WO2006043153A2

WO2006043153A2 - Use of zinc and copper chelators for the treatment of viral diseases

Info

Publication number: WO2006043153A2
Application number: PCT/IB2005/003124
Authority: WO
Inventors: Michel Xilinas
Original assignee: Michel Xilinas
Priority date: 2004-10-20
Filing date: 2005-10-20
Publication date: 2006-04-27
Also published as: WO2006043153A3

Abstract

The Treatment of infections caused by blood borne virus such as HCV, HBV and HIV, using one or more chelators of zinc and/or copper ions is disclosed. The preferred agents are clioquinol and phanquinone.

Description

USE OF CHELATORS OF ZINC AND COPPER IONS FOR THE TREAT¬ MENT OF INFECTIONS CAUSED BY BLOOD BORNE VIRUSES

The present invention relates to the use of chelators of zinc and copper ions for the treatment of infections caused by blood borne viruses, preferably selected among flavoviridae, and in particular hepatitis C virus (HCV), the hepadnaviridae and in particular hepatitis B virus (HBV) or by retroviridae and in particular by Human Immunodeficiency viruses I and 2 (HIV-I and HIV-2).

In a preferred aspect the invention relates to the use of clioquinol and/or phanquinone for the treatment of infections caused by blood borne virus selected among HBV, HCV and HIV.

In another aspects the invention also relates to the use of clioquinol and/or phanquinone for the manufacture of pharmaceutical compositions or the treatment of infections caused by viruses selected among HBV, HCV and HIV.

BACKGROUND FOR THE INVENTION

Among the multiple blood-borne viruses sexually transmitted or transmissible through transfusion or through mother to child or by percuta¬ neous or mucosal exposure to blood or body fluids, hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency viruses (HIV-1/-2) are relatively more important than several other such viruses. Not only do they establish asymptomatic persistent infections with occasional oncogenic sequelas, but they also cause significant morbidity and mortality when sexu¬ ally transmitted or when transmitted through transfusion or contamination of blood, blood products or biological fluids and tissues.

The hepatitis C virus (HCV) is one of the most important causes of chronic liver disease in the western countries. It accounts for about 15 per¬ cent of acute viral hepatitis, 60 to 70 percent of chronic hepatitis, and up to 50 percent of cirrhosis, end-stage liver disease, and liver cancer. Almost 4 million people in the U. S. A, or 1.8 percent of the population, have antibody to HCV (anti-HCV), indicating ongoing or previous infection with the virus. Hepatitis C causes an estimated 10,000 to 12,000 deaths annually in the U.S.A.

A distinct and major characteristic of hepatitis C is its tendency to cause chronic liver disease. At least 75 percent of patients with acute hepati¬ tis C ultimately develop chronic infection, and most of these patients have accompanying chronic liver disease.

Chronic hepatitis C varies greatly in its course and outcome. At one end of the spectrum are patients who have no signs or symptoms of liver di- sease and completely normal levels of biomarkers such as the serum liver enzymes. Liver biopsy usually shows some degree of chronic hepatitis, but the degree of injury is usually mild, and the overall prognosis may be good. At the other end of the spectrum are patients with severe hepatitis C who have symptoms, HCV RNA in serum, and elevated serum liver enzymes, and who ultimately develop cirrhosis and end-stage liver disease. In the middle of the spectrum are many patients who have few or no symptoms, mild to moderate elevations in liver enzymes, and an uncertain prognosis.

Chronic hepatitis C can cause cirrhosis, liver failure, and liver can¬ cer. Researchers estimate that at least 20 percent of patients with chronic hepatitis C develop cirrhosis, a process that takes at least 10 to 20 years. After 20 to 40 years, a smaller percentage of patients with chronic disease develop liver cancer. Liver failure from chronic hepatitis C is one of the most common reasons for liver transplants in the United States. Hepatitis C is the cause of about half of cases of primary liver cancer in the developed world. Men, alcoholics, patients with cirrhosis, people over age 40, and those in¬ fected for 20 to 40 years are more likely to develop HCV-related liver cancer. Hepatitis B virus infection is an important medical problem world¬ wide, and is associated with increased morbidity and mortality. Of the ap¬ proximately 2 billion people who have been infected worldwide, more than 350 million are chronic carriers of HBV. The HBV is a non-cytopathic, envel¬ oped DNA virus that causes acute and chronic liver disease characterized by a necro-inflammatory cell infiltrate that is triggered by the cellular immune response to viral antigens. Approximately 15-40% of infected patients will develop cirrhosis, liver failure, or hepatocellular carcinoma (HCC). HBV infection accounts for 500 000 to 1.2 million deaths each year and is the 10th leading cause of death worldwide. Hepatocellular carcinoma incidence has increased world- wide, and the disease is now the 5th most frequent cancer, killing 300 000- 500 000 people each year. (The World Health Report. 1997. World Health Organization, Geneva, Switzerland, and World Health Organization. Hepatitis B. World Health Organization Fact Sheet 204, Revised October 2000). (WHO Web site. 2000. http://who.int/inf-fs/en/fact204.htmn. HBV results in death or liver failure leading to transplantation in 10 percent of patients. It is important to state that many patients do not toler¬ ate the medications. Almost half of the patients treated for the disease will not have a successful response. According to Centers for Disease Control and Prevention (CDC), it is estimated that 1.25 million people in the United States have chronic HBV and there were an estimated 78,000 new HBV in¬ fections.

The majority of the patients may remain in an inactive phase asso¬ ciated with low viral replication and histological remission; however a sig¬ nificant proportion will develop chronic hepatitis B, which is characterized by the presence of high HBV replication and chronic necroinflammation of the liver. (Lok AS. Chronic hepatitis B. N Engl J Med 2002; 346(22): 1682- 1683).

