Asparaginase-based cancer therapy

Abstract

The present invention relates to improved preparation and delivery of asparaginase for pharmaceutical use (eg, human and veterinary medicine).

Description

[0001] The present invention relates to improved preparation and delivery of asparaginase for pharmaceutical use (eg, human and veterinary medicine). Background Art

[0002] Currently, there are three commercially available preparations of L-asparaginase (hereinafter, asparaginase) for clinical use in human and veterinary oncology: (1) a tetramer derived from Escherichia coli with a molecular weight of about 140 kDa; (2) a tetramer of about the same molecular weight derived from Dickeya dadantii (Erwinia chrysanthemi); and (3) a PEGylated form of (1) with about 400 kDa added to the tetramer, such as polyethylene glycol (PEG). There is also a recombinant asparaginase produced in genetically engineered E. coli.

[0003] The molecular weight of all these molecules exceeds the upper limit of about 70kDa of the molecule that can be cleared by spherical filtration in kidney.The main mechanism that asparaginase is cleared from vascular circulation is via nonspecific immune response (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) in the first few weeks of treatment, and then the specific immune response (antibody-mediated) that starts about 4 weeks after treatment begins.The half-life in vivo of the asparaginase not PEGylated reported in the literature is about 8 to about 40 hours, depends on the delivery mode (iv or im), and is likely to depend on the immune status of the patient.The half-life of PEGylated form is significantly longer, in the range of 2.5 to 12 days.

[0004] Asparaginase for iv infusion or im injection is routinely prepared by dissolving the lyophilized enzyme in water and in most cases by further dilution in normal saline.

[0005] Due to their large molecular weight as tetramers, asparaginase molecules are confined to the vascular circulation if delivered by intravenous (iv) infusion. If delivered by intramuscular (im) injection, asparaginase is also transported to the vascular system via lymphatic flow, albeit at a slower rate, so the elimination time is significantly longer. In both delivery modes, the drug does not enter the interstitial fluid space, but enters in trace amounts, which greatly limits its anticancer effectiveness. Modification by PEGylation prolongs the half-life of the drug, but makes it even less likely to be discharged from the vascular system.

[0006] Cancerous cells (e.g., leukemia or lymphoma cells) are present in the vascular system only temporarily and as a very small part of the total population. Therefore, due to the continuous influx of asparagine into the interstitial space from protein conversion and asparagine synthesis by almost all cells in the body, the effectiveness of asparaginases confined to the vascular circulation in consuming asparagine in the interstitial fluid is limited. The diffusion of asparagine into the vascular system is limited, and the molecular transport carried out by convection between the lymphatic circulation and the vascular circulation is very limited (modest). Therefore, the mode of applying the tetrameric form of asparaginase of the present invention cannot achieve the deep consumption of asparagine in the medium around the cancerous cells outside the vascular system and / or in the cancerous cells.

[0007] Asparaginase monomers have a molecular weight of approximately 35 kDa and are enzymatically inactive. However, we have found that administration of asparaginase monomers under appropriate conditions, such as simultaneous insulin infusion accompanied by glucose infusion at an appropriate rate (the so-called insulin-glucose clamp), can mediate the transport of monomers out of the vascular system into the interstitial fluid and, most likely, to most cells surrounding or present in this volume. After entering the interstitial fluid, the monomers can bind and form enzymatically active tetramers. The transport mechanisms promoted and / or stimulated by insulin have not yet been fully elucidated, but they may involve endocytosis, by which the insulin molecule itself is transported into the responding cells.

[0008] Brief description of the invention

[0009] The present invention solves the problem of effectively delivering asparaginase to its target by temporarily reversibly dissociating the tetramer prior to infusion, which is enhanced by the insulin-glucose clamp, which promotes the extravasation of asparaginase monomers, thereby preventing their elimination by the kidneys. Rebinding into the tetramer within the interstitial fluid brings the drug into the immediate vicinity of the target cancerous cells, greatly increasing its efficacy.

[0010] Therefore, a first aspect of the present invention relates to an asparaginase preparation for pharmaceutical use, wherein the preparation comprising asparaginase in dissociated form, in particular in monomeric form, is administered to a subject in need thereof.

[0011] A further aspect of the invention relates to an asparaginase preparation comprising asparaginase in dissociated form, in particular in monomeric form, wherein the preparation is an aqueous preparation comprising urea, in particular in a concentration between about 3 mol / l and about 8 mol / l, more particularly in a concentration between about 4 mol / l and about 6 mol / l, for example about 5 mol / l.

