Asparaginase-Based Cancer Therapies

Abstract

The present invention relates to improved formulations and delivery of asparaginase for use in medicine, including human and veterinary medicine.

Description

[Technical field]

[0001] The present invention relates to improved formulations and delivery of asparaginase for use in medicine, including human and veterinary medicine. [Background technology]

[0002] Currently, there are three commercial preparations of L-asparaginase (hereafter asparaginase) for clinical use in both human and veterinary oncology: (1) a tetramer from Escherichia coli with a molecular weight of about 140 kDa; (2) a tetramer from Dickeya dadantii (Erwinia chrysanthemi) with a similar molecular weight; (3) a pegylated version of (1) with a molecular weight of about 400 kDa attached to the tetramer as poly-ethylene-glycol (PEG). There is also recombinant asparaginase produced in genetically engineered E. coli.

[0003] The molecular weights of all these molecules exceed the upper limit for molecules that can be estimated by glomerular filtration in the kidney (about 70 kDa). The main mechanism of clearance of asparaginase from the vascular circulation is via a non-specific immune response in the first week of therapy (Van der Meer, LT, et al., In Vivo Imaging of Antileukemic Drug Asparaginase Reveals a Rapid Macrophage-Mediated Clearance from the Bone Marrow, The Journal of Nuclear Medicine, Vol. 58, No. 2, February 2017), followed by a specific immune response (antibody-mediated) starting about 4 weeks after the start of therapy. The in vivo half-life of non-pegylated asparaginase reported in the literature is about 8 to about 40 hours, depending on the mode of delivery (iv or im) and possibly on the immune status of the patient. The half-life of the pegylated form is much longer, ranging from 2.5 to 12 days.

[0004] Conventional formulation of asparaginase for iv infusion or im injection is performed by dissolving the lyophilized enzyme in water, most often followed by further dilution in saline.

[0005] Due to their large molecular weight as tetramers, asparaginase molecules are restricted to vascular circulation when delivered by intravenous (iv) infusion. When delivered by intramuscular (im) injection, asparaginase is also transported to the vascular system via lymphatic flow, albeit at a slower rate, and therefore has a significantly longer clearance time. In any delivery mode, the drug only enters the interstitial fluid space in trace amounts, which severely limits its anticancer efficacy. Modification by pegylation extends the drug's half-life, but makes it even more unable to be cleared from the vascular system.

[0006] Cancer cells (e.g., leukemia or lymphoma cells) are present in the vasculature only temporarily and as a very small fraction of the total population. Thus, the efficiency of asparaginase restricted to the vascular circulation to deplete asparagine in interstitial fluid is limited due to the constant influx of asparagine into the interstitial space by protein turnover and asparagine synthesis by virtually all cells in the body. Diffusion of asparagine into the vasculature is limited and convective molecular transport between lymphatic and vascular circulation is very low. As a result, severe depletion of asparagine in cancer cells present in the media surrounding the cancer cells and / or outside the vasculature cannot be achieved with current modes of administration of asparaginase in tetrameric form. Summary of the Invention [Problem to be solved by the invention]

[0007] Asparaginase monomers have a molecular weight of approximately 35 kDa and are not enzymatically active. However, we have found that administration of asparaginase monomers under appropriate conditions, for example by simultaneous infusion of insulin with an appropriate rate of glucose infusion (the so-called insulin-glucose clamp), can mediate transport of the monomers from the vasculature into the interstitial fluid and presumably into most cells surrounding or residing within that volume. After entering the interstitial fluid, the monomers can associate and form into enzymatically active tetramers. The transport mechanism facilitated and / or stimulated by insulin is not fully understood at present, but is thought to involve endocytosis, whereby the insulin molecule itself is transported into responsive cells. [Means for solving the problem]

[0008] The present invention solves the challenge of effective delivery of asparaginase to its targets by transient and reversible disassociation of the tetramer prior to infusion, augmented by an insulin-glucose clamp, thereby promoting extravasation of asparaginase monomers and preventing their elimination through the kidney. Reassociation into tetramers in the interstitial fluid brings the drug into close proximity to the target cancer cells, greatly increasing its efficacy.

