ES2393184T3 - Method for the treatment of cancer and compositions for use therein - Google Patents

Abstract

A pharmaceutical composition for use in a method of treating ovarian cancer in a regional administration subject, in which the composition comprises a lipid carrier and a carbamate mustzimidazole which is albendazole or a sulfoxide or sulfone thereof.

Description

Method for the treatment of cancer and compositions for use therein

FIELD OF THE INVENTION

The present invention relates to compositions for the treatment of tumors, particularly the treatment of ovarian cancer.

BACKGROUND OF THE INVENTION

Hepatocellular carcinoma (HCC; hepatoma) is one of the most common malignant tumors and one of the leading causes of death worldwide (1–3). If left untreated, the HCC usually has a daunting prognosis. Surgical resection remains the main basis for the treatment of HCC and provides the only consistent long-term tumor-free survival (4). However, resection has been limited mainly by low rates of resection capacity and recurrent disease. Systemic chemotherapy has a limited value as a primary treatment modality for HCC, because only a small part of patients will obtain significant relief with the drugs and regimens currently available (2, 4, 5) and because Often, the toxicity of currently available chemotherapeutic agents outweighs their limited benefits (6). In addition, the liver is the most common site for metastatic colorectal carcinoma, which is itself the leading cause of cancer death in nonsmokers in the developed world (7).

Albendazole (ABZ; 5-propylthio-1H-benzimidazol-2-yl-carbamate methyl) is a benzimidazole carbamate (BZ) anthelmintic developed as a veterinary product in 1975. BZs are currently important broad-spectrum drugs for the control of helminth parasites in mammals. These are effective against pulmonary worms and gastrointestinal nematodes, tapeworms and hepatic fasciolas (8). The intrinsic anthelmintic action of benzimidazole compounds on the parasite is based on a progressive disruption of basic cellular functions as a result of their binding to the parasite tubulin and depolymerization of microtubules. However, a number of other mechanisms have also been described for these compounds, including interruption of uptake and glucose metabolism (9–11).

Pourgholumi et al. (Cancer Letters 2001, 165, 43–49) describes in vitro and in vivo suppression of hepatocellular carcinoma cell growth by albendazole. The use of albendazole to treat ovarian cancer is not described.

SUMMARY OF THE INVENTION

The inventors of the present invention tested BZ and, in particular, albendazole against a variety of colorectal (C-170, HT-29 and LOVO) and liver cancer (HepG2, Hep3B, PLC / PRF) cell lines / 5, SKHEP – 1, Hep1–6, HTC, Novikoff). The results obtained show a potent and dose-dependent inhibition of the 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, the incorporation of [3 H] thymidine was reduced by more than 80% (range 81.6 - 99 , 4%) in all these cell lines.

Accordingly, the present invention provides a pharmaceutical composition for use in a treatment method, as described in claim 1.

DESCRIPTION OF THE FIGURES

Figure 1. These figures are presented together with reference examples, for a useful understanding of the present invention. The incorporation of [3H] thymidine [expressed as counts per minute (CPM)] in SKHEP-1 cells was measured either (a) immediately after a 1-day treatment with albendazole, or (b) after 1-day treatment with albendazole followed by a 4-day treatment with the medium alone (not containing the drug). The data points are the mean ± SEM.

Figure 2. These figures are presented together with reference examples, for a useful understanding of the present invention. Effects over time of albendazole on the number of SKHEP – 1 cells. Cells growing in 6-well plates were treated for 1, 3 or 5 days with albendazole (0, 100, 500 or 1,000 nM) and the number of viable cells was counted using the trypan blue exclusion method. The data points are the mean ± SEM.

Figure 3. These figures are presented together with reference examples, for a useful understanding of the present invention. Effect of albendazole on the cell cycle phase of SKHEP – 1 cells. The cells were treated with different concentrations of albendazole (0, 100, 250, and 1,000 nM) for 3 days, stained with propidium iodide and analyzed for DNA content by flow cytometry. A total of 10/000 cores were analyzed from each sample. The data points are the mean ± SEM of the percentage of cells within the G0 – G1, S and G2 – M phases of the cell cycle.

