US20090099134A1

US20090099134A1 - Treatment of dna damage related disorders

Info

Publication number: US20090099134A1
Application number: US12/334,209
Authority: US
Inventors: Michael B. Kastan; Christopher J. Bakkenist
Original assignee: St Jude Childrens Research Hospital
Current assignee: St Jude Childrens Research Hospital
Priority date: 2002-11-27
Filing date: 2008-12-12
Publication date: 2009-04-16
Also published as: WO2004050842A2; US20030157572A1; US6916627B2; US20060241139A1; US20050032834A1; WO2004050837A3; US20050148020A1; US20080020462A1; WO2004050842A3; US7160692B2; US7279290B2; AU2003297595A8; US20070009919A1; AU2003293099A1; WO2004050837A2; AU2003297595A1; AU2003293099A8

Abstract

The present invention provides methods and compositions for prophylaxis and treatment of a variety of disorders including DNA damage related disorders, cancer, ischemia, oxidative stress, atherosclerosis, and stroke using a chloroquine compound.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation-in-part of attorney docket 29202-710.831 filed May 26 2004, the US national phase of PCT US03/37838 filed Nov. 26, 2003, which is a continuation-in-part of 10/351,733 filed Jan. 24, 2003 and is also a continuation-in-part of U.S. Ser. No. 10/307,077, filed Nov. 27, 2002, all of which are incorporated herein by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in the course of research sponsored by the National Institutes of Health (NIH Grant Nos. CA71387). The U.S. government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Cancer is now the second leading cause of death in the United States. Over 1 million new cases of cancer are expected to be diagnosed in 2003 and over 500,000 people are expected to die of cancer.
Cancer is typically treated with one or a combination of three-types of therapies: surgery, radiation, and chemotherapy. Overall costs for cancer, including treatments, were approximately $170 billion dollars in 2002. The cancer treatments are not only expensive; they are ineffective most of the time and also have many side effects. Hence, there is a demand for more effective cancer prevention and treatment agents, as well as for the prevention and treatment of DNA damage related conditions.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods of prophylaxis of cancer. The methods comprise administering to a subject at risk of cancer, an effective amount of a chloroquine compound, provided the subject has been free of a localized skin epithelialoma or carcinoma for at least a year before commencing administration of the chloroquine compound. Optionally, the subject has never suffered from a localized skin epithelialoma or carcinoma basal cell before commencing administration of the chloroquine compound. Optionally, the subject has never suffered from skin cancer.
In some methods, the subject is monitored for development of a cancer after administration of the chloroquine compound. Optionally, the monitoring comprising monitoring the subject for a cancer other than a localized skin epithelialoma or carcinoma basal cell. Optionally, the monitoring comprising taking a sample of a body fluid, or performing a scan of an internal organ.
In some methods, the subject is at risk of cancer by having a precancerous tissue. In some methods, the subject has a first cancer, and is at risk of a second cancer. In some methods, the subject has a cancer and is undergoing radiation therapy to treat the cancer, the radiation therapy putting the subject at risk of a second cancer; and wherein the administration effects prophylaxis of the second cancer. In some methods, the chloroquine is administered before or during the radiation therapy. In some methods, the subject has a cancer and is undergoing chemotherapy to treat the cancer, the chemotherapy placing the subject at risk of a second cancer, and wherein the administration effects prophylaxis of the second cancer. In some methods, the chloroquine compound is administered before or during the chemotherapy.
In some methods, the subject is at risk of cancer due to a genetic variation associated with increased risk of cancer. In some methods, the subject is at risk of cancer due to viral infection. In some methods, the subject is at risk of cancer due to exposure to a carcinogen or irradiation. In some methods, the subject is at risk of cancer due to exposure to X-rays.
In some methods, there is an additional step of determining presence of a genetic variation in an ATM gene of the subject associated with cancer. In some methods, there is an additional step of administering a chemopreventive agent other than the chloroquine compound to the subject. In some methods there is a further step of determining the risk of cancer in a subject before administering the chloroquine compound.
In some methods, the chloroquine is administered intravenously. In some methods, the chloroquine is administered orally.
In some methods, the subject is free of diseases of the immune system, infectious diseases, and neurological diseases. In some methods, the subject is free of psoriasis, malaria, protozoal infections, Alzheimer's disease, Parkinson's disease, lupus erythematosus, rheumatism, hypercalcemia, multiple sclerosis, and migraine.
In some methods, the prophylaxis is effective to prevent detectable development of cancer for at least six months after administering the effective dosage. In some methods, the administering is performed before exposure of the subject to the risk of cancer. In some methods, the administering is performed at regular intervals for a period of at least six months.
In some methods, the chloroquine compound is selected from the group consisting of chloroquine, chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate, hydroxychloroquine sulphate, or enantiomers, derivatives, analogs, metabolites, pharmaceutically acceptable salts, and mixtures thereof. Optionally, the chloroquine compound is chloroquine, chloroquine phosphate or chloroquine diphosphate.
In some methods, the chloroquine compound has a systemic effect. In some methods, the patient is human and the dosage is 0.05 to 1 mg/kg per day. In some methods, the patient is human and the dosage is 0.2 to 0.6 mg/kg per day. In some methods, the patient has been exposed to a carcinogen or radiation, and the dosage is administered at least on the day of exposure and the day following exposure. In some methods, the patient has been exposed to a carcinogen or radiation, and the dosage is administered at least on the day before the exposure, on the day of the exposure, and at least on the day following the exposure. In some methods, the patient is human and has genetic susceptible to cancer, and the dosage is 0.2 to 0.6 mg/kg week. In some methods, the amount of the compound administered is up to about 10 mg/kg/day. In some methods, the amount of the compound administered is more than about 0.1 mg/kg/day. In some methods, the amount of the compound administered is less than about 50 mg/kg/day. In some methods, the amount of the compound administered is less than about 10 mg/kg/day. In some methods, the chloroquine compound is administered more than once a week. In some methods the chloroquine compound is administered daily. In some methods, the chloroquine compound is formulated in a sustained release formulation. In some methods, the patient is human.
The invention further provides methods of therapeutically treating cancer comprising administering to a subject having a cancer an effective amount of a chloroquine compound whereby the cancer is therapeutically treated. Optionally, the cancer is other than a localized skin epithelialoma or carcinoma basal cell. Optionally, the cancer is other than skin cancer. Optionally, the treatment reduces or eliminates further growth of the cancer. Optionally, the treatment shrinks or eliminates the tumor. Optionally, the treatment inhibits invasion of the cancer into tissues of the subject and/or inhibits metastasis of the cancer.
Some methods involve a further step of monitoring changes in the cancer responsive to the administering. Optionally, the monitoring comprises taking a sample of a body fluid, or performing a scan of an internal organ. Optionally, the monitoring comprises identifying a genetic variation in an ATM gene of the subject associated with cancer. Optionally, the methods involve an additional step of administering a chemotherapeutic agent other than the chloroquine compound to the subject. Optionally, the methods involve an additional step of determining presence of the cancer before the administering step.
In some methods, the subject is free of diseases of the immune system, infectious diseases, and neurological diseases.
In some methods, the patient is human and the dosage is 0.05 to 1 mg/kg per week.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Kaplan-Meier survival curve of C57/BL6 mice after exposure to 8 Gy total body irradiation (TBI). Half of the cohort received a dose of chloroquine (dashed line) by either IP injection (1.75 mg/kg or 3.5 mg/kg) or in their drinking water (3.5 mg/kg or 7 mg/kg) the day before the TBI. The one mouse which died in the chloroquine-treated group received 1.75 mg/kg by i.p. injection.

FIG. 2 shows that chloroquine treatment enhances survival after TBI by enhancing recovery of hematopoietic progenitor cells. Five mice received 3.5 mg/kg chloroquine (C) by i.p. injection 24 and 4 hours prior to TBI (bars with diagonal stripes). Five mice received no chloroquine (stippled bars). Fourteen days after irradiation, the cellularity (open bars) of hematopoietic tissues (spleen, thymus, bone marrow) was assessed by a blinded observer on a scale of 0-3 with 3 being normal cellularity. The bars represent the average cellularity of the tissues from the 5 mice in each group.

