tumour wet weight at day 10 was 30% lower than the controls and dry weight was 51% lower (p ⁇ 0.01).
  • ferriprotoporphyrin 40 ⁇ mole/kg
  • Table 3 Effects of zinc protoporphyrin on the development of colon-26 tumours seeded into a polyurethane sponge implanted subcutaneously into mice. Dosing was daily from day 4 after seeding to day 10 when the animals were killed. Data are mean ⁇ s.e.m.
  • ZnDPP zinc deuteroporphyrin IX 2,4 bis glycol.
  • Prelirninary data from colon-26 cells transfected with antisense HO-1 ie HO-1 at greatly reduced expression
  • Tin protoporphyrin SnPP
  • HO-1 inducer Zinc deuteroporphyrin
  • ZnDPP At the lower doses used ZnDPP tended to increase tumour mass. This is consistent with the transfection studies. At the highest dose selectivity for HO may be lost and we are seeing a combination of HO inhibition and cytotoxicity (a known problem with high doses of porphyrins). This explains a reduction in tumour mass at the highest dose of ZnDPP. SnPP is more cytotoxic than ZnDPP; this, together with the higher dose, probably explains why greater effects were seen with SnPP on tumour mass.
  • Tumours can be stressed as a result of out-growing their blood supply (hypoxic stress) and from therapeutic measures such as chemotherapy and radiotherapy.
  • High expression of HO-1 (a stress protein) is therefore likely in a variety of tumours.
  • Tissue damage as a result of chemotherapy and radiotherapy will result in an inflammatory response the effectiveness of which may be limited by the presence of HO.
  • radiotherapy and some chemotherapeutic agents work by creating free radical attack on cells. The radical scavenging activity of HO products could again limit the effectiveness of treatment.
  • the invention proposes HO inhibition as a useful adjunct to existing antitumour therapies, to allow the use of lower concentrations of chemotherapeutic agents or- lower doses of ionising radiation, thus sparing some of the unpleasant side-effects associated with these treatments.
  • some tumours currently resistant to therapy may become susceptible following HO inhibition.

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Abstract

An inhibitor of heme oxygenase is used in treatment of cancers and other tumours, either alone or in combination with another anti-tumour agent or as non-pharmaceutical anti-tumour therapy such as tumour irradiation. An anti-tumour composition comprises an inhibitor of heme oxygenase and a cytotoxic agent.

Description

USE OF HEME-OXYGENASE INHIBITORS TO TREAT CANCER
This invention relates to the treatment of cancers and other tumours, and to pharmaceutical compositions and therapies for such treatment.
Tumours are usually recognizable as a mass of abnormal cells. If these grow in one location and do not spread to other tissues or areas they are usually classified as benign. An in-situ tumour is one that develops in the epithelium, and contains typically small cells showing abnormalities, but which does not invade other tissues. Cancers are fully developed, malignant tumours that invade and destroy adjacent tissues.
In the modern age, cancer is one of the commonest causes of death in western populations and in certain groups of individuals is indeed the commonest. Even non- fatal cancers often have drastic effects on sufferers; as an example, mastectomy is frequently required to prevent spread of cancer from a diseased breast.
It is known to treat cancers by radiotherapy, using low energy X-rays or gamma rays. One disadvantage of this treatment is that therapeutic doses typically kill not only cancer cells but also dividing cells in healthy tissues.
It is further known to treat cancers by chemotherapy. Depending upon the nature of the cancer, this treatment can be aimed at a cure or simply at extending the patient's life. The principle of chemotherapy is that the drugs used are cytotoxic and kill both cancer and healthy cells, though healthy cells can often recover more readily. Major side effects result from death of healthy cells and include bone marrow suppression, impaired immune response, hair loss and impaired reproductive function. Other well known side effects are nausea and vomiting.
Heme (ferri-protoporphyrin-IX) plays a vital role in cellular metabolism, functioning as the prosthetic group of hemeproteins (eg. hemoglobin and cytochromes). Its catabolism is a two-step process. The first, and rate limiting reaction, is the production of biliverdin and carbon monoxide by the microsomal enzyme heme- oxygenase. The second step is the production of bilirubiπ from biiiverdin by the cytosolic enzyme, biliverdin reductase.