Figure 4. These figures are presented together with some reference examples, for a useful understanding of the present

invention. Effect of different doses of albendazole (0, 50, 150 and 300 mg / kg / day in two divided doses administered orally in sesame oil) on the formation and subcutaneous tumor growth of SKHEP– 1 in nude mice. Changes in tumor volumes were measured every 3 days. Each value represents the mean * ± SEM of 10 animals.

Figure 5. Inhibition as a function of the concentration of [3 H] thymidine uptake (ovarian cancer cell line (OVCAR-3) by albendazole in vitro.

Figure 6. These figures are presented together with reference examples, for a useful understanding of the present invention. Serum tumor marker levels (AFP or CEA) in patients with liver tumors (CRC or HCC) undergoing treatment with albendazole (10 mg / kg / day in two or three divided oral doses) for 28 days. The arrow indicates the beginning of therapy.

Figure 7. These figures are presented together with reference examples, for a useful understanding of the present invention. Serum leukocyte count (WCC) in patients with liver tumors (CRC or HCC) undergoing treatment with albendazole (10 mg / kg / day in two or three divided oral doses) for 28 days.

The composition for use in the present invention comprises albendazole:

or a sulfoxide or sulfone thereof. The composition is for use in a method of treating ovarian cancer in a subject.

The method can be used to treat a primary or secondary cancer.

The method may include concomitant treatment with a potentiator of the effect of the benzimidazole compound on cancer. The enhancer can be an isoquinoline compound (for example, praziquantel) or any other compound that increases or adds to the effectiveness of the drug.

Albendazole is poorly absorbed from the gastrointestinal tract and also quickly experiences an extensive first-pass metabolism. At all times after the administration of an oral dose of 400 mg, the concentration of the unchanged drug has been below the detection limits (18). This is mainly due to the rapid and extensive metabolism of the drug in the liver. It has been observed that all of the hydrolysis of the carbamate moiety and the oxidation of the sulfur atom, the aromatic ring and the alkyl side chain take place in man. Five major metabolites have been identified in human urine, of which albendazole sulfoxide is the largest. The sulfoxide is biologically active and contributes to the activity of the drug. It reaches peak plasma concentrations of approximately 200–300 ng / ml and has a plasma half-life of approximately 8–9 hours. Together with other metabolites, it is excreted mainly in the urine, with a small amount being excreted in the feces (18, 19).

As a result of this extensive metabolism, the precursor drug is virtually undetectable in the body, and its anthelmintic effect seems to be exerted in part by the non-absorbed part that remains in the intestine and, in part, by the active sulfoxide metabolite that forms in the liver. However, to be effective in the treatment of HCC, a concentration of more than 100 nM must be available in the immediate vicinity of the tumor cells, which means that, in order to achieve effective and sustained antitumor concentrations of albendazole, they must administered large and frequent doses.

Second, the use of the drug as an anthelmintic has been associated with a number of side effects, including mild and transient epigastric insufficiency, diarrhea, nausea, dizziness, lassitude and insomnia in short-term treatments, and an elevation of transaminase levels. Low reversible grade, jaundice, gastrointestinal symptoms, alopecia, rash or pruritus and leukopenia have been reported in patients undergoing 3-month treatment cycles for hydatidosis. Long-term toxicity studies in animals showed diarrhea, anemia, hypotension, bone marrow depression, liver function abnormalities and fetal toxicity, varying by species (13).

The inventors of the present invention believe that regional administration of the benzimidazole compound to the liver can resolve the limitations mentioned above in the use of the drug in the method of treatment of the present invention. The inventors of the present invention also believe that this benefit can also be obtained through regional administration of the benzimidazole compound to ovarian cancer.

Regional administration of the benzimidazole compound is achieved by administering the compound in a pharmaceutically acceptable formulation. The composition can be administered as a continuous infusion of a solution by means of a pump through the main artery of the diseased organ.

The formulation comprises a lipid. Lipids for which the tumor shows avidity are particularly preferred, such that high concentrations of the drug can be administered to the tumor.

Preferably, the lipid is an oil. Preferably, the formulation comprises an iodized oil. A particularly preferred iodized oil is lipiodol, an iodinated ethyl ester of poppyseed oil.