FIG. 3 shows a Kaplan-Meier survival curve of AT mice after exposure to 8 Gy TBI. Half of the cohort received a dose of 3.5 mg/kg chloroquine (CHL; dashed line) by i.p. injection 24 and 4 hours prior to the TBI.

FIG. 4 demonstrates that chloroquine treatment prevents the development of tumors in Eμ-myc mice. After weaning, a cohort of transgenic mice expressing the c-myc oncogene were started on chloroquine (CHL) at 7.0 mg/kg in the drinking water ((+), solid line). Within 100 days, all of the mice with no drug in the water had died of leukemia, while none of the cohort of mice on drug had succumbed. The latter group of mice was then divided into two groups (timing of this event depicted by heavy arrow), one group of which was taken off of chloroquine ((−), dashed line) and the other group of which was started on i.p. injections of 3.5 mg/kg of chloroquine once a week. Within a month, all of the mice taken off of chloroquine had developed malignancies and all of the mice on the weekly i.p. injections remained tumor-free for months.

FIG. 5 illustrates that chloroquine treatment reduces the development of tumors in mice injected with the potent chemical carcinogen, 3-methylcholanthrene (3-MC). Chloroquine (CHL, 3.5 mg/kg) was given by i.p. injection 24 and 4 hours prior to 3-MC injection in 30 mice and 30 mice received the carcinogen with no chloroquine pretreatment. The percentage of animals remaining tumor-free is plotted. Statistical significance, log rank test P<0.0001.

FIG. 6 demonstrates that chloroquine treatment reduces the development of tumors in mice exposed to ionizing radiation in a protocol that induces thymic lymphomas. Chloroquine (CHL, 3.5 mg/kg) was given by i.p. injection 24 and 4 hours prior to irradiation with 1.75 Gy in four successive weeks and animals were subsequently observed for the development of tumors. Statistical significance, log rank test P=0.0012.

FIG. 7 shows tumor incidence in wildtype mice receiving either placebo or CHQ before 3-MC injection. CHQ markedly protects from tumor development.

FIG. 8 shows tumor incidence in ATM-null mice receiving either placebo or CHQ before 3 MC injection. CHQ does not protect from tumor development.

FIG. 9 shows tumor incidence in p53-null mice receiving either placebo or CHQ before 3 MC injection. CHQ does not protect from tumor development.

FIG. 10 shows protection against coat color loss in mice treated with chloroquine or hydroxychloroquine.

DEFINITIONS

The term “animal subject” or “subject” includes humans as well as other mammals. Nonhuman mammals can include veterinary subjects as well as animal models in which a disorder has been specifically induced to simulate a human disease. For example, a transgenic mouse model of stroke is described in Stroke 27:1124-1129 (1996). Severe combined immunodeficient mice grafted with human skin are useful as models of human skin cancers, including melanomas.
A subject is at risk of a disorder, if the subject presently lacks clinical symptoms of the disorder, but has a statistically significantly higher probability of acquiring the disorder than individuals in the general population. Such risk can be shown by abnormal levels of a marker (e.g., a blood protein) or other sign associated with the disorder. The risk can also arise through exposure to a condition (e.g., radiation) associated with a statistically significant increased risk of the disorder. The risk can also arise through possession of a genetic variation associated with the disorder, or first degree relatives having the disorder. Levels of a marker are considered abnormal if outside the mean plus at least one and preferably at least 2 standard deviations of the level of marker in a population of individuals not known to be suffering from a disorder. Likewise a condition is associated with statistically significant increased risk of the disorder if the number of individuals in a population of individual exposed to the condition acquiring the disorder is greater than the number of individuals in a population of individuals not exposed to the condition acquiring the disorder to a statistically significant extent.
Statistical significance is measure at the level of p≦0.05.
A symptom of a disorder means a phenomenon experienced by an individual having the disorder indicating a departure from normal function, sensation or appearance.
A sign of a disorder is any bodily manifestation that serves to indicate presence or risk of a disorder.
The term “treating” includes achieving a therapeutic benefit and/or a prophylactic benefit.

DETAILED DESCRIPTION OF THE INVENTION

I. General

The present invention provides methods, compositions, and kits for the prevention, prophylaxis and/or treatment of several disorders. These disorders include DNA damage related disorders, cancers, oxidative stress and its many manifestations, stroke, ischemia, and atherosclerosis. Although there is some overlap between these disorders (for example, atherosclerosis is a common cause of ischemia and ischemia often gives rise to stroke, the different disorders are not coextensive. For example, atherosclerosis can cause problems by aneurysm as well as ischemia. Chloroquine compounds are useful for prevention, prophylaxis and/or treatment of these disorders.