It is estimated that about 15-20% of patients with chronic hepatitis B develop cirrhosis within 5 years, and only 55-85% of those with active HBV-related cirrhosis survive for more than 5 years. Moreover, although all patients with chronic HBV infection are at higher risk for hepatocellular car¬ cinoma when compared with the general population, the risk becomes much higher when cirrhosis develops. Thus, it is estimated that over 250 000 pa¬ tients world-wide die annually from HBV-related liver disease. Chronic hepatitis B is diagnosed according to the presence or not of

HBV e antigen (HBeAg) and antibody (anti-HBe) HBeAg-positive chronic hepatitis B occurs during the early phases of chronic HBV infection and is characterized by extremely high HBV replication and persistently or intermit- tently increased aminotransferase levels. The majority of such patients, if left untreated, maintain high HBV replication and severe liver necroinflam- mation, which is associated with worsening fibrosis, the development of cir¬ rhosis and an increased risk of hepatocellular carcinoma. (Mahoney FJ. Up- date on diagnosis, management, and prevention of hepatitis B virus infec¬ tion. Clin Microbiol Rev 1999; 12: 351-366).

HBV is a virus that causes inflammation of the liver and liver cell damage, which can lead to cirrhosis and cancer. HBV is transmitted through direct contact with blood, semen, or vaginal secretions. HBV is found in transmittable levels in body fluids including semen, vaginal secretions, sa¬ liva and blood. HBV has also been found in low concentrations in ears, urine, feces, breast milk and cerebrospinal fluid other body fluids, though these fluids have not been associated with transmission.

Many adults have few or no symptoms. Symptoms may mimic in- fluenza and can include anorexia, malaise, fatigue, nausea, vomiting, ab¬ dominal pain, dark urine, jaundice, rash or arthralgia. Less than 1% of pa¬ tients have a more severe course of illness and may experience sudden and severe liver failure within a short period of time after infection. These people may suddenly collapse with fatigue, have jaundice, and develop swelling in their abdomen. This can be fatal if not treated immediately.

Blood tests can determine whether a person has acute or chronic hepatitis. There are three standard blood tests for HBV, HbsAg or HbeAg (HBV viremia), anti-HBs and anti-HBc. HBV usually takes between 3 weeks to 2 months to show up in the blood. It may take up to two months after in- fection ^'for an HBVtest to be accurate.

Patients with HBeAg-negative chronic hepatitis B, represent a rather late phase in the course of chronic HBV infection. The disease is char¬ acterized by persistent or intermittent elevations in alanine aminotrans¬ ferase activity and necroinflammation and fibrosis on liver histology that are linked aetiopathogenetically to the underlying HBV replication. HBeAg- negative chronic hepatitis B represents a potentially severe and progressive form of chronic liver disease with very rare spontaneous remissions, fre¬ quent progression to cirrhosis and increased risk of the development of hepatocellular carcinoma. (World Health Organization. Hepatitis B Vaccines. WHO Web site. 2003. http://www.who.int/vaccines/en/hepatitisb.shtml).

All patients with either HBeAg-positive or HBeAg-negative chronic hepatitis B require effective therapeutic intervention because of the poor rate of spontaneous remission and the increased risk of cirrhosis and devel¬ opment of hepatocellular carcinoma.

HBV infection is preventable through vaccination. Since hepatitis D can only coexist with HBV, the HBV vaccination also protects the patient against hepatitis D. After receiving the HBV vaccine antibody confirmatory testing is required in order to ensure that the vaccination was successful.

Treatment considerations for HBV vary depending on whether the infection is acute or chronic. Always consult your health care provider for specific recommendations and treatment options. No specific treatment is available for acute HBV infection. In chronic HBV, interferon is used to stop the replication of HBV, with a 40% effectiveness in eliminating chronic HBV infection. Lamivudine is a specific drug in the treatment of chronic HBV. Hepatitis B can cause chronic liver disease, cirrhosis, hepatocellular carci¬ noma and death.

Although, ideally, all patients with chronic hepatitis B should be treated, therapeutic intervention is currently recommended for cases with a relatively satisfactory likelihood of response and/or advanced disease. A re¬ alistic therapeutic approach aims to sustain HBeAg loss and hepatitis B e an¬ tibody (anti-HBe) seroconversion in HBeAg-positive chronic hepatitis B and to sustain biochemical and virological remission in HBeAg-negative chronic hepatitis B. Currently, three drugs are licensed for chronic hepatitis B: inter- feron-alpha, lamivudine and adefovir dipivoxil. In patients with HBeAg- positive chronic hepatitis B, all of these drugs achieve HBeAg loss (24-33%) and anti-HBe seroconversion (12-30%) rates significantly superior to those observed in untreated placebo controls. (Cooksley WG, Piratvisuth T, Lee SD et al. Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B e antigen-positive chronic hepatitis B. J Viral Hepat 2003; 10: 298-305).

In patients with HBeAg-negative chronic hepatitis B, the sustained off-therapy response rate is 20-25% after a 12-month course of interferon- alpha and minimal, if any, after a 12-month course of lamivudine or ade- fovir. Long-term lamivudine induces an initial response in 70-90% of pa¬ tients, but only 30-40% of patients remain in remission after the third year due to progressively increasing viral resistance. Long-term adefovir achieves a response in approximately 70% of patients at 12 months, which is main¬ tained at 24 months with rare drug resistance. Adefovir is also effective against lamivudine-resistant strains. Many other anti-viral agents, immuno¬ modulatory approaches and combination therapies are currently being evaluated in chronic hepatitis B. (de Man RA, Wolters LM, Nevens F et al. Safety and efficacy of oral entecavir given for 28 days in patients with chronic hepatitis B virus infection. Hepatology 2001;4: 578-582).