Compared to systemic administration, regional administration using a lipid such as lipiodol allows the achievement of higher drug concentrations at the tumor site while reducing the degree of exposure of other body organs to non-effect. desired drug and, consequently, the number and severity of side effects is reduced. In HCC, this can be made even more selective and effective by choosing lipiodol as the vehicle for drug administration.

The HCC is described below for the useful understanding of the present invention. When injected into the hepatic artery, the oil is retained by HCC for several weeks to more than a year, but is removed from the parenchyma of a normal liver within 7 days. Without intending to restrict the present invention in any way, one of the hypotheses in trying to explain the retention of lipiodol in HCC suggests that these cells are unable to eliminate lipiodol because they lack a reticulo-endothelial Kupffer cell component. . The inventors of the present invention have previously shown that in vitro vitamin D components, such as 1,25-dihydroxyvitamin D3 dissolved in lipiodol, produce a deep and sustained inhibition effect on HepG2 cells when injected through the hepatic artery of tumor-bearing rats, the drug is retained within the tumor (see WO 98/56337 and WO 99156697.

Based on the experience of the inventors of the present invention with the 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 sustained release of the drug from the oil inside the tumor cells, which leads to the sustained inhibition of tumor cell proliferation.

These unique characteristics of lipiodol coupled with the potency and lipid solubility of albendazole make the combination an attractive formulation for intrahepatic arterial administration in patients with HCC.

Preferably, the benzimidazole compound is present in the composition at a concentration of at least about 0.1 M. 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 an enhancer of the effect of the benzimidazole compound on cancer. The enhancer can be, for example, praziquantel or any other compound that increases the effectiveness of the drug, that has an additive effect with it, or that reduces its side effects.

Throughout the present specification, it will be understood that the word "understand", or variations such as "comprises" or "comprising / comprising", implies the inclusion of an indicated element, integer or stage, or group of elements, integers or stages; but not the exclusion of any other element, integer or stage, or group of elements, integers or stages.

In order that the nature of the present invention may be more fully understood, the invention will be described below with reference to the following non-limiting embodiments.

REFERENCE EXAMPLE 1

In vitro and in vivo suppression of hepatocellular carcinoma cell growth by albendazole

Materials and methods

Cell culture

HepG2, Hep3 – B, Hep1–6, SKHEP – 1, PLC / PRF / 5, and HTC cells were obtained from the European Cell Culture Collection (ECACC; RU.), Novikoff was obtained from the Center for Cancer Research ( DKFZ), Heidelberg, Germany. The cells were cultured in MEM or DMEM supplemented with 10% FBS, 50 units / ml of penicillin, 50 units / ml of streptomycin, 25 IgIml of amphotericin B (Gibco, Grand Island, NY) and kept subconfluent at 37 ° C in humidified incubators containing 5% CO2. Albendazole (Sigma, Australian subsidiary) was dissolved in absolute ethanol at concentrations that were 1,000 times higher than the concentration in the final medium.

[3 H] thymidine incorporation assay

For the study of the incorporation of [3 H] thymidine, adherent cells (5–10 x 104) were placed on 24-well Corning tissue culture plates and exposed to a culture medium (5% FBS) that It contained the vehicle (0.1% ethanol) or different concentrations of albendazole (10–8 to 10–6 M). For Novikoff, a separate rat cell line, 2,500 cells were suspended in 2 ml of DMEM (5% FBS) and maintained under the same condition as for the bound cells. The media were replaced with new media on alternate days. At the end of the treatment period (5 days), cell cultures were evaluated for thymidine incorporation by adding 0.5 ICi of [3 H] thymidine (60 Ci / mmol. ICN Biochem, Irvine, CA) to each well during the last 4 h of cultivation. The amount of radioactivity incorporated into the cells was determined using a scintillation counter �. The results are presented as the percentage of [3 H] thymidine incorporation in relation to the control. For recovery experiments, SKHEP-1 cells were treated for 1 day with different concentrations of albendazole and then either evaluated for incorporation of [3 H] thymidine or the medium was replaced and the cells were treated with a new medium without the drug for another 4 days, at the end of which the [3H] thymidine incorporation test was performed.

Cell counts

SKEP-1 cells (2.5 x 104) were placed 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), the 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 cells treated with albendazole. All counts were obtained in quadruplicate and each experiment was repeated at least twice.