II. Chloroquine Compounds

The term “chloroquine compounds” as used herein means chloroquine-like compounds, chloroquine and enantiomers, analogs, derivatives, metabolites, pharmaceutically acceptable salts, and mixtures thereof. Examples of chloroquine compounds include, but are not limited to, chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate, hydroxychloroquine sulphate, and enantiomers, analogs, derivatives, metabolites, pharmaceutically acceptable salts, and mixtures thereof. The term “chloroquine-like compounds” as used herein means compounds that mimic chloroquine's biological and/or chemical properties.
In a specific embodiment, the invention is practiced with chloroquine. The chemical structure of chloroquine, N⁴-(7-Chloro-4-quinolinyl)-N¹,N¹-diethyl-1,4-pentanediamine or 7-chloro-4-(4-diethylamino-1-methylbutylamino) quinoline, is as follows:
Chloroquine (The Merck Index, p. 2220, 1996) is a synthetically manufactured drug containing a quinoline nucleus. Suitable synthesis techniques for chloroquine are well known in the art. For example see U.S. Pat. No. 2,233,970.
As mentioned above, the chloroquine compounds useful herein include chloroquine analogs and derivatives. A number of chloroquine analogs and derivatives are well known. For example, suitable compounds and methods for synthesizing the same are described in U.S. Pat. Nos. 6,417,177; 6,127,111; 5,639,737; 5,624,938; 5,736,557; 5,596,002; 5,948,791; 5,510,356; 2,653,940; 2,233,970; 5,668,149; 5,639,761; 4,431,807; and 4,421,920.
Examples of suitable chloroquine compounds include chloroquine phosphate; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline (hydroxychloroquine); 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; hydroxychloroquine phosphate; 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine); 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 8-[(4-aminopentyl)amino]-6-methoxydihydrochloride quinoline; 1-acetyl-1,2,3,4-tetrahydroquinoline; 8-[4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride; 1-butyryl-1,2,3,4-tetrahydroquinoline; 7-chloro-2-(o-chlorostyryl)-4-[4-diethylamino-1-methylbutyl]aminoquinoline phosphate; 3-chloro-4-(4-hydroxy-.alpha.,.alpha.′-bis(2-methyl-1-pyrrolidinyl)-2,5-xyl idinoquinoline, 4-[(4-diethylamino)-1-methylbutyl)amino]-6-methoxyquinoline; 3,4-dihydro-1 (2H)-quinolinecarboxyaldehyde; 1,1′-pentamethylenediquinoleinium diiodide; and 8-quinolinol sulfate, enantiomers thereof, as well as suitable pharmaceutical salts thereof.
Additional suitable chloroquine derivatives include aminoquinoline derivatives and their pharmaceutically acceptable salts such as those described in U.S. Pat. Nos. 5,948,791 and 5,596,002. Suitable examples include (S)—N₂-(7-Chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine; (R)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine; N₁-(7-chloro-quinolin-4-yl)-2,N₂,N₂-trimethyl-propane-1,2-diamine; N₃-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,3-diamine; (RS)-(7-chloro-quinolin-4-yl)-(1-methyl-piperidin-3-yl)-amine; (RS)-(7-chloro-quinolin-4-yl)-(1-methyl-pyrrolidin-3-yl)-amine; (RS)—N₂-(7-Chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine; (RS)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine; (S)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine; (R)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine; (RS)-7-chloro-quinolin-4-yl)-(1-methyl-2-pyrrolidin-1-yl-ethyl)-amine; N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-ethane-1,2-diamine; N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-ethane-1,2-diamine; N₃-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,3-diamine; (R)—N₁-(7-chloro-quinolin-4-yl)-N₂,N₂-dimethyl-propane-1,2-diamine; (S)—N₁-(7-chloro-quinoline-4-yl)-N₂,N₂-dimethyl-propane-1,2-diamine; (RS)-(7-chloro-quinolin-4-yl)-(1-methyl-pyrrolidin-2-yl-methyl)-amine; N₁-(7-Chloro-quinolin-4-yl)-N₂-(3-chloro-benzyl)-2-methyl-propane-1,2-diamine; N₁-(7-chloro-quinolin-4-yl)-N₂-(benzyl)-2-methyl-propane-1,2-diamine; N₁-(7-chloro-quinolin-4-yl)-N₂-(2-hydroxy-3-methoxy-benzyl)-2-methyl-propane-1,2-diamine; N₁-(7-chloro-quinolin-4-yl)-N₂-(2-hydroxy-5-methoxy-benzyl)-2-methyl-propane-1,2-diamine; and N₁-(7-chloro-quinolin-4-yl)-N₂-(4-hydroxy-3-methoxy-benzyl)-2-methyl-propane-1,2-diamine; (1S,2S)—N₁-(7-chloro-quinolin-4-yl)-N₂-(benzyl)-cyclohexane-1,2-diamine; (1S,2S)—N₁-(7-chloro-quinolin-4-yl)-N₂-(4-chlorobenzyl)-cyclohexane-1,2-diamine; (1S,2S)—N₁-(7-chloro-quinolin-4-yl)-N₂-(4-dimethylamino-benzyl)-cyclohexane-1,2-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-dimethylamino-benzyl)-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(benzyl)-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(3-chloro-benzyl)-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(2-hydroxy-4-methoxy-benzyl)-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(3,5-dimethoxy-benzyl)-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-methylsulphanyl-benzyl)-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-diethylamino-benzyl)-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(biphenyl-4-yl)methyl-cyclohexane-1,4-diamine; trans-N₁-(7-chloro-quinolin-4-yl)-N₄-[2-(3,5-dimethoxy-phenyl)-ethyl]-cyclohexane-1,4-diamine; cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-methoxy-benzyl)-cyclohexane-1,4-diamine; trans-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-dimethylamino-benzyl)-cyclohexane-1,4-diamine; and trans-N₁-(7-chloro-quinolin-4-yl)-N₄-(2,6-difluoro-benzyl)-cyclohexane-1,4-diamine.
Chloroquine compounds such as chloroquine may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and/or optical isomerism.
The invention covers any tautomeric, conformational isomeric, optical isomeric and/or geometric isomeric forms of the chloroquine compounds, as well as mixtures of these various different forms.
Chloroquine and hydroxychloroquine are generally racemic mixtures of (−)- and (+)-enantiomers. The (−)-enantiomers are also known as (R)-enantiomers (physical rotation) and 1-enantiomers (optical rotation). The (+)-enantiomers are also known as (S)-enantiomers (physical rotation) and r-enantiomers (optical rotation). The metabolism of the (+)- and the (−)-enantiomers of chloroquine are described in Augustijins and Verbeke (1993) Clin. Pharmacokin. 24(3):259-69; Augustijins, et al. (1999) Eur. J. Drug Metabol. Pharmacokin. 24(1):105-8; DuCharme and Farinotti (1996) Clin. Pharmacokin. 31(4):257-74; Ducharme, et al. (1995) Br. J. Clin. Pharmacol. 40(2):127-33. Preferably, the (−)-enantiomer of chloroquine is used. The enantiomers of chloroquine and hydroxychloroquine can be prepared by procedures known to the art.
The chloroquine compounds may metabolize to produce active metabolites. The used of active metabolites is also within the scope of the present invention.
Although an understanding of mechanism is not required for practice of the invention, it is believed that one mechanism of action of chloroquine compounds is to enhance the activity of Ataxia-Telangiectasia Mutated (ATM) kinase. The agonistic properties of chloroquine on ATM kinase have been demonstrated (see US Pub. No. 20030077661 entitled “ATM Kinase Compositions and Methods,” filed Nov. 27, 2003, which is incorporated by reference herein in its entirety). Hence, choroquine-like compounds include compounds that are agonists of ATM kinase. Agonists of ATM kinase include compounds that promote the dissociation of ATM into active monomers and/or compounds that promote phosphorylation of a serine corresponding to the residue 1981 of ATM kinase of SEQ ID NO: 1. Chloroquine compounds may also be effective via one or other mechanisms that do not involve interaction with ATM.

III. Use of Chloroquine Compounds

1. General

The invention provides methods of prophylaxis or therapeutic treatment of an animal subject, including a human. The methods generally involve the administration of effective amounts of chloroquine compounds including chloroquine like compounds for the treatment of one or more the diseases or disorders described in more detail below. The effects of the chloroquine compounds used in the present methods are preferably systemic, but can be local, or topical depending on the mode of administration. Although an understanding of mechanism is not required for practice of the invention, it is believed that chloroquine compounds act in part by protecting normal cells from radiation or radicals and by inhibiting the cellular damage caused by the radiation or radicals to normal cells and enhancing the repair process of the normal cells.

2. DNA-Damage Related Disorders

The methods are generally applicable to “DNA damage related disorders.” This term includes cancer, aging, and other disorders caused by damage to DNA due to exposure to carcinogens, toxins, free radicals, like oxygen radicals, or DNA damaging radiations like ionizing radiation and UV radiation. Cancer and other disorders included in this definition are discussed individually below. Although an understanding of mechanism is not required for practice of the invention, it is believed that administration of chloroquine compounds prevents DNA damage, inhibits the effects of DNA damage, and/or stimulates cellular response to DNA damage, and that the chloroquine compound acts at least in part through agonizing ATM and p53.
The p53 gene spans 20 kbp in humans and has 11 exons, 10 of which are protein coding (see Tominaga et al., 1992, Critical Reviews in Oncogenesis 3:257 282, incorporated herein by reference). The gene produces a 53 kilodalton phosphoprotein that regulates DNA replication. The protein acts to halt replication at the G1/S boundary in the cell cycle and is believed to act as a “molecular policeman,” shutting down replication when the DNA is damaged or blocking the reproduction of DNA viruses (Kastan et al. Cancer Res 51:6304-6311 (1991); Kuerbitz et al., Proc Natl Acad Sci USA. 89:7491-7495 (1992); Kastan, Cell 71:587-597 (1992), Hartwell et al., Science 266:1821-1828 (1994)).
In one embodiment, chloroquine compounds are used as prophylactics to inhibit side effects of frequent exposure to X-rays in individuals, such as athletes. This method is useful for other subject populations that are frequently exposed to DNA damaging radiations, such as X-ray technicians, police officers, astronauts, and the like. It is known that exposure to X-rays causes DNA damage. Administration of chloroquine compounds is contemplated to inhibit the side-effects of frequent exposure to DNA damaging radiations, including inhibit the damage to cells due to damage to DNA.
The prophylactic benefits of chloroquine compounds can be obtained by administering in advance of exposure to the DNA damaging agent to provide the enhancing effect in one embodiment. The amount of time prior to the exposure to the DNA damaging agent that the chloroquine compound is administered can vary from days, hours, to minutes. Also, the chloroquine compounds can be administered during exposure to the DNA damaging agent or after such exposure. Chloroquine compounds can also be administered after exposure to the DNA-damaging agent, in which case, the exposure is preferably within one day, a week or a month. In one embodiment, the effective amount of a chloroquine compound is an amount which reduces DNA damage, reduces DNA mutation or increases survival of cells exposed to a DNA damaging agent when compared to cells exposed to the same DNA damaging agent and not receiving a chloroquine compound.

