COMBINATION OF METHYLOL TRANSFER AGENTS WITH TUMOUR- INHIB I TING PROTEINS OR PEPTIDES AND THE USE THEREOF FOR THE TREATMENT OF CANCER OR TUMOR GROWTH

CROSS-REFERENCE TO RELATED APPLICATION

[ooi] This application claims the benefit of U.S. Provisional Application Serial No. 60/707,953, filed 15 August 2005.

Technical Field

[002] The invention relates to the treatment of cancer.

Background of the Invention

[003] Methylol transfer agents, such as the antibacterial and anti-toxin drug taurolidine and the related product taurultam, have been shown to exert a modifying effect on the toxicity of tumor necrosis factor (TNF) which is used, inter alia, in the treatment of tumors. Furthermore, the action of methylol transfer agents has been shown to be selective in that the growth of normal cell-lines was not significantly inhibited.

[004] Taurolidine acts by transferring three methylol groups at the site of action, taurultam being an intermediate metabolite which itself transfers a single methylol group with liberation of the very well tolerated compound taurinamide. Thus, the two compounds act by essentially the same mechanism. It should be noted that methylol transfer is to be contrasted with methyl transfer which is characteristic of many highly toxic anti-tumor drugs. Taurolidine and taurultam have low toxicity and are not cytotoxic against normal cells.

[005] Programmed cell death is an evolutionary conserved biological principle in the regulation of cell numbers. Sensitive cells contain death receptors which are activated when the appropriate ligands are secreted from neighboring cells. A prominent system in programmed cell death is Fas-ligaήd mediated apoptosis. Fas, also known as CD 95/APO-l, is a cell surface receptor and a member of the tumor necrosis factor receptor superfamily which mediates apoptosis in sensitive cells upon oligomerization by the Fas-ligand (FasL).

[006] Therapeutic proteins, such as monoclonal antibodies, also have been proposed for treatment of cancers.

[007] There remains a need in the art for improved treatments of cancers.

SUMMARY OF THE INVENTION

[008] In accordance with the present invention, pharmaceutical combinations and methods

[007] There remains a need in the art for improved treatments of cancers.

SUMMARY OF THE INfVENTION

[008] In accordance with the present invention, pharmaceutical combinations and methods of treatment are provided for treating, preventing, inhibiting or reducing cancer or tumor growth in a subject utilizing a cancer or tumor-inhibiting methylol transfer agent and a cancer or tumor-inhibiting protein or peptide which comprises a cancer or tumor-inhibiting multiple target inhibitor, a cancer or tumor-inhibiting tyrosine kinase inhibitor, a cancer or tumor-inhibiting small molecule inhibitor, a cancer or tumor-inhibiting small molecule, a cancer or tumor-inhibiting antibody or a combination thereof. The methylol transfer agent and protein or peptide can be administered separately or together.

DETAILED DESCRIPTION

[009] The present invention relates to treatment of cancers and tumors in a subject by administering to the subject a methylol transfer agent such as taurohdine and/or taurultam, in combination with administering to the subject of a cancer or tumor- inhibiting amount of a cancer or tumor-inhibiting therapeutic protein or peptide as described herein.

[010] In accordance with one embodiment, a pharmaceutical combination is provided for treating, preventing, inhibiting or reducing cancer or tumor growth in a subject. In preferred embodiments, the subject is a mammalian subject, most preferably a human subject. A pharmaceutical combination comprises a cancer or tumor-inhibiting methylol transfer agent and a cancer or tumor-inhibiting protein or peptide. The protein or peptide may comprise a cancer or tumor-inhibiting multiple target inhibitor, tyrosine kinase inhibitor, small molecule inhibitor, small molecule, antibody or a combination thereof. The methylol transfer agent and the protein or peptide may be administered to the patient separately, or together as a pharmaceutical composition.

[Oil] Methylol transfer agents include methylol-containing compounds such as taurolidine and taurultam, and their derivatives. The compounds taurolidine and taurultam are disclosed in U.S. Patent No. 5,210,083. Other suitable methylol- containing compounds include taurinamide derivatives and urea derivatives. Examples of derivatives of taurohdine, taurultam, taurinamide and urea useful in the present invention can be found in WO 01/39763A2. Particularly preferred methylol transfer agents for utilization in accordance with the present invention are taurolidine,

taurultam, biologically active derivatives thereof and mixtures thereof.

[012] Alternatively, the compound is a taurinamide derivative, or a urea derivative. Examples of derivatives of taurolidine, taurultam, taurinamide and urea useful in the present invention can be found in WO 01/39763A2.