Cell cycle analysis

SKHEP-1 cells (5 x 104) were placed on six-well tissue culture plates. Samples were treated in triplicate with the indicated concentrations of albendazole (100, 250 and 1,000 nM). The medium was changed every day. After 72 h, the cells of the relevant group 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 was determined using a FACScan flow cytometer (Becton Dickinson FACSort) and a Multifit LT cell cycle analysis software (Verity Software INC.)

Tumor formation in nude mice

Male BALB / c Nu / Nu mice 6 to 10 weeks old (Animal Resources Center, Perth, Australia) were inoculated subcutaneously with 106 SKHEP-1 cells on the right side. 24 hours after inoculation, the animals were randomly assigned to one of the treatment groups (n = 10 per group), receiving 25, 50 or 150 mg / kg of oral albendazole suspended in sesame oil twice daily. for 20 days

The control group was treated with the vehicle (sesame oil). Using vernier calipers, the diameter of the tumor (mm) was measured on day eight and then every three days until day 20 after inoculation of tumor cells. Tumor volumes were calculated using the formula: ab2 / 2, in which a and b are the smallest diameters in millimeters, respectively [13] and a tumor fragment was preserved in paraffin for immunohistochemical determination of the maximum proliferation index (MPI) . In the present case, after fixation, the test sample was processed for the detection of the Ki-67 antigen with the MIB1 monoclonal antibody according to the method described by McCormick [15].

The animal model was chosen based on the fact that SKHEP – 1 is the most tumorigenic human liver cancer cell line in nude mice [16] and previous experience with the model [17]

Statistic analysis

The differences between the different treatment groups were analyzed using ANOVA followed by the Tukey test. P values of less than 0.05 were considered to represent a significant difference.

Results

Inhibition of [3 H] thymidine incorporation by albendazole

The [3H] thymidine incorporation assay was used to determine the effect of albendazole on cell proliferation in a series of human HCC cell lines (HepG2, Hep3 – B, PLC / PRF / 5, SKHEP – 1), of rat (HTC and Novikoff) and mouse (Hep1–6). The results obtained show that, in all cell lines examined, albendazole effectively reduces the incorporation of thymidine (Table 1). When treated with the concentration of 100 nM albendazole, compared to other cell lines, SKHEP – 1 showed the highest level of sensitivity to albendazole (p <0.01 compared to the control), while the cell line of HTC rat was the least sensitive of all. Treatment with the concentration of 1,000 nM albendazole reduced the incorporation of thymidine to less than 20% of the control values (p <0.001) in all cell lines and to less than 5% in SKHEP-1 and HepG2. In the present case, again the SKHEP-1 cells showed the highest level of sensitivity to albendazole. In these cells, thymidine incorporation was reduced to 0.6 ± 0.1% of the corresponding control values with a 99.4% inhibition. For this reason, SKHEP – 1 was used for all additional investigations. Exposure of SKHEP-1 cells at different concentrations of albendazole for 1 day revealed that concentrations of 250 nM and more of albendazole still produce a profound inhibition of thymidine incorporation (Figure 1a). The elimination of the drug and the treatment of the cells with the medium

Normal for 4 more days led to the recovery of thymidine incorporation by cells (Figure 1b). Except for concentrations of 500 and 1,000 nM, cells exposed to all other concentrations of albendazole were able to recover from the effect of drug inhibition.

Table 1: Effect of albendazole on the incorporation of [3H] thymidine in HCC cell lines.

Cellphone line

10 [albendazole] nM 100 500 1,000

HepG2 Hep3 – B SKHEP – 1 PLC / PRF / 5 Novikoff HTC Hep1-6

93.1 ± 5.6 105.7 ± 8.6 89.6 ± 6.1 92.7 ± 4.7 96.5 ± 8.8 98.4 ± 7.5 97.7 ± 4.3 72.9 ± 6.3 68.5 ± 5.7 63.7 ± 3.1 69.4 ± 5.2 71.6 ± 5.9 86.0 ± 6.9 79.50 ± 3.2 12.2 ± 2.9 22.6 ± 1.8 4.5 ± 1.1 26.9 ± 2.7 29.2 ± 3.3 28.5 ± 2.2 28.1 ± 2.5 3.5 ± 0.4 9.3 ± 0.7 0.6 ± 0.1 18.4 ± 2.1 10.3 ± 1.8 11.4 ± 1.3 5.6 ± 0.6

Cells were treated with different concentrations of albendazole (10–1,000 nM) for 5 days at the end of which the incorporation of [3 H] thymidine was measured. The values (% of control) represent the mean ± SEM of several determinations.