3. Cancer

The chloroquine compounds are useful in the prophylaxis, including prevention, and therapeutic treatment of cancers caused by toxins, carcinogens, DNA damaging radiations, and/or genetic mutations, among other causes. For example, chloroquine compounds are useful in the treatment of cancers caused by exposure to toxins and carcinogens like aromatic hydrocarbons, cigarette smoke, acetyl amino fluorine, and MTBE. Also, chloroquine compounds are useful in prophylaxis or therapeutic treatment of cancers caused by DNA damaging radiations like UV and ionizing radiation. The ionizing radiation includes both natural and therapeutic radiation exposures. Examples of ionizing radiations are X-rays for diagnostics and radiation therapy used for tumors. For example, chloroquine compounds can be used in the prophylaxis or treatment of one or more of the following cancers: melanomas, prostate cancer, breast cancer, colon cancer, lung cancer, non-Hodgkin's lymphoma, retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer.
Chloroquine compounds can be used in prophylaxis of subjects at risk of cancer. Cancer is the uncontrolled, proliferation (growth and multiplication) of a single type of cell, occurring in any tissue of the body. Malignant cancers are those that invade surrounding tissues and metastasize (spread) to other body sites via the blood and lymphatic circulations. Metastasized cancers usually remain the same type of cell as the initial site of cancer development; for example, if breast cancer metastasizes to a lung, the cancer in the lung consists of breast cells. Benign cancers do not invade other tissues or spread, have a slower growth rate than malignant cancers, and in most cases are not fatal. The methods are particularly useful for prophylaxis or therapeutic treatment of malignant cancers.
Examples of subjects at risk of cancer include subjects having a primary cancer who are at risk of a second cancer, subjects having been exposed to a carcinogen (e.g., asbestos, benzene) or radiation, including X-rays, radioactivity, nuclear bombs, dirty bombs, excessive exposure to sun rays, or ultraviolet irradiation, subjects have a genetic variation associated with cancer, subjects having first degree relatives with a cancer known to be hereditary, subjects showing a biochemical marker associated with cancer (e.g., carcinoembryonic antigen), subjects having an abnormal cellular morphology (precancerous tissue) associated with development of cancer (e.g., melanocytic naevi), subjects infected with an oncogenic virus (e.g., certain forms of HPV, HBV, KSHV and EBV) or helicobacter pylori, and subjects who smoke or engage in excessive drinking. An exemplary class of subjects at risk of cancer are subjects who have previously had a melanoma and are risk of developing a further melanoma. A further exemplary class of subjects at risk of metastatic melanoma are subjects having a nonmetastatic melanoma and at least two family members with a history of melanoma. Another exemplary class of subjects at risk of melanoma are those having congenital melanocytic naevi. They are brown or black moles which are present at birth, or which develop in the first month or so of life.
Genetic risk of cancer can be associated with either homozygous or heterozygous variations in genes. The variations can be present in the germline (and other cells of a patient) or may appear only in precancerous or cancerous tissue (i.e., somatic variations). Generally, the most commonly occurring allele in a population is referred to as the wildtype allele, and other less common alleles are referred to as variant alleles. Some variant alleles in some genes are associated with cancer. Variation can result in cancer either by increased or altered function (e.g., oncogenes) or reduced or lack of function (e.g., tumor suppressor genes). Variant forms associated with cancer can be recognized by comparing alleles in populations with and without a cancer (usually the individuals in the population with cancer have the same type for purposes of analysis). Alleles occurring significantly more frequently in the population having cancer are associated with cancer. A causative relationship can be conferred by transforming the allele into cells or transgenic animals or knocking out an endogenous allele and determining whether the allele causes cancer in the transformed cell or animal.
Many variations in many genes have already been characterized as being associated with cancer. Examples of genes in which variation causes increased risk of cancer include RB1 retinoblastoma, the WT1 gene for Wilms' tumor, the NF1 and NF2 genes for neuroblastomatosis, types 1 and 2, the VHL gene for renal cancer tumors association with Von Hippel-Lindau disease, the APC gene for adenomatous polyposis coli, and the BRCAI and II genes for breast and ovarian cancer, PTEN and HRAS-1 both associated with breast cancer (see, e.g., Mulligan et al., Nature Genet. 6, 70-74 (1994); Knudson, Cancer Res. 63, 1888-1891 (1989); Miki et al., Sciences 266, 66-71 (1994); Bronner, Nature 368, 258-261 (1994), Leach, Cell 75, 1215, Lieberman, Amer. J. Gastro. 87, 1085-1093 (1992), Easton, Breast Cancer Research 1, 14-17 (1999), Peto et al., J. Natl. Cancer Inst 91, 943-949 (1999), Kronitiris, N. Engl. J. Med. 329, 517-523 (1993); Tirkkonen et al. Cancer Research 57(7), 1222-1227 (1997)).
ATM and/or p53 are further examples of genes having variations associated with cancer (see, e.g., Garber et al., Cancer Research 51, 6094-6097 (1991); Olsen et al., J. Nat. Cancer Inst. 93, 121-127 (2001)). As noted these genes are also thought at least in part to effect the prophylactic and therapeutic benefits of chloroquine compounds. Optionally, subjects can be screened for variations in these genes before commencing treatment. Subjects having wildtype forms of ATM or p53, or heterozygous mutations or homozygous mutations leaving residual activity of p53 or ATM are preferred for treatment with chloroquine compounds. Subjects having mutations that effectively eliminate ATM or p53 function are less preferred for treatment with chloroquine compounds.
For example, retinoblastoma is a cancer affecting young children in which tumors develop in the retina of one (unilateral, 75% of cases) or both (bilateral, 25% of cases) eyes. In all cases, retinoblastoma seems to be caused by a mutation in the RB1 gene located on chromosome 13, which is responsible for controlling retinal cell division. Although retinoblastoma may occur at any age, it most often occurs in younger children, with 80% of cases diagnosed before the age of 5 years. Retinoblastoma presents in both hereditary (40%) and nonhereditary (60%) forms. The hereditary form may manifest as unilateral or bilateral disease. Most unilateral disease is not hereditary, whereas all bilateral disease is hereditary. For persons carrying the hereditary form of retinoblastoma, the disease demonstrates with high penetrance (about 90% will manifest disease). Patients with the hereditary type of retinoblastoma have a markedly increased frequency of additional malignancies. The cumulative incidence is about 26+/−10% in nonirradiated and 58+/−10% in irradiated patients by 50 years after diagnosis of retinoblastoma. Most of the excess cancers were osteosarcomas, soft tissue sarcomas, or melanomas. The carcinogenic effect of radiation increased with dose. In irradiated patients, two thirds of the second cancers occur within irradiated tissue and one third outside the radiation field. Treatment of patients having a genetic variation of the RB1 gene can prevents or reduce the frequency of both retinoblastomas and/or the additional malignancies.
Methods for determining presence of genetic variations individuals are described in attorney docket 100/1046-20, serial no. [unassigned], “Improvements to Analysis Methods for Individual Genotyping”, filed Feb. 24, 2004, U.S. Ser. No. 10/768,788, “Apparatus and Methods for Analyzing and Characterizing Nucleic Acid Sequences”, filed Jan. 30, 2003., and U.S. Ser. No. 10/042,819, “Genetic Analysis Systems and Methods”, filed Jan. 7, 2002, and EP 0 730 663, each of which is incorporated by reference.
The presence of risk of cancer in a subject can be determined by biochemical analysis, cytological examination to detect precancerous or cancerous tissues (e.g., Pap smear), genetic analysis to detect genetic variation, family history of hereditary cancer, subject history of exposure to a carcinogen or radiation, or knowledge that the subject will be exposed to a carcinogen or radiation. If the risk of cancer is due to an event that has not yet happened (e.g., occupational exposure to radiation or a carcinogen, or exposure to radiation through treatment of an existing cancer), chloroquine treatment is preferably administered before the event creating the risk, and or during the event, and/or as soon as possible after the event. Administration can then be continued, preferably at regular intervals, thereafter. If the risk of cancer is due to an event that has already occurred (e.g., past exposure to a carcinogen or radiation or genetic mutation), chloroquine treatment is preferably administered as soon as possible after the event, and continued at regular intervals thereafter.
In some methods, the subject is monitored to determine response of the subject to administration of the chloroquine compound. If the subject is at risk of cancer but does not have cancer when the administration is begun, the subject is monitored to determine whether a cancer develops. The monitoring can be performed by similar techniques to those used in assessing risk of cancer. In addition, cancers or lack thereof can be monitored by performing scans (e.g., X-rays, CAT scans, MRI) of internal organs or visuals scans of the skin. If the subject does already have a cancer when the administration is begun, the subject is monitored to determine changes in the cancer responsive to the administration of the chloroquine compound. Preferably, the cancer does not show further increase in size, shrinks in size or disappears. Alternatively, administration can inhibit or stop further growth of the cancer. Monitoring of an existing cancer is performed by visual observation, scanning (e.g., MRI) or biopsy followed by cytological or biochemical analysis.
Prophylactic administration of a chloroquine compound can be accompanied by administration of another chomopreventive agent (e.g., tamoxifen (Nolvadex), Sulindac (Clinoril), or aspirin) or regime designed to decrease the risk of the subject developing cancer. Likewise, therapeutic administration of a chloroquine compound can be accompanied by administration of a chemotherapeutic agent (e.g., busulfan, cisplatin, cyclophosphamide, methotrexate, daunorubicin, doxorubicin, melphalan, cladribine, vincristine, vinblastine, and chlorambucil) or radiation or other regime designed to treat the tumor.
The chloroquine compounds can be used to prevent secondary cancers, i.e., cancers that are caused by radiation therapy and/or chemotherapy used to treat the primary cancer. For example, the chloroquine compounds are used to prevent the occurrence of breast cancer in subjects receiving radiation therapy for Hodgkin's lymphoma (or other cancers). Also, in these subjects the chloroquine compounds can be used to inhibit the cellular damage caused by the radiation therapy to normal cells and enhance the repair process of the normal cells. The chloroquine compounds are also suitable for prevention of the reoccurrence of cancers in subjects who have had prior incidences of cancer.
The chloroquine compounds can be administered prior to, during, or after treatment with radiation. In this embodiment, the beneficial effect of the chloroquine compounds is contemplated to be not solely limited to a beneficial effect on pathological skin conditions like skin carcinomas and dermatoses. The use of chloroquine compounds in combination with radiation therapy is contemplated to protect the normal cells and inhibit the cellular damage caused by the radiation therapy to normal cells and enhance the repair process of the normal cells.
The chloroquine compounds can be used in immunosuppressed subjects, like transplant subjects, who have an increased risk of cancer. In immunosuppressed subjects, the chloroquine compounds can be used to prevent cancers. The chloroquine compounds can be used to prevent Epstein Barr virus induced lymphoproliferative syndrome.

