US20050124653A1

US20050124653A1 - Treatment of dna damage related disorders

Info

Publication number: US20050124653A1
Application number: US10/496,685
Authority: US
Inventors: Michael Kastan; Christopher Bakkenist
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-11-27
Filing date: 2003-11-26
Publication date: 2005-06-09
Also published as: US20030077661A1; US20050014785A1; US7108992B2

Abstract

The present invention provides methods and compositions for the treatment of DNA damage related disorders. One embodiment of the invention is a prophylactic method for the prevention of cancer using a chloroquine compound. Another embodiment is a method for the inhibition of side effects associated with chemotherapeutic and radiotherapeutic agents using chloroquine compounds.

Description

RELATED APPLICATIONS

This application claims the benefit of priority from U.S. patent application Ser. No. 10/351,733 filed Jan. 24, 2003 and Ser. No. 10/307,077, filed Nov. 27, 2002 which are incorporated herein by reference in their entirety.
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.

SUMMARY OF THE INVENTION

The present invention provides compositions, methods, and kits for the treatment of DNA damage related disorders. In one embodiment, a chloroquine compound is administered for the prevention of DNA damage related disorders, like cancer. In another embodiment, the chloroquine compound does not prevent a localized skin cancer alone. In yet another embodiment, the cancers prevented by the chloroquine compounds are a localized skin cancer and a cancer that is not a localized skin cancer. Also described herein are methods of inhibiting the side effects of chemotherapeutic and/or radiotherapeutic agents using chloroquine compounds.

BRIEF DESCRIPTION OF THE FIGURES

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 in four successive weeks and animals were subsequently observed for the development of tumors. Statistical significance, log rank test P=0.0012.