Albendazole inhibits cell proliferation, which leads to a drop in cell numbers

The count of viable cells treated with different concentrations of albendazole for 1, 3 or 5 days resulted in a dose-dependent drop in cell numbers, showing the profound inhibition of SKHEP-1 cell proliferation by the drug ( figure 2).

This was evident from day 3 at the concentrations of 500 and those of 1,000 nM. In comparison with the control, the cells exposed to the concentration of 1,000 nM of the drug were significantly reduced in number (p <0.001).

Effect on dose of albendazole on cell cycle kinetics

Cytometric flow analysis of albendazole-treated cells revealed that the drug induces a dose-dependent effect on the cell cycle kinetics of SKHEP-1 HCC cells. Figure 3 shows the changes that are induced in the distribution of the cells in the different phases of the cell cycle following a 3-day treatment with different concentrations of albendazole. From the above, it is clearly evident that the exposure of the cells to the concentration of 250 nM results in the accumulation of the cells in the G0-G1 phase and with the above was associated a reduction in the percentage of cells both in the S phase as in the G2 – M of the cell cycle. The changes induced by the 500 nM concentration of the drug were identical to those of the 250 nM concentration (data not shown). However, as shown in the same figure, the treatment of cells with the concentration of 1,000 nM albendazole leads to a completely different pattern of changes. In the present case, the stopping and accumulation of cells in the G2 – M phase of the cell cycle was accompanied by a drastic reduction in the percentage of cells in the G0 – G1 phase, while the percentage of cells in the S phase It remained unchanged.

Effect of albendazole on tumor growth in vivo

In control animals, SKHEP – 1 tumors developed to an average volume of 87.9 ± 12.3 mm3 at 20 days after inoculation. In animals that received 50 and 150 mg / kg per day, tumor growth slowed slightly but not significantly. However, tumor growth was profoundly suppressed in animals that received the dose of 300 mg / kg of albendazole (Figure 4) with a mean tumor volume of 12.0 ± 7.8 (p <0.001). The results from the immunohistochemical analysis of the tumors revealed that the tumors from the animals that received the dose of 50 and 150 mg / kg of albendazole had reduced the MPI from 22.54 ± 1.53 (mean ± SEM) and 13.36 ± 3.04 respectively compared to 34.2 ± 3.13 for the control. There was not enough tissue for MPI analysis in tumors of mice that received the dose of 300 mg / kg / day.

Effect on dose of albendazole on cell cycle kinetics

Albendazole was also shown to exhibit a dose-dependent inhibition of ovarian cancer cell line proliferation (OVCAR-3) in vitro, (see Figure 5).

Discussion

The results of cell proliferation studies clearly showed that all human liver, rat and mouse cell lines examined are deeply inhibited by albendazole. This was evidenced by the significant reduction of thymidine incorporation following treatment with albendazole doses of 100 nM and more. An analogous treatment of SKEP-1 cells with albendazole led to a reduction depending on the dose and time of the number of cells. The reason behind the higher sensitivity of SKHEP – 1 to albendazole is not clear at this stage. However, the lack of the enzymes necessary for the conversion of the drug to less active or inactive metabolites may partly explain this observation [14]. Cytometric analysis of cell cycle flow revealed that albendazole results in a dose-dependent effect on the kinetics of the SKHEP-1 cell cycle. The accumulation of cells in the G0 – G1 phase following treatment with albendazole concentrations of up to 500 nM with an associated drop in the percentage of cells in the S and G2 – M phases, indicates that progression outside of the G1 phase. Many natural triggers for programmed cell death, including glucocorticoid hormones act in the G1 – G0 transition and cells die in a process that is described as ‘premature aging’ [18]. However, following treatment with the concentration of 1,000 nM albendazole, the pattern of cell distribution was reversed, which leads to the accumulation of cells in the G2-M phase of the cycle. The above indicates that the primary effect of albendazole at the present concentration can be mediated by a transition delay through G2 – M or mitosis.