[0009] Thus, a first aspect of the invention relates to an asparaginase formulation for use in medicine, wherein the formulation comprises asparaginase in a dissociated form, in particular a monomeric form, to be administered to a subject in need thereof.

[0010] A further aspect of the invention relates to an asparaginase formulation comprising asparaginase in a dissociated form, particularly in monomeric form, wherein said formulation is particularly an aqueous formulation comprising urea at a concentration of about 3 mol / l to about 8 mol / l, more particularly about 4 mol / l to about 6 mol / l, e.g., about 5 mol / l.

[0011] A further aspect of the invention relates to a method for the treatment of cancer comprising administering to a subject in need thereof a therapeutically effective amount of asparaginase in a dissociated form, particularly in monomeric form. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention is based on the observation that asparaginase can be delivered into vascular circulation in a dissociated form, in particular as a monomer. Asparaginase monomers can be produced by reversible dissociation of asparaginase tetramers in chaotropic agents such as urea (Shifrin S and Parrot CL, In vitro Assembly of L-Asparaginase Subunits, The journal of Biological Chemistry, Vol. 249, No. 13, pp. 4175-4180, 1974). When dissolved in, for example, about 8 mol / l urea, the monomers dissociate and denature. When urea is diluted to less than about 2.5 mol / l, the monomers begin to refold and reassociate into tetramers without losing activity.

[0013] This is the scientific basis of the invention disclosed herein. The immediate dilution of urea upon administration, for example by infusion (concentration in plasma is only 2.5-7 mmol / l), initiates a refolding process (completed within about 30 seconds) that then reassociates in a characteristic time of minutes. During this time, some monomers are lost via the kidney, but the majority are cleared from the vasculature by the action of insulin. The reassociation process in the interstitial fluid delivers the active form of the enzyme to the immediate vicinity of and inside the cancer cells.

[0014] This mode of formulation and delivery can greatly increase the antitumor efficacy of this very important drug (Metayer, LE, et al., Mechanisms of cell death induced by arginase and asparaginase in precursor B-cell lymphoblasts, Apoptosis, 2019, 24:145-156). Even the currently used one is credited with saving the lives of thousands of patients since its introduction in widespread clinical use in the 70s, especially in those with childhood acute lymphoblastic leukemia (Camargos Rocha, JM, et al., Current Strategies for the Detection of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia, a Review in Mediterranean Journal of Hematology and Infectious Diseases, 2016:8; e2016024). Asparaginase is also used to treat acute myeloid leukemia and non-Hodgkin's lymphoma in humans and many lymphoma types in dogs.

[0015] According to the present invention, asparaginase can be used in combination with chemotherapy during the induction and, optionally, the maintenance phase, but can also be administered as a monotherapy.

[0016] Recently, there have been several publications suggesting that asparaginase may have antitumor activity even in some solid tumors if the enzyme is delivered to a target (Kim, K., et al., L-Asparaginase delivered by Salmonella typhimurium suppresses solid tumors, Molecular Therapy-Oncolytics, 2015, 2, 15007; doi:10.1038 / mto.2015.7). The delivery modes disclosed herein may be suitably used in these new indications for asparaginase.

[0017] Asparaginase is on the World Health Organization's list of essential medicines, which are the most effective and safe medicines needed in the health system.

[0018] According to the present invention, a formulation of asparaginase in a dissociated form, particularly in a monomeric form, is provided. The formulation is typically a solution, particularly an aqueous solution. In a particular embodiment, the formulation comprises dissociated asparaginase (e.g., as a monomer) and a chaotropic agent to maintain the asparaginase in a dissociated state. The chaotropic agent is present in a concentration sufficient to maintain the dissociated form of asparaginase. A preferred chaotropic agent is urea, which may be present in a concentration of about 3 mol / l to about 8 mol / l, particularly about 4 mol / l to about 6 mol / l, for example about 5 mol / l. In addition to the chaotropic agent, further pharma- ceutically acceptable excipients, such as buffers, surfactants, and the like, may be present.