Combination Therapies for Chronic Hepatitis B: Up until this point, combination therapies for HBV have not been shown to improve virologic re- sponse. Specific therapies that have been examined include lamivudine plus telbivudine and adefovir plus lamivudine. However, in a study (Werle- Lapostolle B, Bowden S, Locarnini S, Wursthom K, Petersen J, Lau G, Trepo C, Marcellin P, Goodman Z, Delaney WE 4th, Xiong S, Brosgart CL, Chen SS, Gibbs CS, Zoulim F. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroen¬ terology. 2004;126: 1750-8) the the efficacy of adefovir plus emtricitabine evaluated. Thirty treatment-naive, HBeAg-positive patients with serum ALT > 1.3 times the upper limit of normal were randomized to either adefovir (10 mg a day) plus emtricitabine (200 mg a day) or adefovir (10 mg a day) plus placebo for 48 weeks'. Combination therapy produced a reduction of 3.14 vs 2.16 for adefovir alone at 24 weeks and 3.48 vs 2.22 at 48 weeks. There was no difference in HBeAg seroconversion between the groups. Lar¬ ger studies will be required to determine whether there is a difference in se¬ roconversion. This was the first time that a combination therapy regimen showed improved virologic response over monotherapy. It is important to keep in mind that the primary benefit of combination therapy will likely not be an improved virologic response, but rather a decreased rate of viral resis¬ tance. There are a number of studies looking at the combination of pegy- lated interferon and lamivudine (Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, Lu ZM, Piratvisuth T, Germanidis G, Yurdaydin C, Di- ago M, Gurel S-, Lai MY, Button P, Pluck N; Peginterferon Alfa-2a HBeAg- Negative Chronic Hepatitis B Study Group. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg- negative chronic hepatitis B. N Engl J Med. 2004;351: 1206-17). Patients re¬ ceiving pegylated interferon alfa-2a, with or without lamivudine, had statis¬ tically better results for each of the measured end points than those receiv- ing lamivudine alone. Data suggest that pegylated interferon alfa-2a has ef¬ ficacy in patients with HBeAg-negative chronic hepatitis B and that the addi¬ tion of lamivudine adds little benefit. No resistance data are provided to in¬ dicate whether the combination therapy protected against the development of lamivudine resistance. The use of pegylated interferon for 48 weeks provided excellent re¬ sults in this group of HBeAg-positive chronic hepatitis B patients, and was well tolerated. The 48-week duration is longer than the 16 to 24-week dura¬ tion typically used with standard interferon alfa-2a and -2b. Lamivudine added no additional benefit. No resistance data were provided. Janssen et al (Janssen HL, van Zonneveld M, Senturk H, Zeuzem S,

Akarca US, Cakaloglu Y, Simon C, So TM, Gerken G, de Man RA, Niesters HG, Zondervan P, Hansen B, Schalm SW; Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet. 2005 Jan 8;365: 123-9) studied the combination of pegylated interferon alfa'-2b (100 meg/week for 32 weeks, then 50 meg/week for 20 weeks) in combination with lamivudine 100 mg/day (n = 130) or placebo (n = 136) in HBeAg-positive chronic hepatitis B. On the ba¬ sis of these studies, pegylated interferon looks promising in both HBeAg- positive and HBeAg-negative chronic hepatitis B, particularly for those pa- tients with low-baseline HBV DNA and high-baseline ALT levels. The optimal duration of treatment appears to be at least 1 year, particularly in the HBeAg-negative patients. Lamivudine appears to add no benefit, except per¬ haps in the subset of patients with genotype D disease ~ but this finding will require confirmation.

Since HBV is a major healthcare problem and patients are at in¬ creased risk for progression to cirrhosis and end-stage liver disease and for the development of hepatocellular carcinoma there is a pivotal need for new treatment options. Interferon has been used to treat HBV infection since the mid-1980s with limited results. The efficacy of pegylated interferon is now being actively investigated. The development and availability of nucleoside and nucleotide analogues has greatly altered the management of patients with chronic hepatitis B. Unfortunately, the increased use of these drugs, particularly when used as monotherapy, has produced mutations that confer viral resistance.

There is no radical treatment at present for AIDS, but pharmacol¬ ogical agents can slow down the rate at which HIV inactivates the immune system. As with other diseases, early detection offers more options for treatment and preventing complications.

The term AIDS refers to. an advanced stage of HIV infection, when the immune system has sustained substantial damage. Not all individuals who have HIV infection develop AIDS. When HIV invades the CD4+ T cells, the damaged immune system loses its ability to defend against diseases caused by infective agents. A substantial decline in CD4+ T cells also leaves the body vulnerable to certain cancers.

When HIV progresses to AIDS, however, it has proved to be a fatal illness. Few patients survive five years from the time they are diagnosed with AΪDS, although this is' increasing with improvements in treatment tech¬ niques.

It is estimated that about half the patients with HIV will develop AIDS within 10 years after becoming infected. This time varies greatly and depends on several factors. A patient is said to have AIDS when he or she has signs or symp¬ toms specified in guidelines formulated by the U.S. Centers for Disease Con¬ trol and Prevention (CDC). This revised definition of HIV infection, which ap¬ plies to any HIV (e.g., HIV-I or HIV-2), is intended for public health surveil- lance only. It incorporates the reporting criteria for HIV infection and AIDS into a single case definition. The revised criteria for HIV infection update the definition of HIV infection, the revised HIV criteria apply to AIDS-defining conditions for adults and children, which require laboratory evidence of HIV. AIDS therefore is the name of the fatal clinical condition that results from in¬ fection with the HIV, which progressively damages the body's ability to pro¬ tect itself from disease organisms. Thus, many AIDS deaths result from pneumonia, tuberculosis or diarrhea; death is not caused by HIV itself but by one or more of these infections. The CDCs definition of AIDS includes:

All HIV-infected persons with fewer than 200 CD4+ T cells per cubic millimetre of blood

Patients with HIV infection who have at least one of more of AIDS- associated conditions that are the result of HIVs attack on the immune sys- tern.

All a nti retroviral medications currently approved to treat HIV infec¬ tion target two viral enzymes. used by the virus to replicate itself. These en¬ zymes, reverse transcriptase and protease, are involved in different stages of viral replication. Three classes of a nti retroviral drugs have been developed to inter¬ fere with the activity of these viral enzymes and slow down the multiplica¬ tion of the virus. These are:

Nucleoside analogue reverse transcriptase inhibitors (NRTIs). NRTIs interrupt an early stage of HIV replication by interfering with the activity of reverse transcriptase. AZT (zidovudine), the first drug approved for treating HIV infection, is an NRTI, as are zalcitabine (ddC), didanosine (ddl), stavudine (d4T), lamivudine (3TC), and abacavir;

Non-nucleoside reverse transcriptase inhibitors (NNRTIs). NNRTIs also work by hindering the action of reverse transcriptase. This class of drugs includes delavirdine, nevirapine, and efavirenz; Protease inhibitors. Protease inhibitors interrupt a later stage of vi¬ ral replication. This class of drugs includes saquinavir, indinavir, ri- tonavir, nelfinavir, and amprenavir.