Data from work in nude mice suggests that, at the highest dose of 300 mg / kg / day, albendazole presumably reaches the necessary concentrations required to suppress tumor formation. The very high rate of albendazole metabolism in mice and the poor blood supply to the subcutaneous tumor are among a number of factors that could explain 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 the rate of tumor proliferation. The Ki-67 antigen used in the present assay is strongly linked to proliferation and has been used in a large number of studies to estimate the tumor growth fraction [15].

REFERENCE EXAMPLE 2

Albendazole in patients with an advanced malignant tumor

Patients and methods

The study was monocentric, open and uncontrolled. Nine patients (8 males and 1 female) with either an advanced CRC and liver metastases or with HCC were included in the present study. A patient with neuroendocrine cancer and mesothelioma was also treated compassionately. Patients aged 38–79 years were not operable and with them had failed existing chemotherapy and also, except in two, had measurable and growing tumor markers. The majority had already failed liver artery chemotherapy. The diagnosis of CRC or HCC was made by ultrasonic examination, CT or MRI, confirmed by histology and by the determination of CEA or AFP levels for CRC or HCC, respectively. Only patients with survival expectancy of more than one month were enrolled in the study. The characteristics of the patients are presented in Table 2. The study was approved by the Human Ethics Committee for Research of SESAHS. The protocol and the end of the study were clearly explained to each patient and their informed consent was obtained. The duration of the present study was four weeks, but I had to stop if leukopenia (WBC <2 x 106) or severe hepatocellular lesion (ALT or AST 2 x upper limit) developed. All patients received albendazole (400 mg slotted tablets, Smith Kline Beecham, Australian subsidiary) 10 mg / kg orally in two or three divided doses with a planned duration of four weeks. This is the clinical dose of albendazole used in the treatment of parasitic diseases. Patients were evaluated every three days by clinical examination along with whole blood tests to monitor tumor markers, hematopoiesis, liver and kidney function toxicity. A partial response (PR) was defined as a decrease of 50% or more in the value of the markers. In addition, there should be no new injuries or progression of any other injury. A 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 a progressive disease (PD) was an increase of 25% or more in the value of tumor markers or the appearance of any new lesions.

Table 2. Characteristics of the 9 patients with non-operable liver tumors, with whom chemotherapy had failed, who participated in the phase 1 trial of albendazole.

Patient

Sex Age Kind Duration of treatment (days) Comments Site of metastatic disease

one

F 38 HCC 19 neutropenia; drug withdrawal MBL, lung and liver

2

M 66 CRC 14 neutropenia; drug withdrawal Liver and lung

3

F 62 * 28 days Pleura and liver

4

M 66 CRC 28 days MBL and bone

5

M 56 CRC 28 days MBL and brain

6

M 66 CRC 28 days Liver and lung

7

M 74 CRC 28 days Liver and lung

8

M 54 CRC 28 days neutropenia withdrawal MBL

9

M 79 CRC 28 days MBL and peritoneum

MBL = multiple bilobular liver * Mesothelioma and carcinoid tumor.

Results

Albendazole treatment led to the stabilization of the disease in three patients, and the progression of the disease in the other two (Figure 6). In the remaining four, either the drug had to be withdrawn (2) or the tumor markers were not measurable (2). In patient number 1, who suffered from HCC, treatment with albendazole led to the stabilization of the disease, but due to the development of neutropenia, treatment with the drug was stopped in this patient on day 19. The patient visited another city after stop albendazole and died of neutropenic sepsis, which was almost certainly related to albendazole therapy. He had suffered neutropenia with previous chemotherapy. Patient number 2 (CRC), who was showing the greatest response to albendazole therapy, also developed neutropenia and thus albendazole was also withdrawn in this patient. Patient number 3 (carcinoid tumor and mesothelioma) had no evaluable serum tumor markers. However, the patient was monitored for adverse effects. There may be a short-term control of tumor markers in patients 4 and 5, but these began to rise again during treatment. In patients 6, 7 and 8, albendazole therapy was associated with stable CEA levels. In patient 9, CEA levels were less than 10 Ig / l and thus remained the duration of treatment (4 weeks).