4. Oxidative Stress

Normal metabolism produces free radical molecules. Free radicals are atoms or molecules which have at least one unpaired electron in the outer orbital. These radicals are the same as generated by external radiation and include hydrogen peroxide and superoxide. Mitochondria are the main source of oxygen free radicals under normal conditions. Free radicals can react with any biological molecule (proteins, lipids, sugars, DNA) altering its structure and often also its function. Therefore living organisms are provided with a rich system of antioxidant defenses whose main purpose is to prevent the free radicals attack to other molecules. Antioxidants can also be supplied in the form of nutrition (e.g., phytochemicals in fruit and vegetables). Oxidative stress arises from an imbalance of these radicals and antioxidants as a result of which unneutralized radicals damage DNA and other macromolecules.
Oxidative stress contributes to a number of disorders including cancer, coronary disease, atherosclerosis, stroke, cataracts, macular degeneration, depression, neurodegenerative disease, and premature aging. Oxidative stress occurs to some extent in everyone. However, levels of oxidative stress that substantially exceed the mean plus a standard deviation in a population of disease free individuals can be recognized as a cause of present or future symptoms, including cancer, coronary disease, atherosclerosis, stroke, cataracts, macular degeneration, depression, neurodegenerative disease, and premature aging.
The level of oxidative stress can be quantified by measuring the level of a series of prostaglandin-like compounds termed isoprostanes. These compounds are formed by perodixation of arachidonic acid. The level of these compounds in the plasma or urine is a measure of oxidative stress in a subject (see Morrow, Am. J. Resp. Crit. Care Med. 166, S25-S30 (2002)). Subjects having an abnormal level of oxidative stress can be recognized by a level of at least one of isoprostane that exceeds the mean plus a standard deviation, preferably, at least two standard deviations, and more preferably at least five standard deviations of the mean level in a population of individuals free of disease. Usually, a determination of the level of oxidative stress is performed before commencing administration of a chloroquine compound. Levels of oxidative stress can be measured at intervals thereafter and compared with a baseline measurement before beginning treatment. Preferably, the level decreases after beginning treatment, preferably toward a normal level, or at least the level does not further increase. The subject can also be monitored for symptoms of oxidative stress, such as macular degeneration, cataracts or premature aging, cancer, heart disease and improvement therein responsive to administration of the chloroquine compound.
The chloroquine compound can be administered in a regime that includes one or more other measures to combat oxidative stress. Such measures including changes in diet, for example, increased intake of fruit and vegetables, and supplementation of the diet with phyotochemicals or antioxidants, such as vitamin B12. Such measures also include increased exercise, and decreased occupational stress. Such measures also include administration of drugs with antioxidant activity such as methylprednisolone, 21-aminosteroids, 2-methylaminochromans, pyrrolopyrimidines and thiazolidinones. Although an understanding of mechanism is not required for practice of the invention, it is believed that administration of a chloroquine compound serves to stimulate the cellular response to DNA damage and promote the repair of the cells exposed to radicals generated by oxidative stress.

5. Ischemia-Reperfusion

Ischemia refers to a disorder caused by an imbalance between supply and demand of oxygen to tissue, usually caused by a reduction in blood flow to the tissue. Organs such as the heart and brain are most vulnerable to ischemia due to their high extraction of oxygen. Reperfusion is the process of restoring blood flow to the tissue. Ischemia and reperfusion result in different damage to the tissues deprived of oxygen. The reduction of blood flow decreases the production of high energy phosphates. The energy failure causes membrane depolarization and uncontrolled release of excitatory amino acids, such as glutamate, in the extracellular space (excitotoxicity). Glutamate acts on various types of receptors, e.g. NMDA and AMPA, eventually causing calcium overload of neuronal cells. Calcium activates proteolytic enzymes that begin to degrade both intracellular and extracellular structures, and other enzymes, i.e. phospholipase A2 and cyclooxigenase, which can produce free radicals. Neuronal nitric oxide synthase is also calcium dependent and produces nitric oxide, which is able to react with superoxide generating the highly reactive radical peroxynitrite. Secondary to ischemia proinflammatory genes are expressed and several inflammatory mediators are released, such as tumor necrosis factor, interleukin 1β. Adhesion molecules are also expressed and therefore neutrophils, monocytes and macrophages start to bind the endothelium causing microvascular occlusion and cross the vascular wall penetrating in the brain. These inflammatory cells can also produce free radicals.
Restarting blood flow after more than about ten minutes of ischemia is typically more damaging than the ischemia itself because the ischemia sets the stage for oxygen to generate free-radicals. Arachidonic acid conversion to eicosanoids can lead to production of the superoxide radical when oxygen is available. Lipid peroxidation chain reactions occur in the membranes of neurons as well as of astrocytes. Free radical damage to blood vessels is particularly severe. Ischemia results in large amounts of ATP being broken-down to xanthine. Reperfusion allows the endothelial enzyme xanthine oxidase to convert xanthine plus oxygen to superoxide and uric acid. Liberated iron and zinc ions further increase free radical damage. Superoxide increases the adhesion of leucocytes to vessel walls. Normally, nitric oxide can combine with superoxide to produce peroxynitrite in small amounts, reducing the adhesion. During ischemia, impaired nitric oxide production accompanies increased leukocyte adhesion. Reperfusion increases the amount of nitric oxide produced by the endothelial cells. During reperfusion, abnormally high amounts of superoxide converts almost all available nitric oxide to perxoynitrite, which is regarded as the agent causing most of the damage to brain capillary endothelial cells.
Neutrophils which have accumulated in blood vessels due to the ischemia can also release oxygen-rich free radicals with the availability of oxygen from reperfusion.
Administration of a chloroquine compound to a subject having or at risk of ischemia is effective in prophylaxis or treatment of tissues damage caused by ischemia and/or reperfusion, particularly tissue damage or death resulting from the generation of free radicals following reperfusion.
Subjects at risk of ischemia include those having previously had heart disease, those having elevated biochemical markers of the disease (e.g., protein. C), those identified as having blockage of blood vessels by angioplasty or MRI imaging, and those undergoing a surgical procedure requiring temporary obstruction of blood vessels. The presence or absence and the amount of myocardial damage resulting from prolonged ischemia can be assessed by a number of different means, including pathologic examination, measurement of myocardial proteins in the blood, ECG recordings (ST-T segment wave changes, Q waves), imaging modalities such as myocardial perfusion imaging, echocardiography, contrast ventriculography or positron emission tomography (see, e.g., Hanninen et al., Int. J. Bioelectromagnetism No. I Vol. 2 (2000); Alpert, J. Am. College. Cardiol. 36, 959-69 (2000)). Myocardial necrosis results in and can be recognized by the appearance in the blood of different proteins released into the circulation due to the damaged myocytes: myoglobin, cardiac troponins T and I, creatine kinase, and lactate dehydrogenase. The response of the subject to treatment with a chloroquine can be monitored by any of these tests. Preferably, the amount of pathological damage or level of a marker associated with the same shows a reduced increase, does not increase or even is reduced followed administration of a chloroquine compound relative to a placebo.
In some methods, a chloroquine compound is administered to a subject having or at risk of ischemia in combination with a second agent effective in prophylaxis of damage resulting from ischemia and/or reperfusion. Such agents include antibodies to adhesion molecules such as L-selectin, or CD18, tissue plasminogen activator (see EP-B 0 093 619), activase, alteplase, duteplase, silteplase, streptokinase, anistreplase, urokinase, heparin, warfarin and coumarin. Additional thrombolytic agents include saruplase and vampire bat plasminogen activator.