[0019] The asparaginase formulation of the present invention can be prepared from any available asparaginase, for example asparaginase produced in the cells of bacteria such as Escherichia coli or Dickeya dadantii (including recombinant asparaginase), by dissolving said asparaginase in a chaotropic agent under conditions that provide the asparaginase in a dissociated form, particularly in monomeric form. The concentration of asparaginase in the formulation can be adjusted according to the particular application. For example, the concentration of asparaginase can be about 1,000 IU / ml to about 100,000 IU / ml, for example about 2,000 IU / ml to about 10,000 IU / ml. The dose administered to a human subject in need thereof is typically in the range of about 500 to about 5,000 IU asparaginase / kg body weight per day, depending on the subject and the disorder to be treated. Typically, the dose administered to a human patient is about 2,500 IU / kg / day. For dogs, the dosage is approximately double, i.e., about 1,000 to about 10,000 IU asparaginase / kg body weight per day, typically about 5,000 IU / kg / day.

[0020] The formulation may be administered by intravenous infusion, for example, for a period of about 4 to about 144 hours, depending on the subject and the disorder to be treated. Other modes of administration may also be used.

[0021] In certain embodiments, the asparaginase is administered under conditions that promote transport of proteins, particularly asparaginase monomers, from the vasculature to the interstitial fluid. This can be accomplished, for example, when the subject to whom the asparaginase formulation is administered is provided with insulin and a sufficient level of glucose to promote insulin activity. The insulin can be any type of insulin, such as human insulin, animal insulin, or an insulin analog, including short and long half-life insulin analogs. Glucose can be provided to the subject, for example, by infusion, to maintain normal levels of plasma glucose concentration. In certain embodiments, the asparaginase formulation is co-administered by an insulin-glucose clamp, i.e., simultaneous administration of insulin and glucose. The administration of glucose can be accompanied by administration of a potassium salt, such as KCl, to compensate for potassium ions entering the cells when high doses of glucose are administered.

[0022] Treatment may be supported by concomitant administration of essential amino acids, electrolytes, fluids and / or antibiotics.

[0023] The preparation of the present invention is suitable for cancer treatment, for example in human medicine or veterinary medicine, for example in the treatment of cats and dogs.The preparation can be administered to a human patient suffering from leukemia or lymphoma, for example acute lymphoblastic leukemia, acute myeloid leukemia or non-Hodgkin's lymphoma, or to an animal patient suffering from leukemia or lymphoma.However, the preparation can also be used for the treatment of solid tumors, such as colon cancer, breast cancer, and pancreatic cancer (Kim, K., et al., L-Asparaginase delivered by Salmonella typhimurium suppresses solid tumors, Molecular Therapy-Oncolytics, 2015, 2, 15007; doi:10.1038 / mto.2015.7).

[0024] The present invention will now be described in more detail with reference to the following examples. EXAMPLES

[0025] Example 1 - Case Report A 12 kg, 3 year old French bulldog diagnosed with high grade stage 3 lymphoma confirmed by lymph node biopsy was treated with E. coli asparaginase prepared and delivered according to the present invention.

[0026] 90,000 IU of asparaginase was prepared in batches of 10,000 IU by dissolving in 6 ml of 8 mol / l urea and further diluting to 4 mol / l and delivered by continuous infusion over 48 h together with short half-life insulin (6 IU / kg / day) and glucose as needed to maintain normal levels of plasma glucose concentrations (50% glucose, 12–33 ml / h).

[0027] Antibiotics were delivered by sc injection once daily. Antiemetics were also given once daily by adding into the infusion line. No food was given during these two days. Fluid balance was maintained by administering Ringer's solution by infusion. Replacement infusions of potassium chloride and / or potassium phosphate were given as needed by adding the respective salts to Ringer's solution.

[0028] All infusions were delivered through a central venous catheter using two syringe pumps (for insulin and asparaginase) and two peristaltic pumps (for glucose and Ringer's solution). Asparaginase was delivered through a separate infusion line to prevent dilution of the urea before it entered the catheter. Blood and urine samples were taken twice daily.