Heme-oxygenase is found in liver, kidney, spleen and skin, and has also been localised to specific cell types, notably fibroblasts and macrophages. The enzyme exists in at least two isoforms, one constitutive and the other inducible. Heme, heavy metal ions (eg. tin, gold, platinum and mercury) and transition metal ions (eg. iron, cobalt, chromium and nickel) can all induce heme-oxygenase. In addition, heme- oxygenase is induced as part of a generalised stress response to stimuli such as thermal shock (hence the alternative name heat-shock protein 32: hsp32), oxidative stress and cytokines such as interleukin-1 (IL— 1), tumour necrosis factor and 1L-6. The stress response is seen as beneficial in that it results in protection of vulnerable cell enzymes from inactivation.
Known compounds that inhibit heme-oxygenase are structural analogues of ferriprotoporphyrin (FePP), which itself induces the enzyme, and some examples include tin protoporphyrin (SnPP) and tin mesoporphyrin (SnMP).
U.S. Patent No. 4657902 relates to the use of tin mesoporphyrin and compositions containing it to inhibit heme metabolism in mammals to control the rate of tryptophan metabolism in mammals, and to increase the rate at which heme is excreted by mammals.
U.S. Patent No. 5010073 relates to liposomal metalloporphyrin preparations for targeting the spleen for inhibition of heme-oxygenase activity in the spleen.
Reduction in or amelioration of side effects in known radiotherapy or chemotherapy regimes would be of benefit to the cancer patient, as would any improvement in the prognosis of cancer sufferers.
The provision and development of further treatments for cancers, and other tumours, whether those treatments -are to be used alone or in conjunction with known treatments, is thus a continuing concern.
It is an object of the invention to provide means of treating cancers and other tumours. Another object is to provide pharmaceutical compositions, of use in treating cancers and other tumours. A further object is to provide methods of therapy against cancers and other tumours.
The present invention is based on the observation of reduction in tumour growth and tumour dry mass following administration of a heme oxygenase inhibitor.
Accordingly, a first aspect of the invention provides the use of an inhibitor of heme oxygenase in the manufacture of a medicament for the treatment of a tumour.
By reference to an inhibitor of heme oxygenase, it is hereby intended to include compounds that directly inhibit heme oxygenase and those whose inhibitory action is indirect but nevertheless induce a reduction in the amount of heme oxygenase activity - the reduction can be due to a reduced amount of heme oxygenase, a reduced amount of active heme oxygenase or to heme oxygenase of reduced efficiency. Preferably, the inhibitor inhibits an inducible form of HO-1.
Agents suitable for inhibiting heme-oxygenase (for example, inducible heme- oxygenase in monocytes and macrophages) are typically structural analogues of FePP (nb. which induces heme-oxygenase) in which the Fe ion is replaced by another metal ion, or the PP is replaced. EΞxamples are:
SnPP SnMP SnDPP CrPP CrMP CrDPP
ZnPP ZnMP ZnDPP MnPP MnMP MnDPP where:
Sn=tin, Cr-chromium, Zn=zinc, Mn=manganese, PP=protoporphyrin,
MP=mesoporphyrin and DPP=diiododeuteroporphyrin
Suitable dosage amounts of these agents range from 0.1 to 50 μmoles/kg of body weight of, for example, a human. For example, where tin protoporphyrin (SnPP) is used, suitable dosage amounts of SnPP would be from 0.07mg/kg to 46mg/kg of body weight of a human.
Route of administration of a heme oxygenase inhibitor according to the invention is optionally via any conventional pharmaceutical route, these including but being not limited to oral, rectal, subcutaneous, parenteral, intravenous, intraperitoneal and topical. It is further explained below how certain specific formulations also represent further aspects of the invention; though, generally, the preparation of pharmaceutical compositions comprising a heme oxygenase inhibitor for oral use is described in US patents numbers 4657902 and 5010073, and to which the skilled person is referred for further guidance on the formulation and preparation of such compositions.
In a specific embodiment of the inventions, hereinafter described, the FePP analogue tinprotoporphyrin (SnPP) was used to treat a tumour of colon-26 cells. SnPP was administered subcutaneously and over a period of 7 days, at the end of which period significant reduction in tumour was observed.
Antagonists of prostaglandin A receptors are further agents suitable for decreasing heme-oxygenase (HO) activity.