Studies have found that various combinations of a nti retroviral drugs are more effective in suppressing HIV than a nti retroviral drugs used alone. Experts refer to one common treatment approach, usually involving a protease inhibitor combined with two other a nti retroviral drugs, as "highly active a nti retroviral therapy" or HAART.

Drug combinations, also can help reduce the risk that drug- resistant HIV will develop. When drug resistance occurs, medications that initially succeeded in suppressing the replication of HIV in the patient's body lose their effectiveness.

Antiretroviral drugs have side effects that can limit their use in cer¬ tain patients. AZT, for example, may result in a loss of blood cells. Protease inhibitors can cause nausea, diarrhoea, and other symptoms.

Clioquinol (iodochlorhydroxyquin, 5-chloro-7-iodo-8- hydroxyquinolin- e, MW 305.5) is a USP drug that chelates zinc [K(Zn)=12.5, K(Cu)=15.8, K(Ca)=8.1, K(Mg)=8.6], is hydrophobic, has a low general toxicity profile, and crosses the blood brain barrier (Padmanab- han et al., 1989). It has been demonstrated that clioquinol is rapidly absorbed from the gut of rats and mice where blood levels reached about 1-10 μM within one hour of ingestion (Kotaki et al., J Pharmacobiodyn, 6(ll):881-887 (1983)). Since the drug is hydrophobic, it passes rapidly into the brain, and then is rapidly excreted, so that a bolus dose of clioquinol is almost com- pletely removed from the brain within three hours. It appears to be safe in many mammalian species, including rat and mouse (Tateishi et al., 1972; Tateishi et al., 1973), and is still used as a veterinary antibiotic.

Clioquinol was withdrawn from use as an oral anti-infective for hu¬ mans in the early 1970's when its ingestion in Japan was linked to a condi- tion named subacute myelo-optic neuritis (SMON), a condition that resem¬ bles subacute combined degeneration of the cord caused by vitamin B_n dep¬ rivation.

Clioquinol has a relatively safe profile in mice, and there is a large literature on its pharmacology in this animal. It is disclosed herein data re¬ garding its ability to specifically chelate zinc from A beta deposits in vitro (induced aggregates and brain samples). Based on the in vitro data, it is reasonably expected that the_. low concentrations of clioquiηol shown to be effective in resolubilizing A beta. The addition of vitamin B_i2 in the present invention or monitoring of the blood vitamin B₁₂ levels may avoid the ad¬ verse SMON effect noted above.

Thus, given its strong chelating effects on zinc, its oral high absorp¬ tion and excellent distribution in all body tissues and fluids as well as its other pharmacological properties, clioquinol holds promise as an effective agent in the treatment of HCV infection in humans. Clioquinol has been marketed by CIBA-GEIGY as ENTEROVIOFORM as well as by many other pharmaceutical companies.

Phanquinone (4,7-phenanthroline-5,6-dione) has hitherto been used for the treatment of various disorders, such as amoebiasis. However, the treatment or prevention of hepatitis C virus disease has not been sug¬ gested previously. Thus, given its strong chelating effects on zinc, its^' oral high absorption and excellent distribution in all body tissues and fluids as well as its other pharmacological properties, phanquinone holds promise as an effective agent in the treatment of HCV infection in humans. Phanquinone has been marketed by CIBA-GEIGY as ENTOBEX.

DESCRIPTION OF THE INVENTION

It has surprisingly been realized that infections caused by blood borne viruses can be efficiently treated using chelators specific for copper and/or zinc.

Thus, in one aspect the invention relates to the use of one or more chelators of zinc and/or copper ions for the treatment of infections caused by a blood borne virus.

The term "chelators of zinc and/or copper ions" is in the present description and claims intended to mean a compound capable of binding zinc and/or copper ions with high affinity. It will be appreciated that chelators generally have affinity for several metal ions and therefore will chelators of zinc and/or copper ions also is some degrees have affinity for other metal ions. The chelators for zinc and/or copper ions according to the present in- vention may have a high affinity to zinc ions and a lower affinity to copper ions, a high affinity to copper ions and a lower affinity to zinc ions or a high affinity to both zinc and copper ions. Several chelators are known within the area as well as their affinity to zinc and/or copper ions, or if necessary can the affinity to zinc and/or copper ions be determined by the skilled person using routine experimentation. It is therefore within the skills of the average practitioner to select suitable chelators of zinc and/or copper ions.

As preferred examples of chelators of zinc and/or copper ions can be mentioned clioquinol, phanquinone and mixtures thereof.

The term "blood borne virus" is intended to be understood in the usual way as viruses mainly being transferred from an infected human being to a non-infected human being carried by blood or other bodily fluids such as semen and vaginal fluids. Examples of blood borne viruses include members of the flavoviridae group, the hepadnaviridae group and the retroviredae group.

The term flavoviridae is intended to be understood in the usual way of viruses that belong to the Apoi virus in the taxonomy and include the he- pactivirus, amongst which a member is the HCV virus.

The term hepadnaviridae is intended to be understood in the usual way and includes the orthόhepadnaviridae, amongst which a member is the HBV.

The term retroviridae is intended to be understood in the usual way as a RNA virus, where the RNA genome in a part of its infectious cycle is re¬ verse transcribed into one or more DNA molecules by the action of a reverse transcriptase enzyme. Several of such retro virus are known, amongst which members are Human Immunodeficiency virus 1 and 2 (HIV-I and HIV-2).

The virus is according to the invention preferably a sexually or through transfusion or mother to child transmission or through contamina- tion from blood or other body fluids a blood borne virus belonging to the family of flaviridae or the family of hepadnaviridae or the family of retroviri- dae.

Most preferred is the virus according to the invention selected to be the Hepatitis C virus (HCV), Hepatitis B virus (HBV) or of Human Immunode¬ ficiency viruses (HIV-I and HIV-2).

It should be understood that the expression "infection caused by a blood borne virus" is intended to include any infection with such a virus where the virus is stable maintained within the individual in question, whether or not the individual expires any physiologically symptoms of the infection.