[0029] Vital signs, hematology and biochemistry showed no abnormalities during treatment and there was no evidence of hypersensitivity. On the second day, the enlarged lymph nodes were clearly reduced. Two days after treatment, the lymph nodes were excised and histopathology was negative. No other drugs were administered before or after asparaginase. After 4 weeks of treatment, the dog had no clinical signs of lymphoma.

[0030] Asparaginase activity was measured in serum samples. Compared to another dog not dissolved in urea but treated with a 6-fold lower dose of asparaginase / kg body weight, the activity in serum was 2-fold lower. This strongly indicates that asparaginase delivered dissolved in urea was removed from the vascular circulation as intended. Some activity was also measured in urine, as expected, contrary to findings from previous uses of asparaginase (asparaginase monomers filtered by the kidney could reassociate in urine). Plasma concentrations of asparagine fell below detection by conventional amino acid analysis.

[0031] Approximately 70% of lymphoma cases in dogs are known to respond to asparaginase. Our original in vitro study comparing arginase to asparaginase (Wells, J., et al., Arginase Treatment Prevents the Recovery of Canine Lymphoma and Osteosarcoma Cells Resistant to the Toxic Effects of Prolonged Arginine Deprivation, PLOS One, January 2013, Vol. 8, Issue 1, e54464) is broadly consistent with this clinical experience.

[0032] There is therefore great urgency to treat human and veterinary patients, especially if this modification of asparaginase can lead to a successful monotherapy and therefore reduce the incidence of long-term side effects currently known to result from conventional chemotherapy treatments of childhood leukemia (Science, special report, March 15, 2019).

[0033] Example 2 - Clinical Trial Protocol Based on the encouraging results shown in Example 1, a clinical trial protocol was developed.

[0034] The goal of this clinical trial is to test whether modified asparaginase and its delivery to cancerous dogs (i.e. canine lymphoma patients) can result in longer survival than established standard therapies. The native molecule of asparaginase is a tetramer with a molecular weight of 140 kDa. Molecules of this size are unable to leave the circulation and access and attack cancer cells. Administration of reversibly dissociated asparaginase monomers in patients simultaneously treated with an insulin-glucose clamp results in extravasation of asparaginase molecules, which then at least partially reassociate in the extracellular space and directly attack cancer cells.

[0035] [Study design] I) Preliminary Test Dogs are examined (physical exam, blood CBC and biochemistry), fine needle aspiration (FNA) and / or biopsy.

[0036] II) Eligibility Criteria Only dogs meeting the following criteria are eligible for treatment: 1. Diagnosis of lymphoma where any current treatment (surgery, chemotherapy, radiotherapy, (neo)adjuvant chemotherapy or other therapies) is unlikely to achieve tumor control, functional and cosmetic outcome. 2. Not previously treated with any form of conventional or experimental drug that may impair normal organ and bone marrow function. 3. Age ≥ 1 year and < 10 years. 4. Life expectancy greater than 6 months at the start of the study (if healthy). 5. Normal laboratory and physical parameters as defined below: -Platelets: ≧150,000 / μl -Glucose: ≦6mmol / l - Creatinine: ≦100μmol / l -Liver enzymes AST (SGOT) / ALT (SGPT): ≤ 5 × institutional upper limit of normal. -Temperature: 38.3~39.2℃ - Pulse rate: Small breeds 90~160; Medium breeds 80~130 - Exhalation during sleep: 10-30 / min

[0037] III) Exclusion Criteria 1. Have not recovered from chemotherapy or radiation therapy within 4 weeks (6 weeks for nitroso-ureas or mitomycin C) prior to study entry, or from an adverse event due to a drug administered prior to 4 weeks. 2. Previous treatment with any other investigational agent or steroid therapy within 2 weeks of sampling. 3. History of allergic reactions to agents used in this study due to similar chemical compounds or biological compositions, including enzyme allergy. 4. History of diabetes. 5. Uncontrolled intercurrent illness, including but not limited to ongoing or active infection, symptomatic congestive heart failure, cardiac arrhythmias or anxiety. 6. Pregnancy and breastfeeding. 7. Esophageal disorders (e.g. esophageal stricture, esophagitis, and megaesophagus) or after esophageal foreign body removal or esophageal surgical procedures. 8. Presence of ileus, significant GI bleeding, hemodynamic instability, uncontrollable vomiting and decreased level of consciousness.

[0038] IV) Treatment Protocol 1. Administration of a standard dose of asparaginase (10,000 IU) to test tumor sensitivity and potential hypersensitivity of the dog to asparaginase. 2. Placement of a central venous catheter (CVC) during general anesthesia. 3. Monitoring recovery from anesthesia. 4. The next day, start of drug infusion (asparaginase, insulin and glucose) via the previously prepared infusion pumps. 5. 24 / 7 monitoring for up to 96 hours. 6. Stopping infusion. 7. Return to normal life with owner(s). Dogs are fasted prior to general anesthesia and throughout the procedure. Parenteral food (glucose, amino acids) and fluids are provided. Dogs are under general anesthesia during CVC insertion and treated with pain medication (NSAIDs) as needed. Each dog will be under treatment for 96 hours: 12 hours for preparation, 72 hours for the therapy itself, 12 hours for the tapering off protocol and follow-up. There are 10 dogs in total. Dogs are constantly monitored. Any signs of pain, discomfort and symptoms are monitored, recorded and treated.

[0039] [Preparing the dog] I) Lymphoma diagnosis Diagnostic evaluation, including signalment, medical history and physical examination, is performed according to Withrow & MacEwen, (Canine Lymphoma and Lymphoid Leukemias, Third Edition). In addition, a complete blood count (CBC) with differential cell count, platelet count and serum biochemistry profile is obtained. Whole lymph nodes, e.g., prescapular or popliteal lymph nodes, are removed and histopathologically evaluated by an independent pathologist. In addition, chest and abdominal radiographs and ultrasound, including ultrasound-guided fine-needle aspiration cytology and / or needle biopsy, are performed.

[0040] II) Staging of Lymphoma After diagnosis, the extent of the disease is determined and correlated with the clinical stage of the disease. Most dogs present with aggressive stages (III-IV) according to the WHO Clinical Staging System for Lymphosarcoma in Domestic Animals.

[0041] III) Catheter Central venous catheters (CVCs) and standard cephalic venous catheters (e.g., Surfl oi / v. catheter, 20G, 1 1 / 4'', Terumo).

[0042] IV) Clistir An oral gut irrigation (WBI) is performed 24 hours before the start of the procedure. One day before WBI, the dogs are given antibiotics (a combination of vancomycin and gentamicin) for gut sterilization.

[0043] [Chemicals] I) L-Asparaginase L-asparaginase (Kyowa Kirin) is administered at a dose of 4,000 IU / kg / day (or 10,000 IU dissolved in 5 ml of 5 mol / l urea at 2 ml / kg / day) via a CVC and Injectomat Agilia® syringe pump.

[0044] II) Insulin 2 ml (200 IU) insulin (Novo Nordisk) is diluted in 48 ml saline (200 IU / 50 ml = 4 IU / ml) and administered via the Injectomat Agilia® at a dose of 6-12 IU / kg / day.

[0045] III) Glucose Insulin immediately reduces blood glucose levels unless a sugar source is provided. A 50% glucose formulation enriched with KCl is administered via an Agilia® Large Volume Pump initially at a dose of 14 g / kg / day (30 ml / h for a 25 kg dog) and then according to glycemia.

[0046] IV) Essential Amino Acids Amino acid solution for parenteral nutrition, such as Aminoven (Fresenius Kabi), is administered at a dose of 1 g / kg / day (i / v) via an Agilia® Large Volume Pump.

[0047] V) Antibiotics Vancomycin (Vancocin®, Eli Lilly) 5 mg / kg / day and gentamicin 2 mg / kg / day are given through a feeding tube after intestinal irrigation for alimentary canal sterilization. A cephalosporin such as cefotaxim (Claforan®, Aventis) 25-50 mg / kg every 12 hours or cefazolin sodium (Kefzol®, Eli Lilly) 1 g equivalent, divided into 4 doses, are given i / v throughout the entire treatment (6 days) as prophylactic antibiotics.