[013] Other methylol-containing compounds suitable for inducing apoptotic death of cancer cells include but are not limited to l,3,-dimethylol-5,5-dimethylhydantoin, hexamethylene tetramine, or noxythiolin. By derivative of taurolidine or taurultam is meant a sulfonamide compound which possesses at least 10% of the neoplastic activity of taurolidine or taurultam, respectively. A sulfonamide compound is one having a R2N-SO2R' formula. Derivatives of the compounds described herein may differ structurally from a reference compound, e.g., taurolidine or taurultam, but preferably retain at least 50% of the biological activity, e.g., induction of apoptotic cell death, of the reference compound. Preferably, a derivative has at least 75%, 85%, 95%, 99% or 100% of the biological activity of the reference compound. In some cases, the biological activity of the derivative may exceed the level of activity of the reference compound. Derivatives may also possess characteristics or activities not possessed by the reference compound. For example, a derivative may have reduced toxicity, prolonged clinical half-life, or improved ability to cross the blood-brain barrier.

[014] While the invention herein is sometimes described in connection with administration of taurolidine and/or taurultam to a subject, it is to be understood that other methylol transfer agents may be equally applicable.

[015] Additionally, while the present invention sometimes is herein described in connection with the administration of antibodies, including monoclonal and polyclonal antibodies, it is to be understood that the invention is applicable to other cancer or tumor-inhibiting therapeutic proteins or peptides, including those described herein.

[016] Certain tumor-inhibiting proteins and peptides utilized in accordance with the present invention inhibit the receptors for VEGF, PDGF, Flt-3 and other receptors. The invention also may utilize epidermal growth factor receptor (EGFR)-inhibitors or EGFR- antibodies, as well human epidermal growth factor receptor 2 (HER2) inhibitors or HER2-antibodies.

[017] The invention may utilize without limitation, cancer or tumor-inhibiting therapeutic proteins or peptides as described herein, including, but not limited to, rituxirnab, erlotmib, capecitabine, bevacizumab, trastuzumab, ibandronate, alemtuzumab, exemestan, sunitinib, tamoxifen, cetuximab, ibritumomab, vatalanib,

tositumomab, ixabepilon, imatinib, sorafenib, panitumumab, gefitinib, lapatnib and AG-Ol 3736.

[018] The combination therapy of the present invention is particularly advantageous in the enhancement and prolongation of the efficacy of cancer or tumor-inhibiting therapeutic proteins or peptides as described herein, such as antibodies, in the combination with methylol transfer agents such as taurolidine and/or taurultam, because of their dual mechanism of action, mainly induction of apoptosis and inhibition of angiogenesis.

[019] Known antibodies presently only have a responder rate of approximately 20- 30%. Known antibodies generally suffer from a loss of efficacy during lengthy administration periods, including fading, tachyphylaxis and enhancement of tumor cell growth. Moreover, many therapeutic proteins or peptides as described herein, including monoclonal antibodies, have toxicity problems and show many undesirable side effects. Generally, antibodies only act very specifically against only one parameter of tumor growth, e.g., against VEGF. Thus, the efficacy of the therapeutic antibodies weaken during the course of treatment while the serious adverse effects of tumors remain substantially unchanged. One advantage of utilizing methylol transfer agents such as taurolidine and/or taurultam are that such methylol transfer agents show practically no relevant adverse events during administration. Thus, the dosage of tumor-inhibiting therapeutic proteins or peptides such as monoclonal antibodies may be reduced, and the safety administering them significantly improved, by combining their administration with methylol transfer agents such as taurolidine and/or taurultam. No formal drug interactions of therapeutic proteins or peptides such as monoclonal antibodies, and methylol transfer agents such as taurolidine and/or taurultam, have been reported.

[020] Thus, according to one embodiment, the invention is a method of reducing toxicity and side-effects of a cancer or tumor treatment in a patient with a pharmaceutical combination as described herein. In accordance with this method, the toxicity and side-effects of tumor-inhibiting proteins or peptides are reduced. A cancer or tumor-inhibiting protein or peptide as described herein is administered to a patient during a treatment period, wherein such protein or peptide administration is capable of causing toxicity and side-effects in the patients. The method further comprises administering to the patient a toxicity-reducing and side-effects-reducing amount of a methylol transfer agent as described herein, such as taurolidine, taurultam or a combination thereof, during the treatment period, so as to reduce the toxicity and side-effects of the cancer or tumor-inhibiting protein or peptide in the

patient. Additionally, the dosage of the cancer or tumor-inhibiting protein or peptide may be reduced in the patient without loss of efficacy.

[021] Toxicity and side-effects which may be reduced by the present invention include the following:

Very common: Dyspnea, nausea, vomiting, allergic skin eruptions, (> 10%) proteinuria, hematuria, edema, increased liver function values, transaminases increased, alkaline phosphatase increased, flue-like symptoms.

Common: Leukopenia, thrombozytopenia, anemia, loss of (> 1 - < 10%) appetite, headache, sleep disturbance, sweating, itching, hair loss, mucosal ulceration, fever, chills, increased bilirubin, tachycardia, hypotonia, anxiety, depression, neuropathy, tremor.

Uncommon: Pulmonary edema, lung damage (infiltration, fibroses,

(> 0.1% - < 1%) ARDS) asthma, cough, rash.