6. Stroke

Stroke is a sudden loss of brain function resulting from interference with the blood supply to the central nervous system. Acute stroke can be classified either as ischemic (80% of stroke cases), which can be further classified to extra-cranial embolism and intracranial thrombosis, or a hemorrhagic stroke (20% of stroke cases), which can be further classified to intracerebral hemorrhage and subarachnoid hemorrhage. Stroke is a leading cause of morbidity and mortality, particularly in the elderly. Its incidence and prevalence sharply increase with age: in the United States 72% of the subjects suffering a stroke are age 65 and older and up to 88% of stroke deaths occur in this population. Moreover stroke is an important cause of long-term disability: up to 40% of survivors are not expected to recover independence with self-care and 25% become unable to walk independently. Ischemic stroke accounts for 70 to 80% of all strokes and haemorregic stoke accounts for the remainder. Free radicals play an important role in the pathogenesis of stroke, especially superoxide interact with NO, leading to peroxynitrite formation. Although practice of the invention is not dependent on an understanding of mechanism, it is believed that administration of a chloroquine compound to a subject having or at risk of stroke effects prophylaxis or treatment by mitigating the effects of free radicals, and the response of cells to damage by the free radicals.
Subjects at risk of stroke can be determined by presence of one, and usually at least two of the following risk factors: high blood pressure, heart disease, high cholesterol levels, sleep apnea, previous occurrence of stroke, smoking, excessive alcohol consumption and excessive weight. Alternatively, transcranial doppler (TCD) testing uses sound waves to measure the speed with which blood flows through the large blood vessels within the head. The test can detect constriction (narrowing) of blood vessels as well as blood flow abnormalities related to cerebrovascular disease. Damage to tissue from stroke can be monitored by MRI and/or by cognitive testing. Monitoring of tissue damage, if any, can be performed following administration of treatment.
Administration of chloroquine can be accompanied by administration of other agents to treat stroke. These include the same agents discussed for treating ischemia and oxidative stress as noted above.

7. Atherosclerosis

Atherosclerosis is a process in which deposits of fatty substances, cholesterol, cellular waste products, calcium and other substances build up in the inner lining of an artery. This buildup is called plaque. It usually affects large and medium-sized arteries. Some hardening of arteries often occurs when people grow older. Plaques can grow large enough to significantly reduce the blood's flow through an artery. But most of the damage occurs when they become fragile and rupture. Plaques that rupture cause blood clots to form that can block blood flow or break off and travel to another part of the body. If either happens and blocks a blood vessel that feeds the heart, it causes a heart attack. If it blocks a blood vessel that feeds the brain, it causes a stroke. And if blood supply to the arms or legs is reduced, it can cause difficulty walking and eventually gangrene. Thus, atherosclerosis as well as being a disorder in itself, is a risk factor for ischemia and stroke. Insofar as atherosclerosis causes ischemia and/or stroke, it generates free radicals as these specific diseases and can also be subject to prophylaxis or treatment with a chloroquine compound
Risk factors of atherosclerosis are high blood pressure, high LDL, obesity, high cholesterol levels and smoking. Atherosclerosis can be detected by MRI or B-mode ultrasound methods (see Tang, Am J Cardiac Imaging 6:333-339 (1992)). Subjects can be screened for risk factors before commencing treatment. Tissue damage resulting from atherosclerosis and its sequelae can be monitored following treatment as for ischemia or stroke.
Administration of a chloroquine compound can be combined with other agents conventionally used in prophylaxis or treatment of atherosclerosis. These include antiplatelet agents, lipid lowering agents, bile acid sequestrants, fibrinates, HMG-CoA reductase inhibitors, nocotinic acid derivatives, and blood pressure lowering agents.

9. Optional Features

Optionally, the methods of the invention are not practiced on subjects who have presented with a skin carcinoma or epithelioma, like basal cell epithelioma and squamous within one year of beginning treatment with the chloroquine compound. Examples of cancers that are not localized skin carcinomas or epitheliomas include, but are not limited to, melanomas, lymphomas, prostate cancer, breast cancer, colon cancer, lung cancer, retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer. Optionally, the subject has never had a localized skin carcinoma or epithelioma. Optionally, the subject has not suffered from Burkitt's lymphoma, or skin pathologies caused by harmful radiation before administering the chloroquine compound. Optionally, the subject lacks factors predisposing the subject to Burkitt's lymphomas, such factors being one or more of malarial infection, EBV infection (as manifested by antibodies to capsid proteins in the serum detectable by ELISA) and HIV infection. Optionally, the methods are practiced on subjects that are free of diseases of the immune system, infectious diseases, neurological diseases, and multidrug resistance (i.e. resistance to multiple drugs for treatment of the same conditions, such as two anti-cancer drugs, or two antibiotics) before commencing administration of the chloroquine compound. Optionally, the methods are practiced on subjects free of psoriasis, malaria, protozoal infections, Alzheimer's disease, Parkinson's disease, lupus erythematosus, rheumatism, hypercalcemia, multiple sclerosis and migraine before administering the chloroquine compound.

IV. Therapeutic and Prophylactic Benefits

Chloroquine compounds are used as prophylactic agents. For prophylactic benefit, the chloroquine compound can be administered to a subject at risk of developing one of the above disorders but not presently showing symptoms of the disorder being treated. A prophylactic benefit is achieved when a disorder is delayed, reduced in severity or prevented from afflicting a subject. A prophylactic benefit can include a result in which the subject is inflicted with a milder form of the disorder than in the absence of treatment or the appearance of fewer or no symptoms of the disorder, or the absence of the disorder in the subject being treated.
Chloroquine compounds can be used for their therapeutic benefits in treating the disorders described above. A therapeutic benefit includes eradication or amelioration of the underlying disorder being treated. For example, in a cancer subject, therapeutic benefit includes eradication, or amelioration of the underlying cancer, or cessation or reduction in its rate of growth. A therapeutic benefit also includes the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For example, administration of a chloroquine compound to a subject suffering from cancer provides therapeutic benefit not only when the subject's tumor marker level is decreased, but also when an improvement is observed in the subject with respect to other complications that accompany the cancer like pain and psychiatric disorders. A therapeutic benefit also includes elimination or reduction of consequences of the underlying disorder, such as the generation of free radicals and the resulting damage to macromolecules and tissue in atherosclerosis, stroke, ischemia and oxidative stress. A therapeutic benefit can also result when administration of a chloroquine compound inhibits or prevents further deterioration in the patient's condition of an existing disorder.