DETAILED DESCRIPTION OF THE INVENTION

Chloroquine Compounds
The present invention provides methods, compositions, and kits for the prevention of DNA damage related disorders. Chloroquine compounds are useful in practicing the invention described herein. 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-xylidinoquinoline, 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 may 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.
Not intending to be limited by one mechanism, it is believed that the chloroquine compounds and chloroquine-like compounds act by enhancing the activity of Ataxia-Telangiectasia Mutated (ATM) kinase. The agonistic properties of chloroquine on ATM kinase have been demonstrated. Hence, it is intended herein that chloroquine-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.
Use of Chloroquine Compounds
In one aspect, the invention provides methods of treating an animal subject, including a human. The term “animal subject” as used herein includes humans as well as other mammals. The methods described herein generally involve the administration of effective amounts of chloroquine compounds and/or chloroquine like compounds for the treatment of DNA damage related disorders. The term “DNA damage related disorders” include, but are not limited to, cancer, aging, disorders caused by damage to DNA due to exposure to carcinogens, toxins, free radicals, like oxygen radical, or DNA damaging radiations like ionizing radiation and UV radiation. The chloroquine compounds are also useful for prevention of tissue injury resulting from ischemia, such as that which occurs following myocardial infarction or stroke. The effects of the chloroquine compounds used in the methods described herein include systemic, local, and topical effects. It is preferred that the effects of the chloroquine compounds in the methods described herein are systemic.
In one embodiment, the chloroquine compounds are used as prophylactics to prevent DNA damage related disorders. The chloroquine compounds are useful in the prevention of cancers caused by toxins, carcinogens, DNA damaging radiations, and/or genetic mutations. For example, chloroquine compounds are useful in the prevention of cancers caused by exposure to toxins and carcinogens like aromatic hydrocarbons, cigarette smoke, acetyl amino fluorine, MTBE, etc. Also, chloroquine compounds are useful in prevention of preventing cancers caused by DNA damaging radiations like UV and ionizing radiation. The ionizing radiations includes both natural and therapeutic radiation exposures. Examples of ionizing radiations are X-rays for diagnostics and radiation therapy used for tumors.
The prophylactic uses for cancer described herein are not envisioned to encompass the prevention solely of localized skin carcinomas like basal cell epithelioma and squamous cell carcinoma, skin carcinomas, Burkitt's lymphoma, or skin pathologies caused by harmful radiation. When used in patients with actinic keratosis, it is envisioned the chloroquine compounds do not solely inhibit basal cell epithelioma and squamous cell carcinoma. In one embodiment, the chloroquine compounds are used to prevent a localized skin carcinoma and at least one cancer that is not a localized skin carcinoma. Examples of cancers that are not localized skin carcinomas include, but are not limited to, melanomas, lymphomas, prostate cancer, breast cancer, colon cancer, lung cancer, retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer.
In a preferred embodiment, chloroquine compounds are used in the prevention of one or more of the following cancers—melanomas, prostate cancer, breast cancer, colon cancer, lung cancer, non-Hodgkins lymphoma, retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer.
The chloroquine compounds can be used to prevent secondary cancers, i.e., cancers that are caused by radiation therapy and chemotherapy used to treat the primary cancer. In one embodiment, the chloroquine compounds are used to prevent the occurrence of breast cancer in patients receiving radiation therapy for non-Hodgkin's lymphoma. Also, in these patients 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 patients who have had prior incidences of cancer.
In one embodiment, the chloroquine compounds are administered to decrease or prevent the side-effects of radiation therapy used to treat 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.
In one embodiment, the chloroquine compounds are used in immunosuppressed patients, like transplant patients. In immunosuppressed patients, the chloroquine compounds can be used to prevent cancers. The chloroquine compounds can be used to prevent Epstein Barr virus induced lymphoproliferative syndrome.
In another embodiment, chloroquine compounds are used as prophylactics to inhibit side effects of frequent exposure to X-rays in athletes. This method would also be useful for other patient 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 present invention also provides methods for preventing DNA damage, inhibiting the effects of DNA damage, and stimulating cellular response to DNA damage by administering an effective amount of chloroquine compounds. Not intending to be limited by one mechanism of action, it is contemplated that cellular responses are enhanced by an agonistic activity on ATM kinase by priming the cell to respond to agents which cause DNA damage. Further details on ATM kinase are provided in U.S. Ser. No. ______, attorney docket number SJ-0042, entitled “ATM Kinase Compositions and Methods,” filed on the same day as the present invention, which is incorporated by reference herein in its entirety.
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. 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.
The prophylactic use of chloroquine includes the prevention of tissue injury resulting from ischemia, such as that which occurs following myocardial infarction or stroke. While not intending to be limited to one mechanism of action, it is believed that the chloroquine compounds prevent cellular death due to oxidative damage during reperfusion and as such can ameliorate tissue injury resulting from ischemic injury.
In one embodiment, chloroquine compounds are used in the treatment of DNA damage related disorders. The chloroquine compounds are used preferably in combination with chemotherapeutic or radiotherapeutic agents to prevent the side-effects associated with the chemotherapeutic agents. It is known that chloroquine compounds can inhibit multiple drug resistance. Hence, it is not intended that the methods described herein produce a beneficial effect on multiple drug resistance alone. In a preferred embodiment, the beneficial effects of chloroquine compounds, when used in combination with chemotherapeutic agents, are due to modulation of ATM kinase activity. It is contemplated that the chloroquine compounds 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.
In one embodiment, the chloroquine compounds are used to treat and/or prevent disorders caused by oxidative damage. The chloroquine compounds can be administered with anti-oxidants, like vitamin B12, to stimulate the cellular response to DNA damage and promote the repair of the cells exposed to the oxidative agents.
Therapeutic and Prophylactic Benefits
In one embodiment, the chloroquine compounds are used as prophylactic agents. For prophylactic benefit, the chloroquine compound may be administered to a patient at risk of developing a DNA damage related disorder like cancer or to a patient reporting one or more of the physiological symptoms of a DNA damage related disorder, even though a diagnosis of such disorder may not have been made. A prophylactic benefit is achieved when a disorder is prevented from afflicting a patient. This prevention can include the affliction of the patient with a milder form of the disorder or the appearance of fewer or no symptoms of the disorder being prevented or the absence of the disorder in the patient being treated.
In addition to a prophylactic benefit, the chloroquine compounds can be used for their therapeutic benefits. In one embodiment, the chloroquine compounds are used to treat DNA damage related disorders. In a preferred embodiment, the beneficial effect of the chloroquine compounds is not due to an inhibition of multiple drug resistance. The term “treating” as used herein includes achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. For example, in a cancer patient, therapeutic benefit includes eradication or amelioration of the underlying cancer. Also, a therapeutic benefit is achieved with 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 patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For example, administration of a chloroquine compound to a patient suffering from cancer provides therapeutic benefit not only when the patient's tumor marker level is decreased, but also when an improvement is observed in the patient with respect to other complications that accompany the cancer like pain and psychiatric disorders.
Effective Amount
A physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the chloroquine compound required in the methods described herein. 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 will depend on the condition being treated and the route of administration. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the disclosure herein.
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 one embodiment, the effective amount can include the dose ranges, modes of administration, formulations, etc., that have been recommended or approved by any of the various regulatory or advisory organizations in the medical or pharmaceutical arts (eg, FDA, AMA) or by the manufacturer or supplier. Effective amounts of chloroquine can be found, for example, in the Physicians Desk Reference.
The daily dosage range of chloroquine, in one embodiment, can vary between about 0.1 mg/kg to about 2 gm/kg body weight. The daily dose of a chloroquine compound may be less than about 2 gm/kg, less than about 1.5 gm/kg, or less than about 1 gm/kg. In one embodiment, the daily dose of a chloroquine coumpound is more than about 0.5 mg/kg, more than about 500 mg/kg, or more than about 1 gm/kg. Preferred daily dosage ranges of a chloroquine compound are about 0.5 mg/kg to about 50 mg/kg or about 1.0 mg/kg to about 10 mg/kg body weight. Preferred doses of chloroquine diphosphate are about 3.5 mg/kg and 7.0 mg/kg.
In one embodiment, the effective amount of chloroquine is administered 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 yet another embodiment, 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.
In one embodiment, 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.
Routes of Administration and Formulation
The compounds useful in the present invention, or pharmaceutically acceptable salts thereof, can be delivered to the patient 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 formulations useful herein can administer the chloroquine compounds topically or systemically. In one embodiment, the formulation of chloroquine compound is administered systemically. In another embodiment, the formulation of chloroquine compound has a systemic effect if administered either topically or systemically.
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 may 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 may be administered in combination with other therapeutic agents. The choice of therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition being treated.
Agents used in accordance with the methods of the invention may be conveniently administered in a pharmaceutical composition containing the active compound in combination with a suitable carrier. Such pharmaceutical compositions may 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 & 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 patient.
Examples of materials which may 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 harmaceutical 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 may 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 may 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 may 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 may, of course, be varied and may 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 will be obtained.
The tablets, troches, pills, capsules, and the like may 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 may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
For administration orally, the compounds may 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) may 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 may 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 may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
A syrup or elixir may 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 may 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 may 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 may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection or a transdermal patch. Thus, for example, the compounds may 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 will depend upon 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 patient 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 1 shows that exposure to chloroquine prior to irradiation increased cell survival by 30%.