Rare: Anaphylactic reactions, myocardial infarction,

(> 0.01% - < 0.1%) coronary insufficiency, respiratory distress syndrome, blistering and ulcer formation of the skin, kidney failure, haemolytic-uremic syndrome, allergic reactions, asthenia, rigidity, paresis, pancreatitis, leukopenia, thrombocytopenia, anemia, decrease of prothrombin, hyperpotassemia, glomerulopathy.

Very Rare: Thrombocytosis (≥ 10%)

[022] Side-effects may include drowsiness, diarrhea, hypertonia, anorexia, mucositis, loss of weight, acne-like skin eruptions, dry skin, paronychia and/or superinfections.

[023] The methylol transfer agent and the protein or peptide as described herein may be co-administered to a subject, or administered sequentially or cyclically.

[024] Treatment of a cancer or tumor, may be carried out by administering to a mammal, e.g., a human patient subject, a methylol transfer agent and a therapeutic protein or peptide as described herein. The combination may be administered systemically, e.g., orally and/or intravenously, or infused or applied directly to the site of the tumor.

[025] Cancers to which the present invention may be applicable include glioma, neuroblastoma, astrocytoma, carcinomatous meningitis, ovarian cancer, prostate cancer, central nervous system (CNS) cancer, lung cancer, gastric cancer, esophageal cancer, urinary bladder cancer, leukemia, lymphoma, melanoma, renal cell cancer,

mesothelioma and metastases thereof. Other cancers against which the method of the present invention is effective include other carcinomas, sarcomas or lymphomas, cancers of the head and neck, liver cancer, breast cancer and pancreatic cancer.

[026] Certain embodiments involve treatment of cancers selected from the group consisting of glioma, neuroblastoma, astrocytoma, central nervous system (CNS) cancer, and liver cancer, as well as inhibition of tumor metastases thereof.

[027] Preferred embodiments involve treatment of cancers including renal cell carcinoma, colon cancer, lung cancer, colo-rectal carcinoma, bronchial carcinoma, abdominal cancer including pancreatic cancer, and ovarian cancer, leukemia including chronic lymphocytic leukemia, prostate cancer, gastric cancer, bone cancer, B-cell chronic lymphatic leukemia, breast cancer, renal cell cancer, non-Hodgkin's lymphoma, solid tumors, pulmonary carcinoma, pancreas carcinoma, Taxane-resistent mamma-pulmonary carcinoma and the like.

[028] It is particularly beneficial to use taurolidine and/or taurultam, at concentrations sufficient to induce apoptosis in cancer cells, to prevent the spread of metastases, especially following surgical removal of tumors.

[029] The invention also includes the use of taurolidine and/or taurultam, at concentrations sufficient to induce apoptosis in cancer cells, and a therapeutic protein or peptide as described herein for the treatment or prophylaxis of tumors in mammalian subjects.

[030] Effective dosage amounts of a methylol transfer agent in accordance with the present invention may comprise pharmaceutical dosage units within the range of about 0.1-1,000 mg/kg subject body weight, preferably 150-450 mg/kg per day, and most preferably 300-450 mg/kg per day. Alternatively, the dosages can be administered on a grams/day basis, from about 2-60 g/day. Preferred doses may be in the range of about 2.5-30 g/day taurolidine, 4-60 g/day taurultam, or a mixture thereof. Most preferred doses are in the range of about 10-20 g/day taurolidine, 20-40 g/day taurultam, or a mixture thereof.

[031] Effective dosage amounts of the tumor-inhibiting therapeutic protein may comprise pharmaceutical dosage units within a range of about 0.001-lOOOmg/kg subject body weight, preferably within a range of about 1-lOmg/kg, or administered within the range of about 1-lOOOmg/m ² subject body surface area.

[032] Suitable formulations for injection or infusion may comprise a pharmaceutically acceptable carrier, such as water for injection, and preferably comprises an isotonic solution containing one or more solubilizing agents, e.g., polyols

such as glucose, in order to provide solutions of increased taurolidine or taurultam concentration. Such solutions are described in EP 253662B1. The concentration of taurolidine or taurultam in such solutions may be in the range 1-60 g/liter.

[033] Methylol transfer agents are generally poorly soluble in water. Thus, it is often required to administer relatively large volumes of aqueous solutions containing taurolidine or taurultam, for example 1Og to 30g of taurolidine and/or taurultam. Preferred solutions for administration in accordance with the present invention contain from about 0.5-2% taurolidine and/or taurultam. It may be convenient to administer these compounds by infusion in view of the relatively large volumes concerned, conveniently at intervals throughout the day.

[034] Administration of the methylol transfer agent, e.g., by infusion, of the total daily dose can be carried out at a consistent rate over 24 hours, or according to a more rapid infusion schedule of the dose in portions, with breaks between each portion of the dose, e.g. infusion of 250 ml of a 2% taurolidine solution (5 g dose) over 2 hours, followed by a brief break of 4 hours, repeated over the course of a 24 hour infusion period to achieve a total daily dose of 20 g. Alternatively, 250 ml of a 2% taurolidine solution may be infused over one hour, with a one hour break between dose portions, and repeated until the daily dose is achieved, such that the total daily dose is provided over the course of less than 24 hours (i.e., approximately half the day), with no infusion occurring during the remainder of the day.