V. Effective Amount

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the chloroquine compound and other optional active ingredients are present in an effective amount. The effective amounts include doses that partially or completely achieve the desired therapeutic, prophylactic, and/or biological effect. The actual amount effective for a particular application depends on the condition being treated and the route of administration. The effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating and/or gastrointestinal concentrations that have been found to be effective in animals.
In some methods, the effective amount includes the dose ranges, modes of administration, formulations, and so forth, that have been recommended or approved by any of the various regulatory or advisory organizations in the medical or pharmaceutical arts (e.g., FDA, AMA) or by the manufacturer or supplier. Effective amounts of chloroquine can be found, for example, in the Physicians Desk Reference.
In some methods, the daily dosage range of chloroquine, can vary between about 0.1 mg/kg to about 2 gm/kg body weight. The daily dose of a chloroquine compound can be less than about 2 gm/kg, less than about 1.5 gm/kg, or less than about 1 gm/kg. In some methods, the daily dose of a chloroquine compound is more than about 0.5 mg/kg, more than about 500 mg/kg, or more than about 1 gm/kg. Some daily dosage ranges of a chloroquine compound are about 0.5 mg/lkg to about 50 mg/kg or about 1.0 mg/kg to about 10 mg/kg body weight. Some daily doses of chloroquine diphosphate are about 3.5 mg/kg and 7.0 mg/kg.
The dosage can vary depending on the subject being treated. For example, a preferred dosage in mice is 3.5 mg/kg once or twice a day. The equivalent dosages in monkeys and humans are shown in the Table 1.

TABLE 1

			Man (60 kg)
Mouse (20 g)	Monkey (3.0 kg)	Man (60 kg)	CHG Equivalent

3.5 mg/kg	0.875 mg/kg	0.292 mg/kg	17.5 mg CHQ
7.0 mg/kg	1.75 mg/kg	0.583 mg/kg	35.0 mg CHQ

Preferred dosages ranges in human are from 0.05-1 mg/kg, more preferably 0.1 to 0.8 mg/kg, more preferably 0.2-0.6 mg/kg or 0.2 to 0.4 mg/kg. The dosage can be administered daily, weekly, monthly or bimonthly (every two months). In patients whose risk to cancer is occasioned by a distinct event (e.g., exposure to carcinogen or radiation), the dosage is preferably administered daily before, during and/or immediately following the event, for an initial period of at least 1 day, 3 days, a week or a month, and at larger intervals thereafter (e.g., weekly). For example, if the risk of exposure is known in advance, an exemplary regime includes administering the chloroquine compound on the day before, the day of exposure and the day after exposure. Another exemplary regime comprises administering the chloroquine compound 24 hours before the exposure to a known risk of cancer. If the risk of exposure is not known in advance, an exemplary regime includes administering the chloroquine compound at least one the day of exposure and the day following exposure. For patients subject to a chronic risk (e.g., through genetic variation), the dosage is preferably administered weekly, monthly or bimonthly for an indefinite period. The dosage range can be lower e.g., 0.05-0.2 mg/kg per day or per week of chloroquine if a purified (−) enantiomer is used. If hydroxychloroquine is used the dosage range is usually higher than if chloroquine is used.
In some methods, the effective amount of chloroquine is administered at regular intervals, such as every other week, once a week, more than once a week, or once a day. The dose of chloroquine can be administered once or more than once a day. In some methods, the effective amount of a chloroquine compound is an amount that produces the intended beneficial effects but does not produce the side-effects associated with chloroquine compounds, like retinoblastoma.

VI. Kits

The invention provides a kit comprising a chloroquine compound packaged in association with instructions teaching a method of using the compound according to one or more of the above-described methods. The kit can contain the chloroquine compound packaged in unit dosage form.

VII. Routes of Administration and Formulation

The compounds useful in the present invention, or pharmaceutically acceptable salts thereof, can be delivered to the subject using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections.
The chloroquine compounds can be administered topically or systemically. Systemic administration is preferred. In some methods, topical administration also has a systemic effect.
The term “pharmaceutically acceptable salt” means those salts which retain the biological effectiveness and properties of the compounds used in the present invention, and which are not biologically or otherwise undesirable. Such salts include salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid. In addition, if the compounds used in the present invention contain a carboxy group or other acidic group, it can be converted into a pharmaceutically acceptable addition salt with inorganic or organic bases. Examples of suitable bases include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine and triethanolamine.
If necessary, the compounds and useful herein can be administered in combination with other therapeutic agents or regimes as discussed. The choice of therapeutic agents that can be co-administered with the compounds of the invention depends, in part, on the condition being treated.
Agents used in accordance with the methods of the invention can be conveniently administered in a pharmaceutical composition containing the active compound in combination with a suitable carrier. Such pharmaceutical compositions can be prepared by methods and contain carriers which are well-known in the art. A generally recognized compendium of such methods and ingredients is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippingcott Williams and Wilkins: Philadelphia, Pa., 2000. A pharmaceutically-acceptable carrier, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, is involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
Examples of materials which can serve as pharmaceutically-acceptable carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; lycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Agents of use in the invention can be administered parenterally (for example, by intravenous, intraperitoneal, subcutaneous or intramuscular injection), topically (including buccal and sublingual), orally, intranasally, intravaginally, or rectally, with oral administration being particularly preferred.
For oral therapeutic administration, the composition can be combined with one or more carriers and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, foods and the like. Also, for oral consumption the active ingredient can be dissolved or suspended in water or other edible oral solutions. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations can, of course, be varied and can conveniently be between about 0.1 to about 100% of the weight of a given unit dosage form. The amount of active agent in such therapeutically useful compositions is such that an effective dosage level is obtained.
The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. The above listing is merely representative and one skilled in the art could envision other binders, excipients, sweetening agents and the like. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac or sugar and the like.
For administration orally, the compounds can be formulated as a sustained release preparation. Numerous techniques for formulating sustained release preparations are described in the following references—U.S. Pat. Nos. 4,891,223; 6,004,582; 5,397,574; 5,419,917; 5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263; 6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379; 5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362; 5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004; 5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177; and WO 98/47491. These references are hereby incorporated herein by reference in their entireties. In a preferred embodiment, the sustained release formulation utilized has an enteric coating.
For administration by inhalation, the active compound(s) can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
A syrup or elixir can contain the active agent, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active components can be incorporated into sustained-release preparations and devices including, but not limited to, those relying on osmotic pressures to obtain a desired release profile. Once daily formulations for each of the active components are specifically included.
The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection or a transdermal patch. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The selected dosage level depends on a variety of factors including the activity of the particular compound of the present invention employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts.
The invention is described in greater detail by the following non-limiting examples.

EXAMPLES

Example 1

Radioprotection Assay

HeLa cells were treated with 2 μg/ml of chloroquine for one hour, washed for one hour, and irradiated at 2 or 6 Gy. Subsequently, 1000 cells were plated and assessed for colony formation. Table 2 shows that exposure to chloroquine prior to irradiation increased cell survival by 30%.

TABLE 2

Treatment	Average Number of Colonies*	Std. Dev.

2 Gy	444	19.5
Chloroquine + 2 Gy	580	21.2
6 Gy	94.6	10.6
Chloroquine + 6 Gy	129	8.6

*Averages were from five individual samples.