There were no significant changes in liver and kidney function during the course of the trial. However, in patients 1, 2 and 8 a significant neutropenia developed, as a result of which the drug had to be withdrawn. The WCC values for all nine patients are presented in Figure 7.

Discussion

In the knowledge of the inventors of the present invention, the present study is the first notified of albendazole (or any other BZ) administered to human subjects for cancer therapy. The patients who participated in the present study had an advanced malignant tumor, which had not responded to the available therapy. The administration of albendazole to the only patient in the study with primary HCC led to the stabilization of 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 stabilized while being treated with albendazole. Compared to other patients, patient number two, who withdrew from the trial on day 19, had the steepest drop in CEA levels.

This report shows for the first time that albendazole, a benzimidazole carbamate with extensive clinical use as a safe antiparasitic drug, may result in the stabilization of tumor markers in patients with HCC or CRC with liver metastases.

EXAMPLE 3

To study the feasibility of the use of albendazole in the treatment of peritoneal disease, the human ovarian cancer cell line NIH: OVCAR-3 was selected. This is a cell line that develops quite slowly but is tumorigenic in nude mice, therefore allowing the drug to be studied under conditions both in vitro and in vivo.

Thymidine Assay

After finding the right conditions for the growth of the cells in culture, the 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 cell proliferation and that at a concentration of 0.25 micromoles / l, approximately 90% cell inhibition is achieved and at 0.5 micromoles / l Cell growth inhibition is 100%.

These results show that albendazole is very effective against this human ovarian cancer cell. The degree of activity of albendazole is approximately 10 times more than that observed for liver or colorectal cancer cells in which the inhibition activity starts at approximately 0.1 micromoles / L.

Cell count

The cells were treated either with the medium alone (control) or with 0.1 and 1 micromoles / L of albendazole for 0, 1, 3, 6 or 10 days and the number of viable cells was counted. It was observed that at both doses used, treatment with albendazole leads to a profound reduction in the number of cells. This was evident from day 1 after treatment.

REFERENCE EXAMPLE 4

Albendazole / albendazole sulfoxide against liver, colorectal and prostate cancer cell lines:

Studies in animals and humans have shown a rapid conversion of albendazole to a sulfoxide (ABS) and then to a sulfone and a series of other metabolites. As an antiparasitic, ABS is considered to be responsible for most or part of the systemic biological activity of albendazole, while the sulfone metabolite is pharmacologically inactive. Several studies have shown that ABS is almost as effective against parasites as albendazole itself. In addition, it is believed that the activity in clinical situations is mainly due to the formation of ABS. So much so that ABS is now being considered as a new drug (ricobendazole) by some researchers. This knowledge, together with some of the observations of the inventors of the present invention in patients with colorectal disease undergoing albendazole therapy, led the inventors of the present invention to test ABS against cancer cells. To date, there has been no bibliography on this issue.

In order to be able to test the ABS, a suitable solvent had to be found. This is because, unlike albendazole, ABS is not soluble in ethanol. After a series of tests, it was found that DMSO (0.5%) was the most suitable solvent for this compound and for this type of investigation. It was found that higher concentrations of DMSO were too toxic for the cells, while lower concentrations led to incomplete solubility of the ABS. Therefore, to allow a direct comparison between the 2 compounds, all subsequent experiments are performed using 0.5% DMSO for both ABS and albendazole. In the cell lines examined, albendazole produced very similar results to those that the inventors of the present invention had previously reported for the drug in liver cancer cells.

Both albendazole and ABS were tested next to each other against several cell lines, as follows:

Liver ---- HepG2, Hep3B, SKHEP – 1

Colorectal ---- C – 170, LOVO, HT – 29

Prostate ---- PC3, LNCap, Du145

Leukemic ---- HL60

The first 9 cell lines were used to compare effectiveness and potency while the last one was used to assess bone marrow toxicity (as an approximate guide).

The cells were treated with various concentrations of each compound over a period of 5 days.