[035] In accordance with one embodiment, four bottles (250 ml each) of 2% taurolidine solution are administered intravenously to patients with cancer, at a rate of 40 drops per minute, one bottle every six hours. The therapy cycle generally is an administration phase of daily infusions for one week, followed by a rest phase of two weeks. Total treatment generally is at least two such cycles. Efficacy of taurolidine 2% solution administered intravenously has been found to be particularly good with 25- 28 bottles of 250 ml taurolidine 2% solution being instilled per cycle.

[036] In accordance with a second embodiment of the invention, the administration phase comprises a daily regimen whereby 250 ml of taurolidine 2% solution is administered over the course of 2 hours, followed by a four hour break, repeated over 24 hours to achieve the total daily dose.

[037] In accordance with a third embodiment of the invention, the administration phase comprises a daily regimen whereby 250 ml of 2% taurolidine solution is infused over one hour, followed by a one-hour break, and repeated until the daily dose is achieved. If the total dose is 20 g (for example), this regimen would provide the daily

dose with four 250 ml infusions of 2% taurolidine over a 7 hour time span. No infusion occurs for the remainder of the day. Infusion rates can be lengthened (e.g., to 250 ml over 90 or 120 minutes) if the patient shows an elevated liver count.

[038] In particularly preferred embodiments, patients are subjected to dosing cycles having an administration phase of at least 3 continuous days, and up to about 8 continuous days, each administration phase being followed by a non-administration phase of about 1 day to about 4 weeks, e.g., 1-14 days, or even 3, 4 or more weeks, during which the methylol-containing compound is not administered to the patient. During each administration phase, the methylol-containing compound is administered each day. For example, administration phases of 3, 4, 5, 6, 7 and/or 8 days can be utilized, and non-administration phases of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and/or 14 days may be utilized. At least 2 dosing cycles are utilized, preferably 5-10 or more dosing cycles are utilized. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequential dosing cycles can be utilized. Such a regimen has shown surprising and unexpected results with patients. In one particularly preferred embodiment, 6 dosing cycles, each with administration phases of 5 days are utilized, with each administration phase separated by a non-administration phase of 2 days. Preferably, during each day of administration, 250 ml of taurolidine 2% solution is intravenously administered to the patient 4 times daily. Such a regimen has surprisingly and unexpectedly resulted in a marked tumor size reduction with disappearance of perifocal edema in a patient with inoperable glioblastoma infiltration of the basal ganglia.

[039] In another embodiment, a non-administration phase may be 1, 2, 3, 4 or more weeks in length, e.g., about 2-4 weeks. For example, in patients with recurrent cancers such as of the stomach and pancreas may be administered sequential dosing cycles having an administration phase of 3-8 continuous days, e.g., 7 days, with, for example, 250 ml taurolidine 2% solution infused 4 times daily, followed by a non-administration phase of 1, 2, 3, 4, or more weeks, e.g., 3 weeks. As in the previous embodiments, at least 2 dosing cycles are utilized, preferably 5-10 or more dosing cycles.

[040] The tumor-inhibiting therapeutic protein may be administered daily, multiple times per day, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, every two weeks, monthly, etc., or any suitable administration regimen.

[041] The invention also includes treating a drug resistant tumor, e.g., a multiple drug resistant (MDR) tumor. The tumor to be treated may be a carcinoma or sarcoma. The drug resistant tumor may be a solid tumor, a non-solid tumor, or a lymphoma.

For example, the drug resistant tumor may be a breast cancer, ovarian cancer, colon cancer, prostate cancer, pancreatic cancer, CNS cancer, liver cancer, lung cancer, urinary bladder cancer, lymphoma, leukemia, or sarcoma.

Example 1: Isotonic Solution 2% Taurolidine

[042] One suitable composition for intravenous drop infusion is shown below. [043] Isotonic sterile solution, 100 ml:

2.O g Taurolidine

5.0 g PVP 16 PF UP aqua dest. ad solut. 100 ml. PH 7.2 - 7.3

Sterile-filtered and steam sterilization.

Example 2: Isotonic Taurolin® Solution 2% Taurolidine with Taurin and electrolytes

[044] Another suitable composition for intravenous drop infusion is shown below. [045] Isotonic sterile solution, 100 ml:

2.0 g Taurolidine 5.O g PVP 17 PF UP 0.5 g Taurin 0.3 g Sodium chloride [046] Sterile-filtered and steam sterilization

Example 3: Isotonic Taurolin® Ringer Solution 2% Taurolidine with Taurin and electrolytes

[047] Another suitable composition for intravenous drop infusion is shown below. [048] Isotonic sterile solution, 100 ml:

2.0 g Taurolidine 5.O g PVP 17 PF UP 0.5 g Taurin 0.26 g Sodium chloride 0.0033 g Potassium chloride 0.004 g Calcium chloride 2H ₂ O 0.003 g Sodium hydrogen carbonate [049] Sterile-filtered and steam sterilization