[0012] A still further aspect of the invention relates to a method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of asparaginase in dissociated form, particularly in monomeric form.

[0013] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention is based on the observation that asparaginase can be delivered to the vascular circulation in a dissociated form, particularly as a monomer. Asparaginase monomer can be produced by reversibly dissociating asparaginase tetramer in a chaotropic agent (chaotropic agent) (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). If dissolved in, for example, about 8mol / l of urea, the monomer will separate and denature. Once urea is diluted to less than about 2.5mol / l, the monomer will begin to refold and recombine into a tetramer without losing activity.

[0015] This is the scientific basis of the invention disclosed herein. Immediate dilution of urea upon administration (e.g. by infusion) (concentrations in plasma are only 2.5 to 7 mmol / l) initiates the refolding process (completed in about 30 seconds), followed by reassociation with a characteristic time of several minutes. During this period, some monomers are lost via the kidneys, but most are removed from the vascular system by the action of insulin. The reassociation process in the interstitial fluid delivers the enzyme in active form to the immediate vicinity of and into cancerous cells.

[0016] This preparation and delivery mode can greatly increase the anti-tumor efficacy of this very important drug (Metayer, LE et al., Mechanisms of cell death induced by arginase and asparaginase inprecursor B-cell lymphoblasts, Apoptosis, 2019, 24: 145-156). As used now, it is believed that it has saved the lives of thousands of patients because it has been introduced in extensive clinical applications in more than seventy patients, most notably 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.

[0017] According to the invention, asparaginase can be combined with chemotherapy during the induction phase and in some cases during the maintenance phase. However, it can also be administered as a monotherapy.

[0018] Several publications in recent years suggest that asparaginase may have antitumor activity even in some solid tumors, as long as the enzyme is delivered to the target (Kim, K. et al., L-Asparaginase delivered by Salmonellatyphimurium suppresses solid tumors, Molecular Therapy-Oncolytics, 2015, 2, 15007; doi: 10.1038 / mto.2015.7). The delivery mode disclosed herein may have good use for asparaginase in these new indications.

[0019] Asparaginase is on the World Health Organization's list of essential medicines, the most effective and safe medicines needed in a health system.

[0020] According to the present invention, a prepared product of asparaginase in dissociated form, particularly monomeric form is provided. The prepared product is typically a solution, particularly an aqueous solution. In certain embodiments, the prepared product comprises, for example, a dissociated asparaginase as a monomer, and a chaotropic agent for maintaining asparaginase in its 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, other pharmaceutically acceptable excipients may be present, such as buffers, surfactants, etc.

[0021] Asparaginase preparations of the present invention can be prepared from any available asparaginase (e.g., asparaginase produced in bacterial cells such as Escherichia coli or Dictyosella davidiansis), including recombinant asparaginase, which is prepared by dissolving the asparaginase in a chaotropic agent under conditions that provide a dissociated form, particularly a monomeric form of asparaginase. The concentration of asparaginase in the preparation can be adjusted according to the specific application. For example, the concentration of asparaginase can be between about 1,000 IU / ml and about 100,000 IU / ml, such as about 2,000 IU / ml and about 10,000 IU / ml. The dosage applied to a human subject in need thereof should generally be about 500 to about 5,000 IU of asparaginase / kg body weight / day, depending on the subject and the condition to be treated. Typically, the dosage applied to a human patient will be about 2,500 IU / kg / day. For dogs, the dosage is approximately double that, ie, about 1,000 to about 10,000 IU asparaginase / kg body weight / day, usually about 5,000 IU / kg / day.

[0022] The preparation may be administered by intravenous infusion, for example, for a period of about 4 to about 144 hours, depending on the subject and the condition to be treated.Other modes of administration may also be used.

[0023] In a specific embodiment, asparaginase promotes protein, particularly asparaginase monomer, to be used under the condition of transporting from the vascular system to the interstitial fluid. This can be realized, for example, in the case of providing insulin and promoting sufficient levels of glucose of insulin activity to the subject of administering the asparaginase preparation. Insulin can be any type of insulin, such as human insulin, animal insulin or insulin analogs, including insulin analogs with short half-life and with long half-life. Glucose can be provided to the subject (for example, by infusion) to maintain normal plasma glucose concentration. In a specific embodiment, the asparaginase preparation is co-administered with insulin-glucose clamp (i.e., co-administering insulin and glucose). The administration of glucose can be accompanied by administration of potassium salts such as KCl, to compensate for the potassium ions that enter the cell when administering high doses of glucose.