Still further agents for reducing HO activity are agents that increase nitric oxide (NO) levels in a patient. Such agents are, optionally, a substrate for nitric oxide synthase (NOS) or a stimulator of this enzyme or a NO donor. A known substrate for NOS is L-arginine and a known NO donor is sodium nitroprusside. Nitric oxide (NO), formed from L-arginine and molecular oxygen by isoforms of the enzyme nitric oxide synthase (NOS EC 1.14.13.39), is involved in a variety of physiological and pathophysiological processes. The reactivity of this molecule, and its capacity to complex with metalloproteins, underlies many of its biological actions. For example, activation by NO of heme-containing soluble guanylate cyclase (EC 4.6.1.2) in vascular smooth muscle results in vasoregulation, whilst in host defence, inhibition of iron-sulphur enzymes causes metabolic dysfunction in invading pathogens. In an embodiment of the invention the medicament is for treatment of cancer. In a preferred embodiment of the invention a heme oxygenase inhibitor is for use in combination with a cytotoxic agent. Optionally, the inhibitor is for use at such concentrations that both inhibit heme oxygenase and are cytotoxic. Alternatively, the inhibitor is for use with a separate agent that is cytotoxic. A further alternative is that the inhibitor is for use in combination with a cytotoxic therapy such as radiotherapy.
As a further embodiment of the invention, the heme oxygenase inhibitor is for use in combination with a cytotoxic agent and elevated levels of nitric oxide in the patient, optionally achieved using a nitric oxide donor or a substrate for NOS. Thus, nitric oxide is used as an adjunct to tumour treatment by a heme oxygenase inhibitor in combination with a cytotoxic agent.
The invention thus provides the use of known pharmaceuticals in the manufacture of a medicament for the treatment of tumours, such as cancerous tumours. The invention offers an alternative therapy for tumours, which therapy has been neither described nor suggested in any prior art. In a specific embodiment of the invention, described in an example hereinafter, the inventors have tested the treatment of tumours according to the invention and have observed useful anti-tumour results.
In a second aspect of the invention, there is provided a pharmaceutical composition comprising a compound that inhibits heme oxygenase and an anti-tumour pharmaceutical that is not an inhibitor of heme oxygenase. The pharmaceutical composition of the second aspect is thus a combination typically of a heme oxygenase inhibitor with a known anti-tumour agent. The pharmaceutical compositions of the invention can be prepared using any suitable pharmaceutically acceptable carrier. In a specific embodiment of the invention the inhibitor is tin protoporphyrin and this is present in a pharmaceutical in an amount of between 0.1 and 50 μmols/kg.
A suitable anti-tumour agent is one that inhibits cell division, for example an anti- mitotic agent. In embodiments of the invention, a pharmaceutical composition comprises an inhibitor of heme oxygenase with an anti-cancer drug selected from alkylating agents, antimetalbolites, alkaloids, cytotoxic antibodies, nitrosoureas and synthetic anti-neoplastic drugs selected from amacrine, carboplatin, cisplatin, crisantaspase, dacarbazine, hydroxyurea, paclitaxei, pentostatin, procarbazine, mitotane, dibromomannitol and razoxane.
In use of embodiments of the second aspect of the invention, a patient with a tumour is administered a combination of two pharmaceutically active agents; the first being an inhibitor of heme oxygenase and the second a cytotoxic drug. An anti-tumour effect is obtained through the combined actions of the two active ingredients.
Another suitable anti-tumour agent, for combination with a heme oxygenase inhibitor, is a latent agent which is activated by radiation either to become a toxic agent or to release a toxic agent. For example, it is known to treat tumours with a latent agent which when activated by ultra violet radiation produces free radicals, the free radicals being damaging to the tumour. The use of such a latent agent can be of particular use as the radiation is targetted to the tumour area, thus producing activation of the latent agent in areas where only healthy tissue is present.
A problem with known anti-tumour agents is that of tachyphylaxis, that is to say the observation that after a period of administration of such an agent it is observed that the anti-tumour agent loses part or all of its effectiveness and must be administered in ever increasing doses. The invention provides a pharmaceutical composition that combines an inhibitor of heme oxygenase with a known anti-tumour agent and offers the possibility of administering a reduced amount of the known agent. A common visible side effect of chemotherapy for the treatment of cancers is both hair loss and weight loss. The opportunity to use reduced amounts of such anti-cancer agents opens the possibilities for amelioration or reduction in these known side effects.