Example 4: Taurolin® Ringer-Lactate 2% Taurolidine with Taurin and electrolytes

[050] Another suitable composition for intravenous drop infusion is shown below. [051] Isotonic sterile solution, 100 ml:

2.0 g Taurolidine 5.0 g PVP 17 PF UP 0.5 g Taurin 0.20 g Sodium chloride 0.013 g Potassium chloride 0.009 g Calcium chloride 2H ₂ O

0.0033 g Sodium lactate 50% solution (Pharmacopeia Europea) [052] Sterile-filtered and steam sterilization

Example 5: Taurultam Solution [053] One preferred solution comprises:

Lactobionic acid 35.83O g

Adenosine 1.340 g

Raffinose Pentahydrate 17.830 g

Hydroxyethyl starch (HES) PL 40/0.5 50.000 g

Glutathione 0.929 g

Allopurinol 0.136 g

Taurultam 10.000 g

KcI 5.200 g

MgSO ₄ 7H ₂ O 1.230 g NaOH 25% GV to pH 7.8 NaOH pellets Merck 6482

Distilled water 900 ml

[054] The solution was sterilized from 16 minutes at 121° C. The pH after sterilization was 7.2, and pH of ready to use solution was 7.47.

Example 6: Inducement of apoptosis

[055] Taurolidine and taurultam were tested for their ability to enhance apoptosis or induce cell death, alone and in combination with the Fas-ligand, in human malignant glioma cell lines. The two cell lines LN-18 and LN-229 represent validated model systems for apoptotic cell death with different sensitivities to Fas-ligand

(Schlappbach and Fontana, 1997). These cell lines were therefore used to test the potential interaction of taurultam or taurolidine with the apoptotic pathway.

1) Reagents

[056] Taurolidine (Batch Nr. 41692/7) and taurultam (Batch E/39024/4) were provided by Geistlich Pharma AG, Wolhusen, Switzerland. DME-Culture Medium and fetal bovine serum (FBS) were purchased from Gibco BRL, Basel, Switzerland. The cell proliferation assay WST-I was purchased from Roche Diagnostics, Rotkreuz, Switzerland. Fas-ligand (supernatant from an overexpression system) and the human glioma cell lines LN- 18 and LN-229 were kindly provided by Prof. A. Fontana, Institute of Clinical Immunology, University Hospital, Zurich, Switzerland

2) Cell lines

[057] The cell lines LN- 18 and LN-229 were cultured at 37 ⁰ C and 5% CO2 in DMEM containing 5% FBS and 2 niM glutamin (10 cm plates NUNCLON 15035). In the experiments in which Fas-ligand was tested by itself, about 1x104 cells were plated per well in 96-well plates (NUNCLON 167008) resulting in a confluency of about 60% on the following day (17h incubation). In all other experiments about 1.5xlO ⁴ cells were plated which resulted in a confluency of about 90% on the following day (17h incubation). Fas-ligand was added as supernatant indicated as % volume (vol%) of total culture volume.

3) Cell viability test

[058] LN-18 and LN-229 cells were incubated in 50 μl medium in the absence or presence of either Fas-ligand, taurultam, taurolidine or respective combinations thereof. After a 17h incubation the cell viability was determined by adding 50 μl medium containing a double concentrated WST-I reagent. The coloration resulting from the activity of the mitochondrial succinate reductase, was measured in an ELISA reader at 450 nm using a reference wavelength of 690 nm.

[059] The human malignant glioma cell lines LN-18 and LN-229 were used to test the ability of taurolidine and taurultam to affect cell viability and/or to enhance Fas- ligand induced apoptosis. The two human malignant glioma cell lines, LN-18 and LN- 229 had previously been reported to display different sensitivity to the apoptotic effect of Fas-ligand (Schlappbach and Fontana, 1997).

1) Sensitivity of LN-18 and LN-229 to Fas-ligand

[060] In a first set of experiments it was investigated whether the different

sensitivity of LN-18 and LN-229 to Fas-ligand was reproduced under our experimental conditions. The two cell lines were incubated over night (17h) in 96 well plates containing IxIO ⁴ cells per well with increasing concentrations of Fas-ligand (3.1, 6.25, 12.5, 25.0 and 50 vol.%). In the absence of Fas-ligand the cells reached about 60 % confluency after overnight incubation. In the presence of Fas-ligand LN-18 was extremely sensitive, displaying more than 90% loss of cell viability in the presence of only 6.25 vol. % Fas-ligand. Even at 3.1%, an approximately 85% reduction in cell viability was observed. In contrast, the viability of LN-229 cells was not greatly affected by 6.25 vol. % Fas-ligand (approximately 10% reduction) and was reduced only at higher concentrations with a maximum of 40% cell loss in the presence of the highest concentration of Fas-ligand tested (50 vol. %).

2) Influence of taurultam on Fas-ligand induced apoptosis in LN-18-cells

[061] LN-18 cells were incubated for 17h with increasing concentrations of taurultam (5, 20, 100 μg/ml) in the absence and presence of two concentrations of Fas- ligand (0.4 vol. % and 2.0 vol. %). Taurultam by itself even at the highest concentration tested (100 μg/ml) did not affect the cell viability (an approximately 5% reduction was observed at 5 and 20 μg/ml, and viability actually appeared to increase at 100 μg/ml).