To test the possibility that chloroquine activation of ATM may cause radioprotection, C57/BL6 mice were exposed to 8 Gy IR, a dose which kills approximately 80% of the mice at around two weeks. Death appears to result from hematopoietic toxicities. The day before total body irradiation (TBI), mice were either given an i.p. injection of chloroquine or chloroquine was added to the drinking water (5 mice—i.p. 1.75 mg/kg chloroquine; 5 mice—i.p. 3.5 mg/kg chloroquine; 5 mice—1.75 mg/kg chloroquine in drinking water; 5 mice—3.5 mg/kg chloroquine in drinking water). FIG. 1 shows a Kaplan-Meier survival curve indicating that a dose of chloroquine prior to the TBI provided significant protection from death. Significant protection was not observed in ATM deficient (homozygous) transgenic mice. The experiment was reproduced numerous times and analyses of tissues indicated that the protective effect was due to enhanced recovery of hematopoietic cells (bone marrow, spleen, thymus) following irradiation (FIG. 2). Injection of chloroquine prior to the TBI had no effect on the survival of mice lacking ATM genes (FIG. 3), thus indicating that radioprotection may be dependent on ATM.
Treatment with chloroquine or hydroxychloroquine also provided significant protection against loss of coat color in surviving mice. FIG. 10 shows three pairs of mice subject to 8 Gy total body irradiation. The two control mice on the left of the figure show significant loss of coat color. The pair in the middle which were treated with chloroquine before exposure to total body irradiation show no significant loss of coat color. The pair on the right treated with hydroxyquinolone show an intermediate extent of protection.

Example 2

Cancer Prevention

Transgenic mice expressing the c-myc oncogene under the control of the immunoglobulin enhancer (i.e., Eμ-myc mice) develop B-cell lymphomas and leukemias with relatively short latencies. Chloroquine was added to the drinking water of a cohort of Eμ-myc mice and the mice were observed for the development of B-cell malignancies. FIG. 4 demonstrates that 100% of the control transgenic mice developed malignancies within 100 days of birth while 0% of the transgenic mice on chloroquine developed tumors. After ˜120 days, half of the cohort of chloroquine-treated mice were taken off of chloroquine and the other half were switched to receiving a dose of chloroquine by i.p. injection once a week. Within ˜30 days, all of the transgenic mice taken off of the chloroquine had developed tumors while none of the mice receiving weekly i.p. injections developed cancer. At ˜10 months of age, these mice on weekly chloroquine remained cancer-free and appeared healthy and normal.
The carcinogen 3-methylcholanthrene (3-MC) induces soft tissue sarcomas if injected into muscle and skin carcinomas if applied to the skin (Smart et al., Carcinogenesis 7:1669-1675 (1986); Noguchi, et al., Proc. Natl. Acad. Sci. U.S.A 93:11798-11801 (1996); Horak et al., Br. J. Cancer 49:637-644 1984)). This model system has been used to demonstrate that superinduction of p53 after DNA damage (e.g. in a mouse carrying an extra copy of chromosomal DNA containing the p53 gene) protects mice from the development of cancers induced by chemical carcinogen treatments (Garcia-Cao et al., EMBO J. 21:6225-6235 (2002). Therefore, it was determined whether the protective effect observed in these studies could likewise be achieved by biochemically enhancing p53 induction. As demonstrated herein, ATM kinase activation by chloroquine did not induce strand breaks or induce phosphorylation of substrates that normally get phosphorylated by ATM at the sites of DNA break; however, it did lead to induction and phosphorylation of p53 protein.
In a further experiment, doses of 3.5 mg/kg of chloroquine were given by i.p. injection 24 and 4 hours prior to 3-MC injection in 30 wild type (strain C57B1/6) mice. The occurrence of these tumors was readily apparent by visual inspection and confirmed by histologic assessment. Results are shown in FIG. 5. Treatment with chloroquine significantly increased the percentage of mice surviving tumor free (p=0.0013).
In a further experiment 3-MC was injected into the skin on the leg of a mouse once a week for 4 weeks. Three genetic backgrounds were used: wild-type, ATM-null, and p53-null. One half of each cohort of mice received 3.5 mg/kg of chloroquine (CHQ) via IP injection 24 hours and 4 hours prior to each of the four 3-MC administration. The development of skin carcinomas was followed over time. FIG. 7 shows tumor incidence in mice receiving either placebo or chloroquine prior to 3-MC injection. Chloroquine markedly protected from tumor development. FIG. 8 shows tumor incidence in ATM-null mice receiving either placebo or chloroquine prior to 3-MC injection. Chloroquine does not protect from tumor development. FIG. 9 shows tumor incidence in p53-null mice receiving either placebo or chloroquine prior to 3-MC injection. Again chloroquine did not protect from tumor development. These results shows that that the prophylactic effect of chloroquine is mediated at least in part through ATM and p53.
Multiple exposures to non-lethal doses of ionizing radiation can induce thymic lymphomas in C57BL/6 mice (Boniver et al., In Vivo 4(1):41-3 (1990)). Using a classical, tumor-inducing protocol (Kaplan and Brown, J. Natl. Cancer Inst., 13, 185-206 (1952)), which consists of four weekly whole-body exposures of 1.75 Gy each, the effect of chloroquine administration on thymic lymphoma formation was examined. Chloroquine (3.5 mg/kg) was administered to 4-week old female C57BL/6 mice by i.p. injection 24 hours and 4 hours prior to each of the four doses of radiation described in the protocol. According to the protocol, tumors were expected to appear within 4-6 months after the last dose of irradiation in 90% of control (untreated) mice. FIG. 6 shows the results of this analysis. Treatment with chloroquine significantly increased the percentage of tumor free survival (p<0.0001).
As will be apparent from the foregoing the invention provides for various uses. In particular, the invention provides for the use of a chloroquine compound in the manufacture of a medicament for prophylaxis or treatment of cancer. The invention further provides for the use of a chloroquine compound in the manufacture of a medicament for prophylaxis and treatment of tissue damage resulting from oxidative stress. The invention further provides for the use of a chloroquine compound in the manufacture of a medicament for the prophylaxis and treatment of tissue damage resulting from ischemia and/or reperfusion. The invention further provides for the use of a chloroquine compound in the manufacture of a medicament for the prophylaxis and treatment of tissue damage resulting from stroke. The invention further provides for the use of chloroquine in the manufacture of a medicament for the prophylaxis and treatment of tissue damages resulting from atherosclerosis.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
It will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

1.-61. (canceled)

62. A method for treating atherosclerosis comprising administering to a subject in need thereof, an effective amount of a chloroquine compound.

63. The method of claim 62, wherein the chloroquine compound is selected from the group consisting of chloroquine, chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate, hydroxychloroquine sulphate, or enantiomers, derivatives, analogs, metabolites, pharmaceutically acceptable salts, and mixtures thereof.

64. The method of claim 63, wherein the chloroquine compound is chloroquine diphosphate.

65. The method of claim 62, wherein the chloroquine compound is administered intravenously.

66. The method of claim 62, wherein the chloroquine compound is administered orally.

67. The method of claim 62, wherein the administering is performed at regular intervals for a period of at least six months.

68. The method of claim 62, wherein the chloroquine compound is administered systemically.

69. The method of claim 62, wherein the subject is human.

70. The method of claim 69, wherein the administration dosage of the chloroquine compound is 0.05 to 1 mg/kg per day.

71. The method of claim 70, wherein the dosage is 0.2 to 0.6 mg/kg per day.

72. The method of claim 62, wherein the amount of the compound administered is up to about 10 mg/kg/day.

73. The method of claim 62, wherein the amount of the compound administered is more than about 0.1 mg/kg/day.

74. The method of claim 62, wherein the amount of the compound administered is more than about 1.0 mg/kg/day.

75. The method of claim 62, wherein the amount of the compound administered is less than about 50 mg/kg/day.

76. The method of claim 62, wherein the amount of the compound administered is less than about 10 mg/kg/day.

77. The method of claim 62, wherein the chloroquine compound is administered more than once a week.

78. The method claim 62, wherein the chloroquine compound is administered daily.

79. The method of claim 62, wherein the chloroquine compound is formulated in a sustained release formulation.

80. The method of claim 62, further comprising administering one or more additional active agents to the subject.

81. The method of claim 80, wherein one or more additional agents are selected from the group consisting of antiplatelet agents, lipid lowering agents, bile acid sequestrants, fibrinates, HMG-CoA reductase inhibitors, nocotinic acid derivatives, and blood pressure lowering agents.

82. The method of claim 62, wherein the subject is free of psoriasis, malaria, protozoal infections, Alzheimer's disease, Parkinson's disease, lupus erythematosus, rheumatism, hypercalcemia, multiple sclerosis.