The term treatment is intended to be understood as comprising both a treatment curing the infection of the blood borne virus, a treatment that delay the progress of the infection and a treatment that alleviates the symptoms of the virus infection. Thus, it is not required that the individual infected with the retrovirus expires any symptoms of the infection, but the treatment of a symptom less individual for controlling or eradicating the causative virus is also considered a treatment. Preferably is the treatment a curative treatment. The term HCV infection is intended to be understood as an individ¬ ual with acute or chronic hepatitis C presenting the characteristic symptoms and signs of the disease and having positive biomarkers of the condition such as quantitative or qualitative HCV RNA or positive liver enzyme tests or a positive biopsy. The individual can have advanced or mild liver disease or hepatocellular carcinoma and be either naϊve or non responder to treatment patient.

The term HBV infection in intended to be understood as an individ¬ ual having acute or chronic hepatitis B naϊve or non responder to treatment. The individual can have been diagnosed with occult hepatitis B (HBV DNA positive and absence of HbsAg) or with HbaAg-negative chronic hepatitis B with or without hepatocellular carcinoma.

The term Human Immunodeficiency virus 1 and 2 (HIV-I and HIV- 2) infection is intended to be understood as an individual having an infection following the criteria of the Revised WHO Clinical Staging of HIV/AIDS For Adults and Adolescents (2005) criteria.

According to the invention is one or more chelators of zinc and/or copper ions applied to an individual suspected for having an infection of a blood borne virus in a suitable amount following a suitable dosage regimen.

The actual used amount and dosing regimen is determined by the discretion of the attending physician.

Usually the chelator of zinc and/or copper ions is formulated and administered in a pharmaceutical composition. The pharmaceutical composi¬ tions according to the invention is prepared using techniques well known within the pharmaceutical area.

Thus, another aspect of the invention is the use of one or more chelators of zinc and/or copper ions for the manufacture of a pharmaceutical composition for the treatment of an infection caused by an blood borne vi¬ rus.

Preferably the one or more chelators of zinc and/or copper ions is selected among clioquinol, phanquinone and mixtures thereof.

In general, a suitable therapeutically effective amount of clioquino! or phanquinone in the pharmaceutical composition would be e.g. 50 to 2000 mg of each.

The preferred drug treatment is clioquinol is at a dosage of 250 mg to I g. If a combination is used the preferable quantities are 250 mg to 1 g of clioquinol and 50 mg to 100 mg of phanquinone and the preferred combi- nation ratio is of 250 mg of clioquinol and 50 mg of phanquinone. The dos¬ age schedule is one to three times daily.

It will be appreciated that the amount of clioquinol and phan¬ quinone and optional further active constituents required for said treatment, improvement or prevention will vary according to the route of administra- tion, the disorder to be treated, the condition, age, the case history of the subject, the galenic formulation of the pharmaceutical composition, etc.

The actually administered amounts of clioquinol and phanquinone and optional further active constituents may be decided by a supervising 003124

15 physician. If the pharmaceutical composition in addition to phanquinone comprises further active constituents those may be included therein for ad¬ ministering in combination concurrently, or in different compositions for ad¬ ministering substantially simultaneously but separately, or sequentially. When the pharmaceutical compositions according to the invention includes clioquinol it is preferred that vitamin Bi₂ also is included in the composition in order to avoid SMON.

Therapeutic formulations include formulations suitable for parente¬ ral (including intramuscular and intravenous), oral, rectal or intradermal administration, although oral administration is the preferred route. Thus, the pharmaceutical composition may be formulated as tablets, pills, syrups, cap¬ sules, suppositories, formulations for transdermal application, powders, es¬ pecially lyophilized powders for reconstitution with a carrier for intravenous administration, etc. The one or more chelators of zinc and/or copper ions is preferably administered together with one or more pharmaceutical acceptable car- rier(s). The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the re¬ cipients thereof. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which phanquinone is administered. The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, poly¬ vinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatine, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize ^'starch and the like¹; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; a glidant, such as colloidal silicon diox¬ ide; a sweetening agent, such as sucrose or saccharin; and/or a flavouring agent, such as peppermint, methyl salicylate, or orange flavouring.

Therapeutic formulations suitable for oral administration, e.g. tab- lets and pills, may be obtained by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by mixing the constituent(s), and compressing this mixture in a suitable appa¬ ratus into tablets having a suitable size. Prior to the mixing, the active in- gredients may be mixed with a binder, a lubricant, an inert diluent and/or a disintegrating agent and further optionally present constituents may be mixed with a diluent, a lubricant and/or a surfactant. tn a preferred embodiment, active ingredients in form of. a free- flowing powder is mixed with a binder, such as microcrystalline cellulose, and a surfactant, such as sodium lauryl sulphate, until a homogeneous mix¬ ture is obtained. Subsequently, another binder, such as polyvidone, is trans¬ ferred to the mixture under stirring. Said mixture is passed through granu¬ lating sieves and dried by desiccation before compression into tablets in a standard compressing apparatus.

In a second preferred embodiment, active ingredients in form of a free-flowing powder is mixed with surfactants and/or emulsifying agents, such as Sapamine.RTM. (N-(4'-stearoyl amino phenyl)-trimethylammonium methyl sulphuric acid) and lactose monohydrate until a uniform distribution of the constituents is obtained. A second preparation containing a disinte¬ grating agent, such as maize starch, is added to the mixture under continu¬ ous stirring. Such a second preparation may be obtained by adding excess boiling water to maize starch suspended in cold water. The final mixture is granulated and dried as above and mixed with maize starch and magnesium stearate and finally compressed into tablets in a standard apparatus.

A tablet may be coated or uncoated. An uncoated tablet may be scored. A coated tablet may be coated with sugar, shellac, film or other en¬ teric coating agents.

Therapeutical formulations suitable for parenteral administration include^' sterile solutions or suspensions of the active constituents. An aqueous or oily carrier may be used. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including petroleum, animal, vege¬ table or synthetic origin, such as peanut oil, soy bean oil, mineral oil, sesa¬ me oil and the like. Formulations for parenteral administration also include a lyophilized powder comprising one or more chelators of zinc and/or copper ions and optionally further an active constituent that is to be reconstituted by dissolving in a pharmaceutically acceptable carrier dissolving the active constituents, e.g. an aqueous solution of carboxymethylcellulose and lauryl sulphate.