In the presence of 0.4 vol.% Fas-ligand alone cell viability was reduced by only about 10%, an effect which remained unchanged in the presence of 5 or 20 μg/ml taurultam.

However cell viability was strongly decreased when 0.4 vol.% Fas-ligand was coincubated with of 100 μg/ml taurultam. When the Fas-ligand was added at a higher concentration (2.0 vol. %) apoptosis was induced in 60% of the cells by Fas-ligand alone. This effect was also increased by taurultam at 100 μg/ml but not at 5 or 20 μg/ml. Thus, taurultam is able to enhance the apoptotic effect of Fas-ligand in LN-18 cells at a concentration (100 μg/ml) which by itself did not affect cell viability.

3) Influence of taurolidine on Fas-ligand induced apoptosis in LN-18 cells

[062] LN-18 cells were incubated for 17h with either 0.4 or 2.0 vol.% Fas-ligand in the absence and presence of increasing concentrations of taurolidine (5, 20, 100 μg/ml). Taurolidine by itself did not appreciably affect cell viability yielding a reduction by only 10% at the highest concentration tested (100 μg/ml). In the presence of Fas-ligand alone (0.4% or 2.0%) the cell viability was affected in the same way as described above. The cell viability was further reduced by taurolidine but only at the highest concentration tested (100 μg/ml). Thus, taurolidine was able to enhance the effect of Fas-ligand on LN-18 cells at a concentration (100 μg/ml) which did not

appreciably affect cell viability per se.

4) Influence of taurultam on Fas-ligand induced apoptosis in LN-229 cells

[063] The incubation of LN-229 cells for 17h with taurultam alone had no effect at 5 and 20 μg/ml but reduced cell viability by 35% at 100 μg/ml. When the LN-229 cells were incubated with Fas-ligand alone (10% or 50%) the cell viability was reduced by only about 20% in the presence of a high concentration of Fas-ligand (50 vol. %). When taurultam was added at concentrations which were inactive per se (5 and 20 μg/ml) no change in the effectiveness of the Fas-ligand (10 or 50 vol.%) was observed. It was only at the highest concentration of taurultam (100 μg/ml) that Fas-ligand induced cell loss was further enhanced. Thus, the results with LN-229 demonstrate the ability of taurultam to enhance the destruction of cells in the presence of Fas-ligand.

5) Influence of taurolidine on Fas-ligand induced apoptosis in LN-229 cells

[064] The exposure of LN-229 cells to taurolidine alone for 17h caused a strong loss of cell viability by about 70% at the highest concentration tested (100 μg/ml). Thus, LN-229 cells were more sensitive to taurolidine than LN- 18 cells. When co-incubated with Fas-ligand (10 vol.%) cell destruction was enhanced by taurolidine at 100 μg/ml. At 50 vol.% Fas-ligand the effect was more pronounced and apparent even for taurolidine 20 μg/ml.

Example 7: Use and application of taurolidine and/or taurultam for the treatment and/or prophylaxis of tumors of the central nervous system

1. Tumor cells used for the experiments

For experiments, C6 glial tumor cells, HT22 neuronal tumor cells, U373 human glioma/glioblastoma tumor cells and cells derived from patients with glioblastoma were used.

2. Preparation of patient-derived tumor cells

Tumor cells derived from patients with glioblastoma were obtained intraoperatively. Tumor tissue was stored in RPMI 1640 medium without FCS. Tissue was then sub cultured in 15 ml Falcon flasks; adding 0.025% trypsin with PBS, followed by incubation at 37°C. After this, RPMI 1640 with FCS was added and centrifugation performed. The next step was incubation with DNAse, resuspension and dissociation, followed by washing step in medium to remove DNAse. Cells were then cultured in Falcon flasks.

3. Method of anti-neoplastic action of Taurolidine and/or Metabolites

[065] Ultrastructurally, shrinkage of cytoplasm, condensation and marginalization of chromatin could be observed. These changes were already apparent at 30 minutes of incubation with 0.1 μg/ml taurin and increased strikingly over time and with concentration of taurolidine. Mitochondria were not affected ultrastructurally. Flow cytometry showed an initial increase in the G0/G1 peak and S-phase starting at 30 minutes. These initial changes were followed by a decrease in forward light and side scatter. In addition, concentration-dependent fragmentation of DNA started at 60 minutes. Following 24 hours, fragmentation of the DNA was nearly complete. At concentrations of 2.0 μg/ml taurolidine and more, the changes in cell size was only marginal.

[066] The described results in combination with the results of special dying methods (Leucostat preparation) suggests an apoptotic mechanism of tumor cell death. Normal brain cells were not affected by incubation with taurolidine or taurultam in concentrations of up to 4 μg/ml for up to 5 days.

Example 8: Two-cycle dosing Schedule for Treating Patients with Cancer Using Intervenous Taurolidine 2%

[067] Four bottles (250 ml each) of 2% taurolidine solution are administered intravenously to patients with cancer, at a rate of 40 drops per minute, one bottle every six hours. The dosing cycle consists of an administration phase of daily infusions for one week, followed by a non-administration phase of two weeks, then followed by another administration phase of four bottles per day as previously indicated. Efficacy of taurolidine 2% solution administered intravenously has been found to be particularly good with 25-28 bottles of 250 ml taurolidine 2% solution being instilled per cycle.

Example 9: Four-cycle Dosing Schedule for Treating Patients with Malignant Gliomas Using Intravenous Taurolidine 2%

[068] The treatment comprises a minimum of 4 cycles. Each cycle is 7 days long, and is comprised as follows:

1. First Cycle

[069] a. Intravenous infusion of 250 ml taurolidine 2% and 250 ml full electrolyte solution via the central vein catheter with an infusion time of 60 minutes.

[070] b. If this therapy causes an elevated liver count, it is necessary to increase the

infusion time to 90 or 120 minutes.

[071] c. 60-minute break

[072] d. Repeat the therapies under a or b and c for a total of 6 times per day.

[073] e. At an infusion time of 60 minutes the duration of the daily infusion program per 250 ml of taurolidine is 11 hours, at 90 minutes of infusion time 14 hours, and at 120 minutes of infusion time 17 hours. No drug is administered for the remainder of the time.

[074] f. rest phase

2. Subsequent Cycles

[075] a. Intravenous infusion of 250 ml taurolidine 2% and 250 ml full electrolyte solution via the central vein catheter with an infusion time of 60 minutes.

[076] b. If this therapy causes an elevated liver count, it is necessary to increase the infusion time to 90 or 120 minutes.

[077] c. 60 minute break

[078] d. Repeat the therapies under a or b and c for a total of 4 times per day.

[079] e. At an infusion time of 60 minutes the duration of the daily infusion program per 250 ml of taurolidine is 7 hours, at 90 minutes of infusion time 9 hours, and at 120 minutes of infusion time 11 hours. No drug is administered for the remainder of the time.

Example 10: Therapy of Glioblastoma with Taurolidine (Single Case Observation)

[080] The following is a case involving treatment of a single individual with a single treatment cycle.

[081] Patient: "F.D.," male, 59 years

[082] Diagnosis: large (8 x 8 x 8 cm) malignant glioma bifrontal with affection of the corpus callosum ("butterfly glioma").

[083] Procedure prior to treatment with taurolidine: Patient was referred to Neurosurgical departments in Heidelberg and Wurzburg, operation was refused, radiation and chemotherapy were refused by the patient.

[084] Prior treatment: oral corticosteroids. [085] Planned Treatment: Taurolidine intravenously

[086] Chief complaints on admission: Diffuse headache, urinary incontinence, blurred vision, motor aphasia, gait disturbance, impaired memory.

[087] Neurological examination on admission: Awake -somnolent, alert, impaired vision, nearly complete motor aphasia, apraxia, gait disturbance, urinary incontinence, severe mnesic and concentration deficits

[088] Karnofsky index on admission: 20 -30

[089] MRI at Day 1 of treatment (pre treatment): Bifrontal space occupying lesion (ca. 8 x 8 x 8 cm) with irregular shape and ring like contrast enhancement and destructive affection of the corpus callosum. The marked space occupying effect leads to disappearance of nearly all reserve spaces.

[090] Treatment

[091] Day 1: Informed consent; Blood samples; MRI.

[092] Day 2: Insertion of a central venous line; Chest X-ray.

[093] Days 3-8: Intravenous administration of 4 x 250 ml of 2 % taurolidine/day within 2 hours, followed by an interval of 4 hours; Blood samples twice daily; Substitution of electrolytes.

[094] Day 9: Intravenous administration of 1 x 250 ml of 2 % Taurolidine within 2 hours; Discharge.

[095] Treatment summary:

[096] In total, 25 x 250 ml of 2 % taurolidine (125 g taurolidine) were administered without side effects. Electrolytes and fluid were substituted according to the results of the blood samples.

[097] Chief complaints on discharge: Headache improved, no urinary incontinence, vision improved, gait disturbance improved, motor aphasia slightly improved, impaired memory.

[098] Neurological examination on discharge: Awake, alert, vision improved, motor aphasia slightly improved, gait disturbance improved, apraxia slightly improved, no urinary incontinence, severe mnesic and concentration deficits

[099] Karnofsky index on discharge: 40-50

[0100] In view of the dramatic improvement observed in the patient's condition after a single treatment cycle, it is expected that an infusion regime of at least two cycles will provide the desired therapeutic effect.

Example 11: Treatment of Severe Glioblastoma Multiforme Grade IV

[0101] Prior to treatment the patient exhibited severe glioblastoma multiforme grade IV, left temporal lobe affected. The tumor was prominent in computer tomography pictures of the patient's cranium, prior to treatment. The patient's cranium was imaged in a T2-weighted picture sequence in axial, sagittal and coronary layer orientation as well as Tl -weighted picture sequence in axial layer orientation natively and in axial, coronary and sagittal layer orientation after contrast medium application as well as MR spectroscopy.

[0102] The patient was treated with four treatment cycles each consisting of a seven- day infusion phase of a daily dose of 20 g taurolidine (4 x 250 ml 2% taurolidine solution) and a two-day rest phase. After the four cycles, the patient underwent an additional two-day infusion phase. Regular computer tomography images of the patient's cranium were taken during treatment.

[0103] By the end of the second treatment cycle (200 g taurolidine administered), brain edema was noticeably reduced. By the end of third treatment cycle (300g taurolidine administered), tumor growth had stopped. After the completion of the entire course of treatment (60Og taurolidine administered), the tumor was shown by computer tomography to be almost completely disintegrated. Little or no necrosis was observed during the course of treatment, indicating that the tumor reduction was the result of apoptosis.

Example 12: Treatment of brain tumors with direct application of taurolidine/taurultam

The methylol transfer agent is applied directly to the tumor cavity using taurolidine/taurultam containing tubes consisting of several segments with semipermeable membrane.

[0104] Following total or partial tumor removal, a special tube is implanted in the tumor cavity, so that the end of this tube lies subgaleal. The tube includes various segments of semipermeable material, which contains taurolidine/taurultam and can be refilled via a subgaleal port.

Example 13: Treatment of Inoperable glioblastoma infiltration of basal ganglia

[0105] A forty year old male patient with inoperable glioblastoma infiltration in the basal ganglia was treated with a regimen of 6 dosing cycles, each with administration phases of 5 days, with each administration phase separated by a non-administration

phase of 2 days. During each day of administration, 250 ml of taurolidine 2% solution was intravenously administered to the patient 4 times daily. This regimen surprisingly and unexpectedly resulted in a marked size-reduction of the tumor, and disappearance of perifocal edema.

Example 14:

[0106] Taurolidine is administered by central line four times daily, 250ml of 2% solution has a continuous Lv. infusion. Alternatively, in a case of abdominal cancer such as pancreatic cancer, colon cancer, ovarian cancer or the like, taurolidine is administered intraperitoneally 2 to 4 times daily, 100 - 250ml of 2% solution. Taurolidine may be administered simultaneously with the protein or peptide, or non- concurrently if desired due to differences in administration methods of the agents.

Example 15:

[0107] In combination with any of the regimens set forth in Examples 8-14, a cancer or tumor-inhibiting therapeutic protein or peptide is administered.

Example 16:

[0108] Bevacizumab (Avastin™, Genetech, Roche) 5 mg/kg is given to a cancer patient by intravenous infusion once every 14 days as an i.v. infusion.

[0109] Taurolidine is administered by central line four times daily, 250ml of 2% solution has a continuous i.v. infusion. Alternatively, in a case of abdominal cancer such as pancreatic cancer, colon cancer, ovarian cancer or the like, taurolidine is administered intraperitoneally 2 to 4 times daily, 100 - 250ml of 2% solution. Taurolidine may be administered simultaneously with the protein or peptide, or non- concurrently if desired due to differences in administration methods of the agents.

[0110] Toxicity and side-effects of the therapeutic protein is substantially reduced by the taurolidine. Additionally, the dosage of the therapeutic protein may be reduced without reduction in efficacy.

Example 17:

[0111] Trastuzumab (Herceptin™, Roche) is administered to a cancer patient by intravenous infusion initially 4 mg/kg followed by 2 mg/kg once a week as an i.v. infusion.

[0112] Taurolidine is administered by central line four times daily, 250ml of 2% solution has a continuous i.v. infusion. Alternatively, in a case of abdominal cancer such as pancreatic cancer, colon cancer, ovarian cancer or the like, taurolidine is

administered intraperitoneally 2 to 4 times daily, 100 - 250ml of 2% solution. Taurolidine may be administered simultaneously with the protein or peptide, or non- concurrently if desired due to differences in administration methods of the agents.

[0113] Toxicity and side-effects of the therapeutic protein is substantially reduced by the taurolidine. Additionally, the dosage of the therapeutic protein may be reduced without reduction in efficacy.

Example 18:

[0114] Rrituximab (MabThera™, Roche) is administered to a cancer patient by intravenous infusion 375 mg/m ² of body surface four times weekly as an i.v. infusion.

[0115] Taurolidine is administered by central line four times daily, 250ml of 2% solution has a continuous i.v. infusion. Alternatively, in a case of abdominal cancer such as pancreatic cancer, colon cancer, ovarian cancer or the like, taurolidine is administered intraperitoneally 2 to 4 times daily, 100 - 250ml of 2% solution. Taurolidine may be administered simultaneously with the protein or peptide, or non- concurrently if desired due to differences in administration methods of the agents.

[0116] Toxicity and side-effects of the therapeutic protein is substantially reduced by the taurolidine. Additionally, the dosage of the therapeutic protein may be reduced without reduction in efficacy.