Albendazole sulfoxide was shown to inhibit the proliferation of human liver cancer cells HepG2, SKHEP-1 and Hep 3B.

ABS produced the same type of activity against C-170, LOVO and HT-29 colorectal cell lines. In general, the results obtained for ABS against albendazole show a great degree of similarity between the 2 compounds, with an antiproliferative activity that becomes evident at a concentration of 0.01 micromoles / l and drastically increasing at 0.25 micromoles / l and that approaches a 100 to 1 micromol / l inhibition.

All results obtained with prostate cell lines show that the drug and its major metabolite (ABS) are almost equipotent for the inhibition of the proliferation of human prostate cancer cells PC3, LNCap and Du 145.

REFERENCE EXAMPLE 5

Marking:

To find out how albendazole works as an anticancer drug, a wide variety of tests can be performed. One of the first is to see if albendazole induces apoptosis. This is tested again in several ways, one of the most common is to check the DNA labeling. In the laboratories of the inventors of the present invention, after using some time to determine the correct concentration and the correct treatment period, finally the inventors of the present invention could see clear evidence of the induction of DNA labeling in these cells. of ovarian cancer by albendazole. This shows that treatment with albendazole leads to apoptosis. This is the first time that induction of apoptosis by albendazole has been reported.

Similar results were also obtained with the human LOVO colorectal cell line. The DNA obtained from LOVO cells treated with albendazole 1 IM during 16 hours showed a clear marking, which showed that albendazole induces apoptosis in LOVO cells.

Compendium

The results obtained with the ovary cell line (NIH: OVCAR-3) show for the first time that albendazole is profoundly effective for the inhibition of its proliferation in culture in a dose-dependent manner. This was confirmed by both thymidine uptake and cell count. Additional work clearly shows that these cells undergo apoptosis as a result of treatment with albendazole.

The results obtained in liver and colorectal cell lines confirm the antiproliferative activity of albendazole against these cell lines. The results obtained with the 3 human prostate cell lines indicate that these cells are also quite sensitive to the effect of albendazole in culture. There is no previous report about the antiproliferative activity of albendazole in prostate cell lines, which makes the present the first to show such an activity.

It has been well known for years that the conversion of albendazole in the body takes place quite rapidly in the liver and produces an active metabolite, namely albendazole sulfoxide, which has been shown to be active against parasites. In this regard, the previous results of the inventors of the present invention with sulfoxide have shown that it is as effective as the precursor drug (albendazole) in the 10 different cell lines examined. However, the latest results of the inventors of the present invention cast certain doubts on this, and the compound is currently subject to intensive research in the laboratories of the inventors of the present invention.

REFERENCE EXAMPLE 6

Combination Therapy:

The effect of inhibition of albendazole metabolism on its antiproliferative activity was investigated using a human LOVO colorectal cell line and methimazole as an albendazole inhibitor.

From each of HepG2 and SKHEP-1, 5,000 cells were placed in 24-well 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 tested in the present experiment (0.25, 0.1 and 0.01 µM). However, there was a modest growth inhibition when albendazole and MT were used together. On the other hand, albendazole at a concentration of 0.25 IM profoundly inhibited the growth of SKHEP-1 cells. This response was further enhanced in the presence of MT. MT is an antithyroid agent that acts as a competitive oxidation inhibitor based on the microsomal Flavin Monoxygenase (FMO) of several drugs. It has been shown that MT inhibits the metabolism of drugs dependent on this route. MT-mediated inhibition of hepatic sulfoxidation of albendazole to albendazole sulfoxide has been shown both in vitro and in vivo.

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Claims (3)

1. A pharmaceutical composition for use in a method of treating ovarian cancer in a subject by regional administration, wherein the composition comprises a lipid carrier and a benzimidazole carbamate which is albendazole or a sulfoxide or sulfone thereof. .

A pharmaceutical composition for use according to claim 1, wherein the vehicle is an oil.

3.

 A pharmaceutical composition for use according to claim 2, wherein the vehicle is an iodized oil.

Four.

 A pharmaceutical composition for use according to claim 3, wherein the iodized oil is an iodinated ethyl ester of the poppyseed oil.

A pharmaceutical composition for use according to any one of claims 1 to 4, wherein the composition further comprises methimazole.