When the pharmaceutical composition is a capsule, it may contain a liquid carrier, such as a fatty oil, e.g. cacao butter.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim-milk, glycerol, propylene, glycol, water, ethanol and the like. Said compositions may form solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a supposi- tory, with traditional binders and carriers such as triglycerides.

In one embodiment of the pharmaceutical composition according to the invention, clioquinol and phanquinone and the possible further active constituents, are comprised as separate pharmaceutical entities. The two entities may administered simultaneously or sequentially. The pharmaceutical may comprise further active ingredients for ex¬ ample antiviral active compounds known within the area.

All treatment with either clioquinol or phanquinone or both in com¬ bination may be administered alone or in combination with another suitable treatments such as interferon (interferon (peginterferon aifa-2b and peginterferon alfa-2a) and ribavirin.

Other features and advantages of the invention will be apparent for the skilled person.

For the treatment of affected patients based on the clinical signs and symptoms of the disease, the patient may in one embodiment of the in¬ vention be put on a program of treatment consisting of high dose clioquinol or phanquinone for 6 to 24 months. The high dosage of clioquinol is 1 g to 2 g one to three times daily and 100 mg to 500 mg of phanquinone one to three times daily. The high dosage will be followed by a period of low dose therapy for life-time. The low dosage of clioquinol is 100 mg to 250 mg one to three times daily and of phanquinone 25 mg to 50 mg one to three times daily. All regiments would be accompanied by vitamin B₁₂ concomitant ther¬ apy at a dosage of 0.1 to 5 mg total daily. Another embodiment of the invention would be the treatment of a mildly affected patient. Such a patient would be treated with low dose clio- quinol for up to 2 years. If after 6 months no clinical gains have been made, the patient could then be placed on the high regimen for up to another i year. If improvement is recorded then the patient can continue the low dos¬ age treatment for life-time.

Accordingly, the present invention contemplates compositions such as pharmaceutical compositions comprising an active agent and one or more pharmaceutically acceptable carriers and/or diluents.. The active agent may be clioquinol or phanquinone or a combination of clioquinol and phanquinone or a combination of clioquinol or phanquinone with either ribavirin for HCV infection or of lamivudine for HBV infection or any antiretroviral regimen for HIV infection.

Without wishing to be bound by any theory it is believed that the action of the chelators of zinc and/or copper ions according to the invention is mediated through inhibition of metal containing proteins forming an es¬ sential function during the life cycle of the HCV or H BV or HIV virus.

Tellinghuisen et al have worked on the HCV NΞ5 protein characteri- sation (JBC Papers in Press Published on August 31, 2004 as Manuscript M407787200, The NS5A Protein of Hepatitis C Virus is a Zinc Metalloprotein, Timothy L. Tellinghuisen, Joseph Marcotrigiano, Alexander E. Gorbalenya, and Charles M. Rice). They described the need for better characterization of NS5A is paramount for elucidating the role of this protein in the virus life cy- cle and examined of NS5A using bioinformatics tools suggested the protein consisted of three domains and contained an unconventional zinc-binding motif within the amino-terminal domain. They have also developed a method to produce NS5A and performed limited proteolysis to confirm the domain organization model. The zinc content of purified NS5A and the amino-terminal domain of NS5A were determined, and each of these pro¬ teins was found to coordinate one zinc atom per protein.

There has been indication in the literature that metal ions are im¬ portant for the life cycle of the virus, for example has (Stray SJ, Ceres P, Zlotnick A. Biochemistry 43:9989-98, 2004) zinc ions trigger conformational change and oligomerization of hepatitis B virus capsid protein and it has been shown that zinc ions are necessary for initiation the HBV capsid as¬ sembly. A helicase of HIV NCp7 has been shown to be affected by chelation.

It is therefore assumed that the beneficial effect disclosed in the present de¬ scription can be attributed to the inhibition of these metal proteins. It is known that HIV integrase is completely inhibited by zinc chelation (Haugan IR, Nilsen BM, Worland S, Olsen L, Helland DE. Characterization of the DNA- binding activity of HIV-I integrase using a filter binding assay. Biochem Bio- phys Res Commu 217:802-10, 1995). This is due to that both the N- terminal and the C-terminal regions of the HIV integrase are involved in DNA-binding.

The invention is now further illustrated by the following examples which are provided for illustration only, and should not be considered as lim¬ iting the invention in any aspect.

EXAMPLES

EXAMPLE 1. Preparation of a Pharmaceutical Composition Compris¬ ing Phanquinone.

250 g of phanquinone was mixed with 200 g sapamine. RTM. (N- (4'-stearoyl amino-phenyl)-trimethylammonium methyl sulphuric acid) and 1025 g lactose mono-hydrate for a period of 5 minutes. 300 g of boiling wa¬ ter was added at a time to a mixture of 100 g maize starch in 100 g cold water. The maize suspension, cooled to 40°C, was added to the phaniquinone-containing powder mixture under continuous stirring. The mixture was granulated using a 2.5 mm sieve and desiccated for 18 hours at 40°C. The dry granules were mixed with 400 g maize starch and 20 g mag- nesium stearate. The final mixture was formulated into tablets having a di¬ ameter of 8.0 mm and a weight of 200 mg.

EXAMPLE 2

Preparation of a pharmaceutical composition comprising phan- quinone, clioquinol and vitamin B₁₂.

250 g phanquinone and 250 g of clioquinol were mixed with 200 g Sapamine (N- (4'- steatoryl aminophenyl) - trimethylammonium methyl sul¬ phuric acid) and 1025 g lactose mono-hydrate for a period of 5 minutes. 300 g of boiling water was added in one go to a mixture of 100 g maize starch in 100 g cold water. The maize suspension, cooled to 40°C was added to the phanquinone and clioquinol containing powder mixture under continuous stirring. Subsequently, an aqueous solution of 5 g vitamin Bi₂ was added. The mixture was granulated using a 2.5 mm sieve and desiccated for 18 hours at 40°C. The dry granules were mixed with 400 g maize starch and 20 g magnesium stearate. The final mixture was formulated into tablets having a diameter of 8.0 mm and weight of 200 mg.