Similarly, the dose of an anti-cancer agent is often limited to the maximum dose the patient can withstand without risk to life. The invention also offers the possibility that a similar dose of anti-cancer agent, used in combination with a heme oxygenase inhibitor, may give an enhanced anti-cancer effect without increasing the risk to the patient's life.
In a third aspect, the invention provides a topical pharmaceutical composition comprising an inhibitor of heme oxygenase and a topical, pharmaceutically acceptable carrier. A suitable topical carrier can comprise an oil, a wax, an emulsion of an oil, an emulsion of a wax, a gel or a cream. In use, this topical composition may be applied directly to a tumour, such as one found on the skin of a patient. In a specific embodiment, the pharmaceutical composition is a suppository.
In a fourth aspect of the invention, a pharmaceutical composition comprises a compound that inhibits heme oxygenase in a solution for infusion into a patient. This composition can be administered to a patient with cancer by intravenous drip; this route is convenient for a sleeping or anaesthetized patient, or one not physically capable of taking medicaments orally.
A fifth aspect of the invention provides a composition that inhibits heme oxygenase and has formula: -
X-A
wherein X represents an analogue of FePP and A represents a moiety capable of releasing nitric oxide or of inducing release of nitric oxide. A is optionally at least one arginine molecule; this composition is thus the arginate form of a structured analogue of FePP. In specific embodiments, the composition is tin protoporphyrin arginate or zinc protoporphyrin arginate.
Nitric oxide can be cytoxic at high levels, and this composition thus combines inhibition of heme oxygenase with formation of cytotoxic nitric oxide. A is further optionally a nitric oxide donor, a substrate for NOS or a moiety capable of releasing NO, such as a -N02 group.
In a sixth aspect, the invention provides a pharmaceutical composition comprising a composition according to the fifth aspect and a pharmaceutically acceptable carrier.
In a seventh aspect of the invention there is provided a method of treatment of a tumour in a patient, comprising administering to that patient a compound that inhibits heme oxygenase. In an embodiment of the invention, a structural analogue of FePP which inhibits heme oxygenase is administered in an amount of from 0.1 to 50 μrπols per kilogram of body weight of the patient per day. Typically, a structural analogue of FePP, such as SnPP, is administered orally to a patient is an amount sufficient to exert a therapeutic effect. The amount administered can be in the range of 0.1 -50 μmols/kg body weight of the patient. More specifically, the amount can range from 1.0-30 μmols/kg body weight. In a specific embodiment of the invention described in an example hereafter, the dosing rate is about 14 μmois/kg.
In another embodiment, the method further comprises administering at least two pharmaceutically active agents, one being an inhibitor of heme oxygenase and one being an anti-tumour agent that is not an inhibitor of heme oxygenase. The two agents are administered simultaneously or separately. Though it is preferred that the timing of administration is such that a cytotoxic Ievel of the anti-tumour agent is achieved whilst heme oxygenase activity is depressed by the heme oxygenase inhibitor. With knowledge of the pharmacokinetics of the respective active agents, the timing of administration of each, separately or together, is readily determined.
In an embodiment of the invention a HO inhibitor is administered in combination with an anti-tumour agent selected from alkylating agents, antimetabolites, alkaloids, cytotoxic antibodies, nitrosoureas, and synthetic anti-neoplastic drugs selected from amacrine, carboplatin, cisplatin, crisantaspose, decarbazine, hydroxyurea, paclitaxel, pentostatin, procarbazine, dibromomannitol and razoxane.
In a further embodiment of the invention, the method further comprises providing an elevated Ievel of nitric oxide in the patient, such as by administration of a nitric oxide donor or a substrate for NOS. The invention is now illustrated in the following examples.
Example 1
Methods
The brains and spleens of male Wistar rats (160 ± 20g, Tuck Co. UK) were homogenised using a glass homogenizer, in protease inhibitory buffer; phenylmethylsulfonyl fluoride 1 mM, pepstatin A 1.5mM and leupeptin 0.2mM, in 10mM phosphate buffered saline pH 7.3. Protein determinations were carried by the Bradford method, bovine serum albumin was used as protein standard.