TABLE 1

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. 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.

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., 1986; Noguchi et al., 1996; Horak et al., 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., 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 breaks, however, it did lead to induction and phosphorylation of p53 protein. Thus, chloroquine pre-treatment may prevent/reduce tumor development resulting from 3-MC injections. Accordingly, 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 mice. Results are shown in FIG. 5. The occurrence of these tumors was readily apparent by visual inspection and confirmed by histologic assessment.
Multiple exposures to non-lethal doses of ionizing radiation can induce thymic lymphomas in C57BL/6 mice (Boniver et al., 1990). Using a classical, tumor-inducing protocol (Kaplan and Brown, 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.
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. A method of prophylactic treatment for a DNA damage related disorder comprising administering to an animal subject in need thereof an effective amount of a chloroquine compound, with the proviso that the chloroquine compound does not prevent a localized skin carcinoma alone.

2. The method of claim 1 wherein the chloroquine compound prevents a localized skin carcinoma and at least one cancer that is not a localized skin carcinoma.

3. The method of claim 1 wherein the DNA damage related disorder is aging.

4. The method of claim 1 wherein the DNA damage related disorder is cancer.

5. The method of claim 1 wherein the DNA damage related disorder is caused by radiation, toxins, carcinogens, natural processes or exposure to agents which cause oxidative DNA damage.

6. A method of inhibiting side-effects of radiation therapy comprising administering to an animal subject in need thereof an effective amount of a chloroquine compound.

7. A method of inhibiting development of secondary cancers comprising administering to an animal subject in need thereof an effective amount of a chloroquine compound.

8. A method of inhibiting side-effects of chemotherapy comprising administering to an animal subject in need thereof an effective amount of a chloroquine compound.

9. A prophylactic method of prevention of X-ray related DNA damage comprising administering to an athlete in need thereof an effective amount of a chloroquine compound.

10. The method of claims 1, 6, 7, 8, or 9 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.

11. The method of claim 10 wherein the compound is chloroquine, chloroquine phosphate or chloroquine diphosphate.

12. The method of claims 1, 6, 7, 8, or 9 wherein chloroquine compound has a systemic effect.

13. The method of claims 1, 6, 7, 8, or 9 wherein the amount of the compound administered is at least about 0.1 mg/kg/day.

14. The method of claims 1, 6, 7, 8, or 9 wherein the amount of the compound administered is up to about 10 mg/kg/day.

15. The method of claims 1, 6, 7, 8, or 9 wherein the amount of the compound administered is more than about 0.1 mg/kg/day.

16 The method of claims 1, 6, 7, 8, or 9 wherein the amount of the compound administered is more than about 1.0 mg/kg/day.

17. The method of claims 1, 6, 7, 8, or 9 wherein the amount of the compound administered is less than about 50 mg/kg/day.

18. The method of claims 1, 6, 7, 8, or 9 wherein the amount of the compound administered is less than about 10 mg/kg/day.

19. The method of claims 1, 6, 7, 8, or 9 wherein the chloroquine compound is administered more than once a week.

20. The method claims 1, 6, 7, 8, or 9 wherein the chloroquine compound is administered daily.

21. The method of claims 1, 6, 7, 8, or 9 wherein the chloroquine compound is formulated in a sustained release formulation.