[0024] Treatment may be supported by concomitant administration of essential amino acids, electrolytes, fluids, and / or antibiotics.

[0025] The preparation of the present invention is suitable for treating cancer, for example in human medicine or veterinary medicine, for example in the treatment of cats and dogs. The preparation can be applied to, for example, human patients with leukemia or lymphoma, such as acute lymphoblastic leukemia, acute myeloid leukemia or non-Hodgkin's lymphoma, or to animal patients with leukemia or lymphoma. However, the preparation can also be used to treat 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).

[0026] In addition, the present invention will be explained in more detail by the following examples:

[0027] Example 1 - Case Report

[0028] 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.

[0029] 90,000 IU of asparaginase were prepared in batches of 10,000 IU by dissolving in 6 ml of 8 mol / l urea, further diluted to 4 mol / l, and delivered by continuous infusion for 48 hours, together with short half-life insulin (6 IU / kg / day) and glucose required to maintain normal plasma glucose concentration levels (50% glucose, 12-33 ml / h).

[0030] Antibiotics were delivered by sc injection once daily. Antiemetics were also administered once daily by adding them to the infusion line. No food was provided during these two days. Ringer's solution was administered by infusion to maintain fluid balance. When needed, replacement infusions of potassium chloride and / or potassium phosphate were performed by adding the corresponding salts to the Ringer's solution.

[0031] All infusions were delivered via 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 via its own infusion line to prevent dilution of urea prior to entry into the catheter. Blood and urine samples were collected twice daily.

[0032] During treatment, vital signs, hematology and biochemistry showed no abnormalities and no signs of allergy. On the second day, the enlarged lymph nodes were significantly reduced. Two days after treatment, the lymph nodes were excised - histopathology was negative. No other drugs were administered before or after asparaginase. Four weeks after treatment, the dog had no clinical signs of lymphoma.

[0033] The activity of asparaginase in serum samples was measured. Compared with another dog treated with asparaginase that was 6 times lower in dose per kg body weight but not dissolved in urea, the activity in serum was now two times lower. This is a strong indication that the asparaginase delivered dissolved in urea is removed from the vascular circulation as expected. As expected (asparaginase monomers filtered by the kidneys can be re-bound in urine), but contrary to the findings of conventional use of asparaginase, some activity was also measured in urine. The plasma concentration of asparagine was reduced to a level below the detection level of conventional amino acid analysis.

[0034] It is known that approximately 70% of lymphoma cases in dogs respond to asparaginase. Our own in vitro work comparing arginase and 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.

[0035] Therefore, it is of great urgency to treat human and veterinary patients, especially if this modification of asparaginase can lead to successful monotherapy and thus reduce the incidence of long-term side effects currently known to be caused by conventional chemotherapy treatments for childhood leukemia (Science, special report, March 15, 2019).

[0036] Example 2 - Clinical trial protocol

[0037] Based on the promising results shown in Example 1, a clinical trial protocol was developed.

[0038] The purpose of this clinical trial is to examine whether denatured asparaginase and its delivery to cancerous dogs (i.e., canine lymphoma patients) can lead to a longer survival period than established standard therapy. The native molecule of asparaginase is a tetramer with a molecular weight of 140 kDa. Molecules of this size cannot leave the circulation and approach cancer cells to attack them. Using reversibly dissociated asparaginase monomers in patients treated simultaneously with insulin-glucose clamps will cause asparaginase molecules to extravasate, which will then re-bind and directly attack cancer cells at least partially in the extracellular space.

[0039] Study Design

[0040] I) Preliminary inspection

[0041] The dog will undergo an examination (physical exam, blood CBC and biochemistry), fine needle aspiration (FNA) and / or biopsy.

[0042] II) Eligibility criteria

[0043] Only dogs that meet the following criteria are suitable for treatment:

[0044] 1. Diagnosed with lymphoma with a low probability of achieving tumor control, functional outcome, and cosmetic outcome with any current treatment (surgery, chemotherapy, radiotherapy, (neo)adjuvant chemotherapy, or other treatments).

[0045] 2. No previous treatment with any form of conventional or experimental drugs that may be harmful to normal organ and bone marrow function.

[0046] 3. Age ≥1 year old and <10 years old.

[0047] 4. Greater than six months life expectancy at the start of the study (if they were healthy).

[0048] 5. Normal laboratory and physical parameters are defined as follows

[0049] - Platelets: ≥150,000 / μl

[0050] - Glucose: ≤6mmol / l

[0051] - Creatinine: ≤100 μmol / l

[0052] - Liver enzymes AST (SGOT) / ALT (SGPT): ