Patent Application
20050038022
The present invention is concerned with methods and compositions for the treatment of tumors.
Hepatocellular carcinoma (HCC; hepatoma) is one of the most common malignancies and a leading cause of death worldwide (1-3). Untreated, HCC typically has a dismal prognosis. Surgical resection remains the mainstay for treatment of HCC and provides the only consistent long-term tumor-free survival (4). However, resection has been limited primarily by low resectibility rates and recurrent disease. Systemic chemotherapy as a primary treatment modality for HCC has limited value because only a small portion of patients will obtain meaningful palliation with the presently available drugs and regimens (2, 4, 5,) and because the toxicity of currently available chemotherapeutic agents often outweighs their limited benefits (6). Furthermore, liver is the most common site for metastases of colorectal carcinoma which in itself is the leading cause of cancerous death in non-smokers in the developed world (7).
Albendazole (ABZ; methyl 5-propylthio-1H-benzimidazole-2-yl carbamate) is a benzimidazole carbamate (BZs) anthelmintic developed as a veterinary product in 1975. The BZs are now important broad-spectrum drugs for the control of helminth parasites in mammals. They are effective against lungworms and gastrointestinal nematodes, tapeworms and liver flukes (8). The intrinsic anthelmintic action of benzimidazole compounds on parasite relies on a progressive disruption of basic cell functions as a result of their binding to parasite tubulin and depolymerization of microtubules. However, a number of other mechanisms including disruption of glucose uptake and metabolism have also been described for these compounds (9-11).
The present inventors tested BZs and particularly albendazole against a range of liver (HepG2, Hep3B, PLC/PRF/5, SKHEP-1, Hep1-6, HTC, Novikoff) and colorectal cancer (C-170, HT-29 and LOVO) cell lines. The results obtained show potent and dose dependent inhibition of proliferation of these cells by albendazole (and several other BZs). Albendazole was effective against all human and animal cell lines examined, and over a 5 day treatment period, [3H]thymidine incorporation was reduced by over 80% (range 81.6-99.4%) in all these cell lines.
Accordingly, in a first aspect, the present invention provides a method for the treatment of a tumor in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula I:
R3 is selected from H, substituted or unsubstituted, straight or branch chain alkyl, alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, 5- or 6-membered heterocyclic ring the heteroatom(s) of which are selected from one of more of O, S and/or N, —SR14, —OR15, —SOR16, —SO2R17, —SCN, —C(O)—R18, —OR19, NR20COOR21, where R15, to R21 are each independently selected from H, substituted or unsubstituted, straight or branch chain alkyl, alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl or arylalkyl;
or an analogue or metabolite thereof.
In a first aspect, the present invention provides a method for the treatment of a tumor in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula I:
R3 is selected from H, substituted or unsubstituted, straight or branch chain alkyl alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, 5- or 6-membered heterocyclic ring the heteroatom(s) of which are selected from one of more of O, S and/or N, —SR14, —OR15, —SOR16, —SO2R17, —SCN, —C(O)—R18, —OR19, NR20COOR21, where R15 to R21 are each independently selected from H, substituted or unsubstituted, straight or branch chain alkyl, alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl or arylalkyl;
or an analogue or metabolite thereof.
Preferably R1 substitution occurs in the 5 or 6 position and most preferably in the 5 position.
Where R1, R2 and/or R3 are substituted, the substituent(s) may be independently selected from one or more of alkyl, halo, hydroxy or alkoxy.
Preferably the alkyl substituents are C1-6 alkyl. Preferably the aryl substituent are substituted or unsubstituted phenyl.
Preferably the benzimidazole compound used in the method of the present invention is benzimidazole carbamate of Formula II:
The compound of Formula I may be selected from albendazole, mebendazole, flubendazole, triclabendazole, oxfenbendazole, luxabendazole, cambendazole, oxibendazole, parbendazole, thiabendazole or fenbendazole.
Particularly preferred is albendazole:
or an analogue or metabolite thereof. The metabolite may be a major albendazole metabolite such as a sulphoxide or sulphone.
The method of treatment of the invention may be used to treat primary or secondary cancers. The method of the invention may be particularly suitable for the treatment of hepatoma (primary liver cancer) in a subject.
The method of the invention may also be used to treat other cancers, for example, colorectal cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, renal cancer, sarcoma or secondary metastases thereof, particularly in the liver. In addition, the composition used in the method of the present invention may be used to treat peritoneal disease arising from ovarian, gastric or pancreatic cancers.
The method of the invention may include concomitant treatment with a potentiator of the benzimidazole compound effect on the cancer. The potentiator may be an isoquinoline compound (eg praziquantel) or any other compound which will increase or add to the effectiveness of the drug.
Albendazole is poorly absorbed from the gastrointestinal tract and also rapidly undergoes extensive first pass metabolism. At all times after the administration of a 400 mg oral dose, the concentration of the unchanged drug has been below the detection limits (18). This is mainly because of the rapid and extensive metabolism of the drug in the liver. Hydrolysis of the carbamate moiety and oxidation of the sulphur atom, the alkyl side chain and the aromatic ring have all been observed to occur in man. Five major metabolites have been identified in the human urine of which albendazole sulphoxide is the major one. The sulphoxide is biologically active and contributes to the activity of the drug. It attains peak plasma concentrations of about 200-300 ng/ml and has a plasma half life of about 8-9 hours. Together with other metabolites, it is mainly excreted in the urine with a small amount being excreted in the faeces (18,19).
As a result of this extensive metabolism, the parent drug is virtually undetectable in the body, and its anthelmintic effect seems to be partly exerted by the unabsorbed portion left in the intestine and partly by the active sulphoxide metabolite formed in the liver. However, to be effective in the treatment of HCC a concentration of greater than 100 nM must be available in the immediate vicinity of the tumor cells which means that, to attain effective and sustained antitumor concentrations of albendazole, large and frequent doses must be administered.
Secondly, the use of the drug as an anthelmintic has been associated with a number of side effects including mild and transient epigastric distress, diarrhoea, nausea, dizziness, lassitude and insomnia in short term treatments and reversible low grade transaminase elevation, jaundice, gastro intestinal symptoms, alopecia, rash or pruritus and leukopenia have been reported in patients under 3 month treatment courses for hydatid disease. Long term toxicity studies in animals showed diarrhoea, anaemia, hypotension, marrow depression, liver function test abnormalities, and fetal toxicity, varying by species (13).
The present inventors believe that regional administration of the benzimidazole compound to the liver may resolve the above mentioned limitations in the employment of the drug in method of treatment of the present invention. The present inventors also believe that this benefit may also be obtained through regional delivery of the benzimidazole compound to tumors of other cancers such as colorectal cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, mesothelioma or renal cancer.
Accordingly, in a second aspect the present invention consists in a method of treatment of a tumour in a subject, the method comprising regionally delivering to the site of the tumour a composition comprising a therapeutically effective amount of a compound of Formula I:
R3 is selected from H, substituted or unsubstituted, straight or branch chain all, alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, 5- or 6-membered heterocyclic ring the heteroatom(s) of which are selected from one of more of O, S and/or N, —SR14, —OR15, —SOR16, —SO2R17, —SCN, —C(O)—R18, —OR19, NR20COOR21, where R15 to R21 are each independently selected from H, substituted or unsubstituted, straight or branch chain alkyl, alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl or arylalkyl;
or an analogue or metabolite thereof.
Preferably R1 substitution occurs in the 5 or 6 position and most preferably in the 5 position.
Where R1, R2 and/or R3 are substituted, the substituent(s) may be independently selected from one or more of alkyl, halo, hydroxy or alkoxy.
Preferably the alkyl substituents are C1-6 alkyl. Preferably the aryl substituent are substituted or unsubstituted phenyl.
Preferably the benzimidazole compound used in the method of the present invention is benzimidazole carbamate of Formula II:
The compound of Formula I may be selected from albendazole, mebendazole, flubendazole, triclabendazole, oxfenbendazole, luxabendazole, cambendazole, oxibendazole, parbendazole, thiabendazole or fenbendazole.
Particularly preferred is albendazole:
or an analogue or metabolite thereof. The metabolite may be a major albendazole metabolite such as a sulphoxide or sulphone.
The method of the second aspect is particularly suitable for the treatment of tumor of the liver. The tumor may be a hepatoma (primary liver cancer) or a secondary cancer in the liver. Preferably regional delivery to the liver is via the intrahepatic artery.
The method of the second aspect of the invention may also be used to treat other cancers, for example, colorectal cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, renal cancer or secondary metastases in other organs.
Regional delivery of the benzimidazole compound may be achieved by administering the compound in a pharmaceutically acceptable formulation. The composition may be administered as continuous infusion of a solution via a pump through the major artery of the diseased organ for example hepatic artery for hepatomas. Furthermore, the composition may be administered intraperitoneally as a suspension to treat peritoneal disease arising from ovarian, pancreatic, gastric, or any other cancer.
The formulation preferably comprises a lipid. Particularly preferred are lipids for which the tumor is avid so that high concentrations of the drug may be delivered to the tumor.
Preferably the lipid is an oil. Preferably the formulation comprises an iodised oil. A particularly preferred iodised oil is lipiodol, an iodinated ethyl ester of the poppy seed oil.
Compared to systemic administration, regional delivery using a lipid such as lipiodol allows achievement of higher drug concentrations in the tumor site while reducing the degree of exposure of other body organs to the unwanted effects of the drug and consequently reducing the number and the severity of side effects. In HCC this can be made even more selective and effective by choosing lipiodol as the vehicle for the drug delivery.
When injected into the hepatic artery, the oil is retained by HCCs for several weeks to over a year but is cleared from the normal liver parenchyma within 7 days. Without wishing to restrict the present invention in any way, one of the hypotheses in attempting to explain lipiodol retention in HCCs suggests that these cells are unable to clear lipiodol because they lack a reticuloendothelial kupffer cell component. We have previously shown that in vitro, vitamin D compounds such as 1,25-dihydroxyvitamin D3 dissolved in lipiodol produce a profound and sustained inhibitory effect on HepG2 cells when injected through the hepatic artery of tumor bearing rats, the drug is retained within the tumor (See International Patent Application Nos. PCT/AU98/00440 and PCT/AU99/00323 the disclosure of which is incorporated herein by reference).
On the basis of the present inventors experience with albendazole, lipiodol, and hepatoma cell lines, they believe that administration of albendazole dissolved in an oil such as lipiodol and administered through the intrahepatic artery, will lead to the sustained release of the drug from the oil within the tumor cells leading to sustained inhibition of proliferation of the tumor cells.
These unique characteristics of lipiodol coupled with the potency and lipid solubility of albendazole, makes the combination an attractive formulation for intrahepatic arterial administration in patients with HCC.
In a third aspect, the present invention provides a pharmaceutical composition for use in the treatment of a tumour in a subject, the composition comprising a carrier and an effective amount of a compound of Formula I:
R3 is selected from H, substituted or unsubstituted, straight or branch chain alkyl, alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, 5- or 6-membered heterocyclic zing the heteroatom(s) of which are selected from one of more of O, S and/or N, —SR14, —OR15, —SOR16, —SO2R17, —SCN, —C(O)—R18, —OR19, NR20COOR21, where R15, to R21 are each independently selected from H, substituted or unsubstituted, straight or branch chain alkyl, alkenyl, alkenylalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenylalkyl, aryl or arylalkyl;
or an analogue or metabolite thereof.
Preferably R1, substitution occurs in the 5 or 6 position and most preferably in the 5 position.
Where R1, R2 and/or R3 are substituted, the substituent(s) may be independently selected from one or more of alkyl, halo, hydroxy or alkoxy.
Preferably the alkyl substituents are C1-6 alkyl. Preferably the aryl substituent are substituted or unsubstituted phenyl.
Preferably the benzimidazole compound used in the method of the present invention is benzimidazole carbamate of Formula II:
The compound of Formula I may be selected from albendazole, mebendazole, flubendazole, triclabendazole, oxfenbendazole, luxabendazole, cambendazole, oxibendazole, parbendazole, thiabendazole or fenbendazole.
Particularly preferred is albendazole:
or an analogue or metabolite thereof. The metabolite may be a major albendazole metabolite such as a sulphoxide or sulphone.
Preferably the carrier is a lipid. Preferably the lipid is one for which the tumor is avid. Most preferably the carrier is an oil. An iodised oil is particularly preferred. The iodised oil is preferably lipiodol.
Preferably the benzimidazole compound is present in the composition in a concentration of at least about 0.1M. The concentration of the benzimidazole compound is preferably in the range of about 0.1 to about 10 M.
The composition of the invention may include a potentiator of the effect of the benzimidazole compound on the cancer. The potentiator may be, for example, praziquantel or any other compound which would increase the effectiveness of the drug, have an additive effect with it, or reduce its side effects.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
In order that the nature of the present invention may be more fully understood the invention will now be described with reference to the following non-limiting embodiments.
Materials and Methods
Cell Culture
HepG2, Hep3-B, Hep1-6, SKHEP-1, PLC/PRF/5, and HTC cells were obtained from European Collection of Cell Cultures (ECACC; U.K), Novikoff was obtained from Cancer Research Centre (DKFZ) Heidelberg, Germany. Cells were cultured in MEM or DMEM supplemented with 10% FBS, 50 units/ml penicillin, 50 units/ml streptomycin, 25 μg/ml amphotericin B (Gibco, Grand Island, N.Y.) and maintained subconfluent at 37° C. in humidified incubators containing 5% CO2. Albendazole (Sigma, Australian subsidiary) was dissolved in absolute ethanol at concentrations that were 1000-fold higher than the final medium concentration.
[3H]Thymidine Incorporation Assay
For the study of [3H]thymidine incorporation, adherent cells (5-10×104) were plated onto 24-well Corning tissue culture dishes and were exposed to culture medium (5% FBS) containing the vehicle (0.1% ethanol) or different concentrations of albendazole (10−8 to 10−6 M). For Novikoff, a detached rat cell line, 2500 cells were suspended in 2 ml of DMEM (5% FBS) and kept under the same condition as for attached cells. Media were replaced with fresh media on alternate days. At the end of the treatment period (5 days), cell cultures were assayed for thymidine incorporation by the addition of 0.5 μCi of [3H]thymidine (60 Ci/mmol. ICN Biochem, Irvine, Calif.) to each well for the last 4 h of culture. The amount of radioactivity incorporated into cells was determined using a β-scintillation counter. Results are presented as percentage [3H]thymidine incorporation relative to control. For the recovery experiments, SKHEP-1 cells were treated for 1 day with different concentrations of albendazole and then either assayed for [3H]thymidine incorporation or the medium was replaced and the cells treated with fresh medium with out the drug for a further 4 days at the end of which, [3H] thymidine incorporation assay was performed.
Cell Counts
SKHEP-1 cells (2.5×104) were plated in six well plates. The cell treatment procedure was as described for the thymidine assay. At the end of the treatment period (1, 3 or 5 days), cells were trypsinized and counted with a hemocytometer using the trypan blue exclusion method. In all experiments, cells treated with the medium containing 0.1% ethanol were taken as the control for albendazole treated cells. All counts were obtained in quadruplicate and each experiment was repeated at least twice.
Cell Cycle Analysis
SKHEP-1 cells (5×104) were plated onto six-well tissue culture plates. Triplicate samples were treated with the indicated concentrations of albendazole (100, 250 and 1000 nM). The medium was changed everyday. After 72 h the relevant group of cells were collected, washed twice with phosphate buffer and treated with ribonuclease, Triton X-100 and propidium iodide (Sigma) based on the method described by Taylor [12]. The percentage of cells within the G1, S, and G2-M phases of the cell cycle were determined using a FACScan flow cytometer (Becton Dickinson FACSort) and Multifit LT cell cycle analysis software (Verity Software INC.)
Tumor Formation in Nude Mice
6 to 10 weeks old male BALB/c Nu/Nu mice (Animal Resources Center, Perth, Australia) were inoculated subcutaneously with 106 SKHEP-1 cells into the right flank. 24 hours after inoculation animals were randomly assigned to one of the treatment groups (n=10 per group), receiving 25, 50 or 150 mg/kg twice daily oral albendazole suspended in sesame oil for 20 days.
The control group was treated with the vehicle (sesame oil). Using vernier calipers, tumor diameter (mm) was measured on day eight and then every three days up to day 20-post tumor cell inoculation. Tumor volumes were calculated using the formula: ab2/2 where a and b are the smaller diameters in millimeters, respectively [13] and a piece of the tumor was preserved in paraffin for immunohistochemical determination of maximum proliferation index (MPI). Here, after fixation, the specimen was processed for the detection of Ki-67 antigen with the monoclonal antibody MIB1 according to the method described by McCormick [15].
The animal model was chosen on the basis of SKHEP-1 being the most tumorigenic human liver cancer cell line in nude mice [16] and previous experience with the model [17].
Statistical Analysis
Differences between different treatment groups were analyzed using ANOVA followed by Tukey's test P values of less than 0.05 were considered to represent a significant difference.
Results
Inhibition of [3H]Thymidine Incorporation by Albendazole
[3H]Thymidine incorporation assay was used to determine the effect of albendazole on cell proliferation in a number of human (HepG2, Hep3-B, PLC/PRF/5, SKHEP-1), rat (HTC and Novikoff) and mice (Hep1-6) HCC cell lines. Results obtained show that, in all cell lines examined, albendazole effectively reduces thymidine incorporation (Table 1). When treated with the 100 nM concentration of albendazole, compared to other cell lines, SKHEP-1 demonstrated the highest level of sensitivity to albendazole (p<0.01 compared to control), while the rat cell line HTC was the least responsive of all. Treatment with the 1000 nM concentration of albendazole reduced thymidine incorporation to less than 20% of control values (p<0.001) in all cell lines and to less than 5% in SKHEP-1 and HepG2. Here again SKHEP-1 cells displayed the highest level of sensitivity to albendazole. In these cells, thymidine incorporation was reduced to 0.6±0.1% of the control values corresponding to 99.4% Inhibition For this reason, SKHEP-1 was employed for all further investigations. Exposure of SKHEP-1 cells to different concentrations of albendazole for 1 day, revealed that, concentrations of 250 nM and over of albendazole still produce profound inhibition of thymidine incorporation (
Cells were treated with different concentrations of albendazole (10-1000 nM) for 5 days at the end of which [3H]thymidine incorporation was measured. Values (% control) represent mean ± s.e.m. of several determinations.
Albendazole Inhibits Proliferation of Cells Leading to a Decline in Cell Number
Counting of viable cells treated with different concentrations of albendazole for 1, 3 or 5 days produced a dose dependent decline in the number of cells, showing the profound inhibition of proliferation of SKHEP-1 cells by the drug (
This was evident from day 3 at the 500 and the 1000 nM concentrations. Compared to control, cells exposed to the 1000 nM concentration of the drug, were significantly reduced in number (p<0.001).
Dose-Dependent Effect of Albendazole on the Cell Cycle Kinetics
Flow cytometric analysis of albendazole-treated cells, revealed that, the drug induces a dose-dependant effect on the cell cycle kinetics of SKHEP-1 HCC cells.
Effect of Albendazole on Tumor Growth In Vivo
In control animals, SKHEP-1 tumors grew to a mean volume of 87.9±12.3 mm3 at 20 days post inoculation In animals receiving 50 and 150 mg/kg per day, tumor growth was slightly but not significantly retarded. However, tumor growth was profoundly suppressed in animals receiving the 300 mg/kg dose of albendazole (
Dose-Dependent Effect of Albendazole on the Cell Cycle Kinetics
Albendazole was also shown to exhibit dose dependent inhibition of proliferation of the ovarian cancer cell line (OVCAR-3) in vitro, (see
Discussion
Results from the cell proliferation studies dearly demonstrated that all human, rat and mice liver cell lines examined are profoundly inhibited by albendazole. This was manifested by the significant reduction of thymidine incorporation following treatment with albendazole doses of 100 nM and over. Similarly treatment of SKHEP-1 cells with albendazole led to a dose and time dependent reduction of cell number. The reason behind the higher sensitivity of SKHEP-1 to albendazole is not dear at this stage. However, lacking the enzymes needed for the conversion of the drug to less active or inactive metabolites, may partly account for this observation [14]. Flow cytometric analysis of the cell cycle revealed that, albendazole causes differential dose-dependent effect on the cell cycle kinetics of SKHEP-1. Accumulation of cells in the G0-G1 phase following treatment with albendazole concentrations of up to 500 nM with an associated decline in percentage of cells in S and G2-M phases, indicates that progression out of the G1 phase was blocked. Many natural triggers for programmed cell death, including glucocorticoid hormones act at G1-G0 transition and the cells die in a process described as ‘premature aging’ [18]. However, following treatment with the 1000 nM concentration of albendazole, the pattern of cell distribution was reversed, leading to the accumulation of cells in the G2-M phase of the cycle. This indicates that the primary effect of albendazole at this concentration may be mediated by a transition delay through G2-M or mitosis.
The data from work in nude mice suggests that, at the higher dose of the 300 mg/kg/day, albendazole presumably reaches the necessary concentrations required to suppress tumor formation. The very high rate of metabolism of albendazole in mice and the poor blood supply to the subcutaneous tumor, are amongst a number of factors that could account for the high dose of the drug required to suppress tumor growth in these animals.
The MPI data also confirm the ability of albendazole to reduce tumor proliferation rate. The Ki-67 antigen used in this assay is tightly linked to proliferation and has been used in a large number of studies to estimate the growth fraction of tumors [15].
Albendazole in Patients with Advanced Malignancy
Patents and Methods
The study was single-centre, open and non-controlled. Nine patients (8 male and 1 female) with either advanced CRC and hepatic metastasis or HCC were included in this study. One patient with neuroendocrine cancer and mesothelioma was also treated on a compassionate basis. The patients aged between 38-79 years were inoperable and had failed existing chemotherapy and also, except for two, had measurable and increasing tumor markers. The majority had also already failed hepatic artery chemotherapy. The diagnosis of CRC or HCC was made by ultrasound, CT or MRI scan, confirmed by histology and by determination of CEA or AFP levels for CRC or HCC respectively. Only patients with expected survival of more than one month were enrolled into the study. Patient characteristics are presented in Table 2. The study was approved by the Human Ethics Committee for Research of SESAHS. The protocol and the aim of the study was clearly explained to each patient and informed consent obtained. The duration of this study was four weeks but was to be stopped if leukopenia (WBC<2×106) or severe hepatocellular injury (ALT or AST 2× upper limit) developed. All patients received albendazole (400 mg scored tablets, Smith Kline Beecham, Australian subsidiary) 10 mg/kg orally in two or three divided doses for a planned duration of four weeks. This is the clinical dose of albendazole employed in the treatment of parasitic diseases. Patients were evaluated every three days by clinical examination together with full blood tests to monitor tumor markers, hematopoesis, liver and kidney function toxicity. A partial response (PR) was defined as a 50% or more decrease in the value of the markers. In addition there must be no new lesions or progression of any other lesions. Stable disease was defined as a decrease of less than 50%, or an increase of less than 25% in the value of tumor markers, while progressive disease (PD) was a 25% or more increase in the value of the tumor markers or the appearance of any new lesions.
MBL = multiple bilobar liver
*Mesothlioma and carcinoid tumor.
Results
Treatment with albendazole led to stabilization of the disease in three, and progression of the disease in the other two patients (
There were no significant changes in liver and kidney function tests during the course of the trial. However, in patients 1, 2 and 8 significant neutropenia developed as a result, of which the drug had to be withdrawn. WCC values for all nine patients are presented in
Discussion
To the present inventors' knowledge, this is the first reported study of albendazole (or any other BZ) administered to human subjects for the therapy of cancer. Patients participating in this study had advanced malignancy, which had not responded to available therapy. Administration of albendazole to the only patient in the study with primary HCC led to the stabilisation of the AFP values. However, due to neutropenia, the drug had to be withdrawn. The patient had a recent history of low WCC. In the remaining patients (all with CRC) with measurable CEA levels, three had their tumor markers stabilised while on albendazole treatment Compared to other patients, patient number two, who was withdrawn from the trial on day 19, had the steepest fall in CEA levels.
The present report demonstrates for the first time that, albendazole, a benzimidazole carbamate with extensive clinical use as a safe antiparasitic drug, can cause tumor marker stabilisation in patients with HCC or CRC with liver metastasis.
To study the feasibility of using albendazole in the treatment of peritoneal disease, the human ovarian cancer cell line NIH:OVCAR-3 was selected. This is a cell line that grows quite slowly but is tumorogenic in nude mice therefore allowing for the drug to be studied under both in-vitro and in-vivo conditions.
Thymidine Assay
After finding the right conditions for the growth of cells in culture, cells were treated for 5 days with various concentrations of albendazole.
It was shown that concentrations as low as 0.001 micromoles/L of albendazole have an effect on the cell proliferation and that at a concentration of 0.25 micromoles/L, around 90% cell inhibition is achieved and at 0.5 micromoles/L inhibition of cell growth is 100%.
These results show that, albendazole is very effective against this human ovarian cancer cell. The degree of activity of albendazole is about ×10 more than that observed for liver or colorectal cancer cells where inhibitory activity starts at around 0.1 micromoles/L.
Cell Count
Cells were treated with either the medium alone (control) or 0.1 and 1 micromoles/L of albendazole for 0, 1, 3, 6 or 10 days and the number of viable cells counted. It was observed that at both doses employed albendazole treatment leads to profound reduction in cell number. This was evident from day 1 after the treatment
Albendazole/Albendazole Sulfoxide Against Liver, Colorectal and Prostate Cancer Cell Lines:
Animal and human studies have shown rapid conversion of albendazole to a sulphoxide (ABS) and then to a sulphone and a number of other metabolites. As an antiparasite, ABS is considered to be responsible for most or some of the systemic biological activity of albendazole, whereas, the sulphone metabolite is pharmacologically inactive. Several studies have shown that ABS is almost equally effective against parasites as the albendazole itself. Furthermore it is believed that the activity in the clinical setting is mainly due to the formation of ABS. So much so that ABS has now been considered as a new drug (ricobendazole) by some investigators. This knowledge together with some of our observations in the colorectal patients undergoing albendazole therapy, led us to test ABS against cancer cells. To date, there has been no literature on this issue.
In order to be able to test ABS, a proper solvent had to be found. This is because unlike albendazole ABS is not soluble in ethanol. After a number of tests, DMSO (0.5%) was found to be the most suitable solvent for this compound and for this type of investigation. Higher DMSO concentrations were found to be too toxic to the cells while lower concentrations led to incomplete solubility of ABS. Therefore to allow direct comparison between the 2 compounds, all subsequent experiments were run using 0.5% DMSO for both ABS and albendazole. In the cell lines examined, albendazole produced very similar results to what we had previously reported for the drug in liver cancer cells.
Both albendazole and ABS were tested side by side against various cell lines as follows:
The fist 9 cell lines were used to compare effectiveness and potency while, the last one was used to assess toxicity on bone marrow (as a rough guide).
Cells were treated with various concentrations of each compound for a period of 5 days.
Albendazole sulfoxide was shown to inhibit proliferation of human liver cancer cells HepG2, SKHEP-1 and Hep 3B.
ABS produced the same sort of activity against colorectal cell lines C-170, LOVO and HT-29. In general results obtained for ABS versus albendazole show a great degree of similarity between the 2 compounds with antiproliferative activity becoming evident at 0.01 micromoles/L concentration and dramatically increasing at 0.25 micromoles/L and nearing 100 inhibition at 1 micromoles/L.
Results obtained with prostate cell lines all demonstrate that, the drug and its major metabolite (ABS) are almost equipotent in inhibiting the proliferation of the human prostate cancer cells PC3, LNCap and Du 145.
Laddering:
To find out the way albendazole works as an anticancer drug, a large variety of tests can be performed. One of the first is to see if albendazole induces apoptosis. This again is tested in several ways, one of the most common is to check for DNA laddering. In our laboratories, after spending some time to find the right concentration and the right period of treatment, finally we could see dear evidence of induction of DNA laddering in these ovarian cancer cells by albendazole. This shows that, albendazole treatment leads to apoptosis. This is the first time that the induction of apoptosis by albendazole has been reported.
Similar results were also obtained with the human colorectal cell line LOVO. DNA obtained from LOVO cells treated with 1 μM albendazole for 16 hours showed clear laddering demonstrating that albendazole induces apoptosis in LOVO cells.
Results obtained with the ovarian cell line (NIH:OVCAR-3) show for the first time that, albendazole is profoundly effective in inhibiting its proliferation in culture in a dose-dependent fashion. This was confirmed by both thymidine uptake and cell count. Further work clearly shows that these cells undergo apoptosis as a result of albendazole treatment.
Results obtained in liver and colorectal cell lines confirm the antiproliferative activity of albendazole against these cell lines. Results obtained with the 3 human prostate cell lines indicate that, these cells too, are quite sensitive to the effect of albendazole in culture. There is no previous report on the antiproliferative activity of albendazole in prostate cell lines making this the first to show such an activity.
It is well known for years that, conversion of albendazole in the body takes place quite rapidly in the liver and yields an active metabolite namely albendazole sulfoxide, which has been shown to be active against parasites. In this respect, our earlier results with the sulfoxide has shown it to be as effective as the parent drug (albendazole) in the 10 different cell lines examined. However, our latest results castes some doubt over this, and the compound is now under intensive investigation in our laboratories.
Combination Therapy:
The effect of inhibition of metabolism of albendazole on its antiproliferative activity was investigated using human colorectal cell line LOVO and the methimazole as an inhibitor of albendazole
5000 cells each of HepG2 and S EP1 were plated into 24 wells plates and treated for 5 days with various concentrations of albendazole alone or together with MT.
There was no growth inhibition of HepG2 cells when treated with albendazole for 5 days at the doses (0.25, 0.1 and 0.01uM) tested in this experiment. However, there was modest growth inhibition when albendazole and MT were used together. On the other hand, albendazole at 0.25 μM concentration profoundly inhibited the grout of SKHEP-1 cells. This response was further enhanced in the presence of MT. MT is an antithyroid agent which acts as a competitive inhibitor of microsomal Flavin Mono-oxygenase (FMO) dependent oxidation of several drugs. MT has been shown to inhibit the metabolism of drugs dependant on this pathway. MT mediated inhibition of liver sulfoxidation of albendazole to albendazole sulfoxide has been shown both in-vitro and in-vivo.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2342472 | Mar 2001 | CA | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU02/00339 | 3/20/2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 60278435 | Mar 2001 | US |