[0048] VI) Lactated Ringer's Balance fluid and electrolyte needs with an electrolyte source such as lactated Ringer's. Administer i / v via an infusion pump at a dose of 1.5-3 ml / kg / h. Dosage and total volume of lactated Ringer's are calculated according to the patient's total water and electrolyte needs (60 ml / kg / d).

[0049] VII) Potassium Chloride KCl is added to the 50% glucose solution at a dose of 20 mmol / L.

[0050] VIII) Diuretics Diuresis is maintained as needed with a diuretic such as furosemide (Lasix®) at a dose of 2-6 mg / kg, i / v, 4 times daily.

[0051] IX) Propofol Propofol (Fresenius Kabi) was used for induction of general anesthesia, initially administered at a dose of 1-2 mg / kg (iv) and then at 0.4 mg / kg / h for maintenance of anesthesia.

[0052] [Evaluation of treatment response] Treatment success is objectively assessed using procedures identical to those used to diagnose, grade, and stage the cancer.

[0053] In human oncology, criteria for lymphoma treatment evaluation have been standardized as the International Workshop Criteria (IWC) for response evaluation proposed by Cheson et al. (Report of the International Workshop for Standardization of Response Criteria for Non-Hodgkin's Lymphoma) and include five designations:

[0054] 1. Complete response (CR) is the complete disappearance of all detectable CT evidence of disease and all disease-related symptoms, as well as normalization of biochemical abnormalities and normal bone marrow biopsy (BMB). Previously involved nodes measuring >1.5 cm in greatest axial diameter on CT should regress to <1.5 cm and nodes previously measuring 1.1-1.5 cm should be reduced to <1 cm.

[0055] 2. CRu (indeterminate) corresponds to the CR criteria but with a residual mass measuring more than 1.5 cm in maximum axial diameter and more than 75% regression.

[0056] 3. Partial response (PR) is at least a 50% reduction in the sum of the product of the greatest diameters (SPD) of the six largest nodes with no increase in size of other nodes and no new sites of disease. Splenic and liver nodes must have at least 50% regression of SPD. BMB is irrelevant for determining PR.

[0057] 4. Stable disease (SD) is less common than PR but is not progressive disease.

[0058] 5. Progressive disease (PD) is a greater than 50% increase in the sum of the products of the greatest diameters of any previously abnormal nodes or the appearance of any new lesions during or at the end of therapy.

[0059] 6. Recurrent disease (RD) is the appearance of any new lesion or increase in size of more than 50% of a previously involved site or node in patients who have achieved CR or CRu.

[0060] [Measurement method] CT and MRI are routinely used to measure selected target lesions for response assessment. Cytology and histology can be used to distinguish between PR and CR. All measurable lesions up to a maximum of 5 per organ and a total of 10 lesions, representative of all involved organs, are identified and recorded as target lesions and measured at baseline. Target lesions are selected based on their size (lesions with the longest diameter) and their suitability for accurate repeat measurement (by imaging technique or clinical). The sum of the longest diameter (LD) for all target lesions is calculated and reported as baseline total LD. The baseline total LD ​​is used as a reference to objectively characterize the tumor.

[0061] [Response criteria] I) Evaluation of target lesions * Complete response (CR): Disappearance of all target lesions * Partial response (PR): At least a 30% reduction in the sum of the LD of target lesions using the baseline sum LD as reference. * Progressive disease (PD): at least a 20% increase in the sum of the LD of the target lesions, using as reference the smallest total LD ​​recorded since the start of treatment, or the appearance of one or more new lesions. * Stable Disease (SD): Using the smallest total LD ​​since the start of treatment as reference, there is neither sufficient shrinkage to qualify as PR nor sufficient growth to qualify as PD.