Nitric oxide synthase activity was determined by the citrulline assay and the data calculated as pmol citrulline/mg protein/30min (Vane, J.R. et al (1993) Proc. Natl. Acad. Sci. USA 91 , 2046-2050).
Heme-oxygenase activity was assayed as previously described (Sierra, E.E. et al, (1992) Analytical Biochem. 200, 27-30).
Briefly, the 15μl reaction mixture consisted of 11.2μM [ C] heme (specific activity 54 Ci/mol), 1 mM NADPH, 2mM glucose-6-phosphate, 0.1 units of glucose-6-phosphate dehydrogenase, 3mg/ml liver cytosolic protein, 100-50μg of sample protein and the relevant concentration of test drugs; L-arginine, D-arginine, NG-nitro-L-arginine methyl ester (L-NAME) or sodium nitroprusside. The reaction mixture containing the test drugs was allowed to equilibrate at 37°C for 15 mins before the reaction was started by the addition of the heme. The reaction was terminated after 30 mins by the addition of excess cold heme and bilirubin and placed on ice. The reaction mixture was spotted on to the silica gel thin-layer chromatography sheet by 2, 2μl applications. All samples were run in duplicate. The chromatogram was developed using a 20:1 dilution of chloroform: acetic acid. Spots corresponding to heme and bilirubin were excised and placed in 10ml of scintillation fluid to be counted. The data was calculated as pmois bilirubin formed/mg protein/hour. Statistical analysis of the raw data was carried using Student's unpaired t test. Results expressed as mean ± s.e.mean with P<0.05 considered as significant.
Results
Homogenates of rat brain contained both HO and NOS activity as determined by our assay systems. In comparison, rat spleen homogenates had double the HO activity, but lacked NOS activity. Addition of the NOS inhibitor, L-NAME, resulted in a dose dependent increase in brain HO activity, with 5mM L-NAME significantly increasing activity by 80%. Conversely, addition of L-arginine, the NOS substrate, to brain homogenates, resulted in a dose dependent decrease in HO activity. The highest concentration of L-arginine used, 10mM, reduced HO activity by 75%. The enantiomer of L-arginine, D-arginine, which cannot be utilised as a substrate by NOS, had no significant effect on brain HO activity.
Spleen HO activity, unlike brain activity was not modified by the addition of L-NAME, L-arginine or D-arginine. However the addition of the NO donor sodium nitroprusside resulted in a dose dependent decrease in HO activity in both spleen and brain homogenates. The addition of 10mM sodium nitroprusside resulted in a 75% and 80% decrease in the HO activity of brain and spleen homogenates respectively.
These results demonstrate that NO, generated by NOS, inhibits HO activity. It is important to note that the isoforms of NOS and HO present in the brain under normal physiological conditions are the constitutive forms of the enzymes, cNOS and HO-2 respectively (Bredt, D.S. et al, (1990) Proc. Natl. Acad. Sci. USA 87, 682-685, and Verma, A., et al, (1993) Science 259, 381-384); whereas spleen under normal physiological conditions contains only the inducible form of HO (Maines, M.D. (1988) FASEB J. 2,2557-2568).
The effects of inhibitors and donors of NO on HO activity have been examined in homogenates of rat brain, where endogenous activity of both NOS and HO are high and in rat spleen where HO is higher, but NOS activity lower than in brain. We have found that a substrate for nitric oxide, L-arginine (0.1 -10mM), but not D- arginine, decreased heme-oxygenase activity in rat brain homogenates and that the arginine analogue L-NAME (0.1 -10mM) increased activity in the same tissue. In spleen homogenates where endogenous nitric oxide activity is lower than in brain, these compounds had no effect. The nitric oxide donor sodium nitroprusside (0.001 mM-10mM) reduced heme-oxygenase activity in both brain and spleen.
Example 2
Culture and Characterisation of Colon-26 cells
Coion-26 cells were cultured in DMEM supplemented with 10% foetal calf serum, 1000U/ml penicillin, 1000U/ml streptomycin and 100μg/ml gentamycin. These cells have been shown to produce mRNA for HO-1 by Northern blot and to express HO-1 protein by Western blot and immunocytochemistry.