EXAMPLE 3.

In order to prove the concept of the antiviral effect of clioquinol and phanquinone the substances were sent by Dr. M. Xilinas to the National Institute of Health (NIH) that is running screaning programmes in hepatitis B (HBV) and hepatitis C virus. The HBV research is run by the University of Georgetown (Dr. Brent Korba). The results with HBV were negative at this point because of cytotoxicity of the cell line used. In research performed with the NIH collaboration with the

Southern Research Institute (Dr. Victor Buckwold) I have asked for the screening of clioquinol and phanquinone antiviral activity testing with hepa¬ titis C virus. At this laboratory the effects established the effects of clioqui- nol and phanquinone with HCV were evaluated. The cell line used was Huh7 ET.

Phanquinone was evaluated using single high test concentration (2- micromolar) and an antiviral activity of 83.2% war found with a 10.5% cyto- toxicity. In the retest with dose response curve the dose response of phan- qinone showed no activity against HCV RNA replicons due to a high cytotox¬ icity of the Huh7 ET cell iine.

Clioquinol in the same set of experiments showed to be potentially active with an antiviral activity of 82.3% and a selectivity index of over 1. In the retesting dose response clioquinol exhibited a selectivity index SI 50 over 4.8 and a SI 90 of 14.

The results that are presented in the Tables 1 to 5 and conclude that clioquinol is considered a candidate drug for the treatment of HCV infec¬ tion as described in this patent. As to phanquinone its potential antiviral ac- tivity cannot be exhibited yet because of strain specific cytotoxic effect on the Huh7 ET cell line used.

TABLE 1

Screening Report Antiviral Activity Testing with Hepatitis C Virus

ARB Number 04-5868 Drug Name BA 003837-NX-2 (phanquϊnone) Molecular Weight 210.19 Quantity Received 4.95 mg Sponsor Michael Xilϊnas Company No affiliation Date Received 5/19/2004 Special Instructions None

Assay Performed HCV RNA Replicons Assay Type Single high test concentration (20 micromolar) Cell Type Huh7 ET Date Performed 7/19/2004

Antiviral Activity (luciferase)

Percent inhibition of virus control = 83.2%

Cytotoxicity (actively dividing cells) Percent cell control = ^' 10.5%

Selectivity Index = <1

Comments Toxic

Re comm endatϊons Retest with dose response curve in case its active at much lower concentration

Date Submitted 8/31/2004 Submitted bv Victor E. Buckwold, Ph.D. Southern Research Institute

TABLE 2

Screening Report Antiviral Activity Testing with Hepatitis C Virus

ARB Number 04-5868 Drug Name BA 009837-NX-2-(phanquinone) Molecular Weight 210.19 Quantity Received 4.95 mg Sponsor Michael XiIi nas Company No affiliation Date Received 5/19/2004 Special Instructions None

Assay Performed HCV RNA replicon Assay Type Dose response (Luciferase) Cell Type Huh7 ET Date Performed 8/23/2004

Antiviral Activity EC50 (uM) = 8.80 EC90 (uM) = 74.4

Cytotoxicity (actively dividing cells) IC50 (uM) = <1 IC90 uM) = 50.1

Selectivity Index (Sl = IC/EC) SI50 = <1 SI90 = <1

Comments Cytotoxic

Recommendations Retest in case it is active at much lower concentrations

Date Submitted 9/30/2004 Submitted by Victor E. Buckwold, Ph.D. Southern Research Institute TABLE 3

Screening Report Antiviral Activity Testing with Hepatitis C Virus

ARB Number 04-5868 Drug Name BA 009837-NX-2-(phanquinone) Molecular Weight 210.19 Quantity Received 4.95 mg Sponsor Michael Xilinas Company No affiliation Date Received 5/19/2004 Special Instructions None

Assay Performed HCV RNA replicon Assay Type Dose response (Luciferase) Cell Type Huh7 ET Date Performed 9/20/2004

Antiviral Activity EC50 (uM) = >1 EC90 (uM) = >1

Cytotoxicity (actively dividing cells) IC50 (uM) = >1 IC90 uM) = >1

Selectivity Index (Sl = IC/EC) SI50 = <1 SI90 = <1

Comments No activity against HCV RNA replicons

Recommendations No further testing

Date Submitted 9/30/2004 Submitted by Victor E. Buckwold, Ph.D.

Southern Research Institute TABLE 4

Screening Report Antiviral Activity Testing with Hepatitis C Virus

ARB Number 03-674

Drug Name Clioquinol

Molecular Weight 305.5037

Quantity Received 5 mg

Sponsor Michael Xilinas

Company No affiliation

Date Received 7/30/2004

Special Instructions Practically insoluble in water

Assay Performed HCV RNA replicon

Assay Type Dose response (Luciferase)

Cell Type Huh7 ET

Date Performed 9/27/2004

Antiviral Activity

EC50 (uM) = <1

EC90 (uM) = 5.7

Cytotoxicity (actively dividing cells)

1C50 (uM) = 4.80

1C90 uM) = 77.4

Selectivity Index (Sl = IC/EC) SI50 = >4.8 SI90 = 14

Comments EC50 lower than lowest concentration tested Recommendations Retest at lower concentration

Date Submitted 9/30/2004 Submitted by Victor E. Buckwold, Ph.D.

Southern Research Institute TABLE 5

Screening Report Antiviral Activity Testing with Hepatitis C Virus

ARB Number 03-674 Drug Name Clioquinol Molecular Weight 305.5037 Quantity Received 5 mg Sponsor Michael Xilinas Company No affiliation Date Received 7/30/2004 Special Instructions Practically insoluble in water

Assay Performed HCV RNA Replicons Assay Type Single high test concentration (20 micromolar) Cell Type Huh7 ET Date Performed 9/20/2004

Antiviral Activity (luciferase) Percent inhibition of virus control ^; 82.3%

Cytotoxicity (actively dividing cells) Percent cell control = 73.3%

Selectivity Index = >1

Comments Potentially active

Recommendations Retest with dose response curve

bate Submitted 9/30/2004 Submitted by Victor E. Buckwold, Ph.D. Southern Research Institute

EXAMPLE 4

Screening for HBV antiviral activity

Confluent cultures of 2.2.15 cells (constitutively HBV-producing HepG2 cells) are maintained on 96-weϊl flat-bottomed tissue culture plates in RPMI1640 medium with 2% fetal bovine serum (Korba & Gerin, Antivir.Res.19:55- 70,1992). Cultures (6 per each of 4 test concentrations on two replicate plates) are treated with 9 consecutive daily doses of test compounds. Me¬ dium is changed daily with fresh test compounds. HBV virion DNA in the cul¬ ture medium is assessed 24 hours after the last treatment by quantitative blot hybridization. Uptake of neutral red dye (absorbance of internalized dye at 51OnM [A₅I₀]) is used to determine the relative level of toxicity 24 hours following the last treatment (Korba & Gerin, Antivir.Res.19:55-70, 1992). Values are presented as a percentage of the average A₅₁₀ values for 9 sepa¬ rate cultures of untreated cells maintained on the same plate. Cultures for the toxicity analyses are seeded at the same time with the identical pool of stock cells used for the antiviral analyses and maintained in an identical manner. A total of 3 cultures are treated with each concentration of test compound. Primary screening assays are typically performed, at a maximum concentration of 10-100 μM test compound (with 10-fold serial dilutions). Toxicity analyses usually utilize 10-fold higher levels of test compounds (with 3-fold serial dilutions). CC50, EC50, EC90, and S.I. (CC50/EC90) are calculated. Results are shown in Table 6.

TABLE 6

http://www.niaid-aacf.org/protocols/HBV.htm EXAMPLE 5

At the Katholieke Universiteit Leuven, Rega Institute we have performed anti-HIV data against HIV-I(IIIb) and HIV-2(ROD) strains. Both clioquinol and phanquinone were toxic for the cell lines. The IC50 (micro g per ml) for clioquinol was >2.19 and >1.99 in two experiments. Phanquinone was inac¬ tive in this experimental model.

EXAMPLE 6

Inhibition study of MMPs by clioquinol.

An enzyme assay was conducted with five of the enzymes belonging to the MMP group. Specifically, the assay was conducted for MMP-I, MMP-2, MMP- 3, MMP-7, and MMP-9 at various concentrations. The MMP-I, MMP-3, and MMP-7 were initially pre-incubated in 60 min at 37°C and MMP-2 and MMP-9 were pre-incubated in 60 min at 25⁰C in an aqueous vehicle of 50 mM MOPS, 1OmM CaCI₂.2H₂O, 10 μM ZnCI₂, 0,05% Brij 35, pH 7.2 and. a concentration of clioquinol of 100 μM. A test substrate of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ was subsequently added to obtain a concentration of 25 μM. MMP-I was incubated for 2 hours at 37⁰C, MMP-2 was incubated for 3 hours at 25°C, MMP-3 was incubated for 90 min at 37°C. MMP-7 was incubated for 90 min at 37⁰C, and MMP-9 was incubated for 2 hours at 25°C. The activity of the enzymes was measured by fluoro- metric quantitation of Mca-Pro-Leu-Gly-OH. The results are indicated in Ta- ble 7.

Table 7

The enzyme assay was repeated for MMP-2 except that a 10 and 100 times higher clioquinol concentration was used. At a clioquinol concen- tration of 1 mM the inhibition was 26% and at a clioquinol concentration of 1OmM the inhibition was measured to 101% the inhibition being highly de¬ pendent on clioquinol concentration.

Claims

P A T E N T C L A I M S

1. Use of a chelator of zinc and copper ions for the treatment of an infetion caused by a blood borne virus.

2. Use according to claim 1, wherein the chelator of zinc and copper ions are selected among clioquinol, phanquinone and mixtures thereof.

3. Use according to claim 1 or claim 2, wherein the blood borne vi- rus is selected among flavoviridae, hepadnaviridae and retroviredae.

4. Use according to claim 3, wherein the virus is selected among hepatitis B virus, hepatitis C virus and human immunodeficiency viruses 1 and 2.

5. Use of one or more chelator of zinc and copper ions for the manufacture of a pharmaceutical composition for the treatment of infections caused by a blood borne virus.

6. Use according to claim 5, wherein the virus is selected among flavoviridae, hepadnaviridae and retroviredae.

7. Use according to claim 6, wherein the retro virus is selected among hepatitis B virus, hepatitis C virus and human immunodeficiency vi- rus 1 and 2.

8. Use according to any one of claims 5 to 7, wherein the one or more chelator of zinc and copper ions are selected among clioquinol, phan¬ quinone and mixtures thereof.

9. A method for treating an infection caused by a blood borne virus comprising administrating of one or more chelators of zinc and copper ions to a subject in need thereof.

10. The method according to claim 9, wherein the blood borne virus is selected among flavoviridae, hepadnaviridae and retroviredae.

11. The method according to claim 10, where the virus is selected among hepatitis B virus, hepatitis C virus and human immunodeficiency vi¬ rus 1 and 2.

12. The method according to claim 9, where the chelator of zinc and copper ions is selected among clioquinol, phanquinone and mixtures thereof.

13. The method according to claim 12, wherein the amount of daily dosage of clioquinol is of 50 mg to 4 g and that of phanquinone is of 5 mg to 500 mg.

14. The method according to claim 13, wherein the amount of clio- quinol is 250 mg to 1 g and that of phanquinone is 50 mg to 100 mg.

.

15. The method according to claim 12, wherein the clioquinol of phanquinone is administered for a period of time ranging from one week to life-time.

16. The method according to claim 12, wherein clioquinol is co¬ administered together with an amount of vitamin B_i2 of 1 μg to 2 mg.

17. The method according to any of the claims 9 to 16, wherein clioquinol or phanquinone , are administered simultaneously or separately with interferon or pegylated interferon (peginterferon alfa-2b and peginter- feron alfa-2a), or with ribavirin or with lamivudine or with adefovir.dipivoxil.

18. The method according to any of the claims 9 to 16, wherein clioquinol or phanquinone are administered simultaneously or separately with any combination a nti retroviral regimen.