[0062] II) Evaluation of non-target lesions * Complete response (CR): Disappearance of all non-target lesions and normalization of tumor marker levels * Incomplete Response / Stable Disease (SD): Persistence of one or more non-target lesion(s) and / or maintenance of tumor marker levels above normal limits * Progressive Disease (PD): Appearance of one or more new lesions and / or overt progression of existing non-target lesions

[0063] [confirmation] To assign PR or CR status, changes in tumor measurements are confirmed by repeat assessments, which should occur at least 4 weeks after criteria for response were first met. Longer intervals, as determined by the study protocol, may also be appropriate. In the case of SD, follow-up measurements must meet SD criteria at least once after study entry, at the minimum interval defined in the study protocol (generally at least 6-8 weeks).

[0064] [Overall duration of response] Overall duration of response is measured from the time that metrics are met for CR or PR (whichever state is recorded first) to the first date of objectively documented relapse or PD, using the minimum measurement recorded since the start of treatment as the reference for PD.

[0065] [Duration of stable disease] SD is measured from the start of treatment until criteria for disease progression are met, using as reference the minimum measurement recorded since treatment initiation.

[0066] [Response review] All responses will be reviewed by an independent expert at study completion.

[0067] [Reporting the results] All patients included in the study must be evaluated for response to treatment, even if there are major protocol deviations or they are inadequate. Each patient will be assigned to one of the following categories: 1) Complete response 2) Partial response 3) Stable disease 4) Progressive disease 5) Early death from malignant disease 6) Early death due to toxicity 7) premature death from other causes, or 8) Unknown (not evaluable, insufficient data).

[0068] All patients fulfilling the eligibility criteria must be included in the primary analysis of response rate. Patients in response categories 4 to 8 should be considered as not responding to treatment (disease progression). Therefore, inadequate treatment schedule or drug administration does not lead to exclusion from the analysis of response rate.

[0069] All discussions are based on all eligible patients. Subanalyses may then be performed based on subsets of patients excluding those in whom major protocol deviations were identified (e.g., early death due to other reasons, early discontinuation of treatment, major protocol violations, etc.). 95% confidence intervals are provided.

Claims

1. 1. An asparaginase formulation for use in medicine, comprising: An aqueous formulation containing asparaginase in a dissociated form in urea is administered to a subject in need thereof. formulation.

2. 2. The formulation of claim 1, the formulation comprises asparaginase in monomeric form; formulation.

3. 3. The formulation of claim 1 or 2, Contains urea at a concentration of 3 mol / l to 8 mol / l; formulation.

4. 4. The formulation of claim 3, Contains urea at a concentration of 4 mol / l to 6 mol / l; formulation.

5. 5. The formulation of any one of claims 1 to 4, The asparaginase is an asparaginase produced in bacterial cells, including recombinant asparaginase. formulation.

6. 6. The formulation of any one of claims 1 to 5, The asparaginase is administered by intravenous infusion. formulation.

7. 7. The formulation of any one of claims 1 to 6, The asparaginase is administered to the subject under conditions that subject to an insulin-glucose clamp. formulation.

8. 8. The formulation of any one of claims 1 to 7, For use in the treatment of cancer, formulation.

9. 9. The formulation of any one of claims 1 to 8, For use in human medicine, formulation.

10. 10. The formulation of claim 9, For use in treating human patients suffering from leukemia or lymphoma, formulation.

11. 10. The formulation of claim 9, For the treatment of human patients suffering from acute lymphoblastic leukemia, acute myeloid leukemia or non-Hodgkin's lymphoma, formulation.

12. 9. The formulation of any one of claims 1 to 8, For use in veterinary medicine, formulation.

13. 13. The formulation of claim 12, For use in treating cats or dogs, formulation.

14. 14. The formulation of claim 12 or 13, For use in treating animal patients suffering from cancer, formulation.

15. 14. The formulation of claim 12 or 13, For use in treating animal patients suffering from leukemia or lymphoma, formulation.

16. 1. An asparaginase formulation comprising a dissociated form of asparaginase, The formulation is an aqueous formulation containing urea at a concentration of 3 mol / l to 8 mol / l; formulation.

17. 17. The formulation of claim 16, The concentration of the urea is 4 mol / l to 6 mol / l. formulation.