Transfection of Colon-26 Cells with Murine HO-1 in the Antisense orientation
HO-1 fragment generation
Mouse spleen RNA was isolated using the Clontech RNA extraction kit. First strand cDNA synthesis was performed using the Ready-to-Go kit from Pharmacia and PCR carried out using the following parameters: 1x3' at 94.0°C, (45" at 95.0°C, 45" at 59.1 °C, 1 ' at 72.0°C)x30, 1x10' at 72.0°C. Forward primer: 5' gCCTgA ATCgAggAgAACCA3'; reverse primer: 5' CTTTTggTgAgggAACTgTgTCA3'; expected size of the product was 1 kb.
Subcloning into pRC/RSC expression vector
The fragment was gel-isolated (using the Geneclene BIO 01 system, Anachem), and ligated into PCR 2 cloning vector from Clontech. Screening was carried out in E. coli. XLI-blue (Stratagene). The fragment was checked for orientation using Hind III and the antisense construct was digested with Hind III and Spe I for ligation into the expression vector pRC/RSV (Clontech) which had been digested with the same enzymes. 400ml cultures were processed using the Quiagen maxi-prep kit, providing sufficient DNA for the transfection experiment.
Transfection
Transfection of the colon26 cells was carried out for the antisense construct and the vector alone. Transfection was carried out using Lipofectin (Life Technologies). 5ug of plasmid were used per transfection. Transfection was carried out in a T75 cell culture flask, and the cell were left overnight with the Lipofectiπ-plasmid mix in serum- free medium. The medium was replaced with 10% FCS-DMEM the following day. 24hrs later, the cells were removed from the flask, and re-seeded in a 96-well plate in 10% FCS-DMEM containing 1 mg/ml G418 (Geneticin). Medium was changed once a week.
Screening
The screening procedure involved observation for medium colour-change (indicating cell proliferation), and occurred from about one week following transfection. The cells were transferred to a 6-well plate, and allowed to proliferate until enough cells were available for characterisation and freezing down. All medium contained G418. Diminished HO-1 expression was confirmed by isolating protein and RNA for Western blot and Northern blot respectively. The protein isolation was carried out using the method routinely used in the Department and Westerns were carried out using the Amersham ECL-kit, and the antibody used was SP-895 from stressgen. RNA extraction was carried out using the Trizol method (Life Technologies), and RNA was transferred to a nylon support following electrophoresis in a denaturing formaldehyde gel. Northern hybridisation was performed using the Amersham Rapidhybe solution and protocol provided with it for use with radionuclides. The HO-1 cDNA fragment was used as a probe.
Colon-26 Tumours in Mice
Methods
Sterile polyether polyurethane sponge discs (8mm x 4mm, 5mg) were implanted subcutaneously in the dorsum of anaesthetized BALB/c mice (20-23g). Three days later the sponges were seeded by injection of 10 coion-26 cells in a volume of 50μl sterile saline. Ten days after seeding, animals were killed in line with Home Office guidelines for the use of experimental animals.
Transfection studies
The results of seeding transfected colon26 cells into mice are shown in Table 1. Tumours were excised at 10 days. In comparison to tumours carrying the control vector, there was a significant (p<0.05) rise in tumour dry mass when the tumour cells carried antisense HO-1. These tumours were also of greater mass than control colon26 tumours but this difference did not reach statistical significance.
Group Dry weight (mg)
Normal colon26 139±19
Control vector 93+24
Antisense HO-1 158±14*
Table 1 The effects of control vector and antisense HO-1 on the development of colon-26 tumours in mice. Tumours were excised 10 days after seeding. * p<0.05 comparison with control vector.
Effects of protoporphyrins on tumour development
At day 4 after seeding with normal colon26 cells animals were randomly assigned to groups for dosing. Drugs were freshly prepared each day. The protopophyrins were dissolved in 0.1 N NaOH, mixed with an equal volume of phosphate buffered saline. The pH was adjusted to 7.4. A similar solution but lacking protoporphyrin served as vehicle control. Dosing was once daily subcutaneously in a volume of 0.1 ml. Dosing was daily for 7 days at which point animals were killed and tumours excised. Experiment 1: Iron and tin protoporphyrin.
Group 1. Non-treated controls Group 2. Vehicle-treated controls Group 3. Ferriprotoporphyrin IX 40μmole/kg Group 4. Tin protoporphyrin IX 40μmole/kg
The data from the experiment are shown in Table 2. The wet and dry data are inclusive of the 5mg sponge. Daily dosing with 40μmole/kg tin protoporphyrin from day 4 reduced tumour growth by 48% compared to vehicle treated controls (p<0.05). Tumour wet weight at day 10 was 30% lower than the controls and dry weight was 51% lower (p<0.01). In contrast, there was a tendency for dosing with ferriprotoporphyrin (40μmole/kg) to increase tumour diameter (17% increase) and increase tumour wet weight (30% increase). These changes did not reach statistical significance when multiple comparisons were taken into account. There was no change in tumour dry mass.
Group n Tumour growth Wet weight Dry weight (mm) (mg) (mg)
Non treated 9 1.98±0.29 428+16 74±5
Vehicle 9 2.48±0.33 403+41 91 ±9
FePP 10 2.89±0.28 524±61 92±11 40μmole/kg
**
SnPP 10 1.28±0.33* 281 ±19 45±3 40μmole/kg
Table 2 Effects of protoporphyrins on the development of colon-26 tumours seeded into a polyurethane sponge implanted subcutaneously into mice. Dosing was daily from day 4 after seeding to day 10 when the animals were killed. Data are mean ± s.e.m. FePP=ferriprotoporphyrin IX, SnPP=tin protoporphyrin IX. Experiment 2: Zinc deuteroporphyrin.
Group 1. Non-treated controls
Group 2. Vehicle-treated controls
Group 3. Zinc deuteroporphyrin IX 2,4 bis glycol (ZnDPP) 3μmole/kg
Group 4. ZnDPP 10μmole/kg
Group 5. ZnDPP 30μmole/kg
The data from the experiment are shown in Table 3. The wet and dry data are inclusive of the 5mg sponge. Drug-treated groups were compared with the vehicle- treated controls. There were no statistically significant differences between groups when multiple comparisons were taken into account. However, with the highest dose of ZnDPP (30μmole/kg) a 18% reduction in dry weight just failed to reach statistical significance (p=0.058).
Group n Wet weight Dry weight (mg) (mg)
Non treated 10 677±55 116±11
Vehicle 10 719±48 130+11
ZnDPP 3μmole/kg 10 937±129 155±23
ZnDPP 10μmole/kg 10 724±108 158±28
ZnDPP 30μmole/kg 10 718±47 106±7
Table 3 Effects of zinc protoporphyrin on the development of colon-26 tumours seeded into a polyurethane sponge implanted subcutaneously into mice. Dosing was daily from day 4 after seeding to day 10 when the animals were killed. Data are mean ± s.e.m. ZnDPP=zinc deuteroporphyrin IX 2,4 bis glycol. Prelirninary data from colon-26 cells transfected with antisense HO-1 (ie HO-1 at greatly reduced expression) would indicate that these tumours achieve greater mass than cells transfected with an empty vector or non-treated cells. This might suggest that in the absence of HO-1 the tumour cells are proliferating faster. Consistent with this idea is the finding that HO expression is associated with cells being arrested in GO or G1 , removal of HO may therefore push cells into cycling. Since many chemotherapeutic agents target proliferating cells, inhibition of HO renders cells more sensitive to such agents eg paclitaxel.
Tin protoporphyrin (SnPP) at 40μmole/kg caused a 51% reduction (p<0.01) in tumour dry mass using the above model. Administration of ferriprotoporphyrin (the HO-1 inducer) tended to result in larger tumours although this seemed to be due to increased fluid content since tumour dry weight was unchanged. We have also used zinc deuteroporphyrin (ZnDPP) which is claimed to be a more selective inhibitor of HO and investigated a range of doses.
At the lower doses used ZnDPP tended to increase tumour mass. This is consistent with the transfection studies. At the highest dose selectivity for HO may be lost and we are seeing a combination of HO inhibition and cytotoxicity (a known problem with high doses of porphyrins). This explains a reduction in tumour mass at the highest dose of ZnDPP. SnPP is more cytotoxic than ZnDPP; this, together with the higher dose, probably explains why greater effects were seen with SnPP on tumour mass.
Tumours can be stressed as a result of out-growing their blood supply (hypoxic stress) and from therapeutic measures such as chemotherapy and radiotherapy. High expression of HO-1 (a stress protein) is therefore likely in a variety of tumours. Tissue damage as a result of chemotherapy and radiotherapy will result in an inflammatory response the effectiveness of which may be limited by the presence of HO. In addition, radiotherapy and some chemotherapeutic agents work by creating free radical attack on cells. The radical scavenging activity of HO products could again limit the effectiveness of treatment. The invention proposes HO inhibition as a useful adjunct to existing antitumour therapies, to allow the use of lower concentrations of chemotherapeutic agents or- lower doses of ionising radiation, thus sparing some of the unpleasant side-effects associated with these treatments. In addition, some tumours currently resistant to therapy may become susceptible following HO inhibition.

Claims

1. Use of an inhibitor of heme oxygenase in the manufacture of a medicament for tumour treatment.
2. Use according to Claim 1 wherein the inhibitor is selected from a structural analogue of ferriprotoporphyrin (FePP), an antagonist of a prostaglandin receptor and a compound that increases the Ievel of nitric oxide (NO).
3. Use according to Claim 2 wherein the structural FePP analogue is selected from SnPP, SnMP, SnDPP, CrPP, CrMP, CrDPP, ZnPP, ZnMP, ZnDPP, MnPP, MnMP and MnDPP, wherein PP represents protoporphyrin, MP represents mesoporphyrin and DPP represents diiododeuteroporphyrin.
4. Use according to Claim 2 wherein the compound is selected from a substrate for nitric oxide synthase (NOS), a substance that stimulates NOS and a NO donor.
5. Use according to any of Claim 1 -4 wherein the medicament is for treatment of cancer.
6. A pharmaceutical composition comprising:- a compound that inhibits heme oxygenase; and an anti-tumour pharmaceutical that is not an inhibitor of heme oxygenase.
7. A composition according to Claim 6 comprising a pharmaceutical that inhibits cell division, such as an anti-mitotic agent.
8. A composition according to Claim 6 or 7 comprising an anti-cancer drug selected from alkylating agents, antimetalbolites, alkaloids, cytotoxic antibodies, nitrosoureas and synthetic aπti-neoplastic drugs selected from amacrine, carboplatin, cisplatin, crisantaspase, • dacarbazine, hydroxyurea, paclitaxel, pentostatin, procarbazine, mitotane, dibromomannitol and razoxane.
9. A composition according to Claim 6 comprising a latent anti-tumour agent, said latent agent being activated by radiation to become a toxic agent or to release a toxic agent.
10. A composition according to Claim 9 wherein the latent agent is activated by ultra violet radiation to produce free radicals.
11. A pharmaceutical composition comprising a compound that inhibits heme oxygenase in a solution for infusion into a patient.
12. A topical pharmaceutical composition cornprising:- a compound that inhibits heme oxygenase; and a topical, pharmaceutically acceptable carrier.
13. A composition according to Claim 12 comprising a carrier comprising an oil, a wax, an emulsion of an oil, an emulsion of a wax, a gel or a cream.
14. A composition according to Claim 12 or 13 wherein the composition is a suppository.
15. A composition of formula
X-A wherein X represents an analogue of FePP and A represents a moiety capable of releasing nitric oxide or of inducing release of nitric oxide.
16. A composition according to Claim 15 wherein A is at least one arginine molecule.
17. A pharmaceutical composition comprising a composition according to Claim 15 or 16.
18. A method of treatment of a tumour in a patient, comprising administering a compound that inhibits heme oxygenase.
19. A method according to Claim 18 comprising administering an analogue of FePP in an amount of from 0.1 to 50 μmoles per kg of body weight of the patient.
20. A method according to Claim 18 or 19 comprising (1 ) previous, (2) simultaneous and/or (3) subsequent administration of an anti-tumour agent that does not inhibit heme oxygenase.
21. A method according to any of Claims 18-20 further comprising irradiation of the tumour.
22. A method according to any of Claims 18-21 further comprising administering an agent that increases the Ievel of nitric oxide in the patient.
23. A method according to any of Claims 18-21 for treatment of cancer.
24. Use of an inhibitor of heme oxygenase in manufacture of a medicament for cancer treatment by a combination therapy comprising administration of the medicament and irradiation of the cancer.
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US10888569B1 (en) * 2017-06-09 2021-01-12 The University Of Chicago Methods and compositions for treating cancer

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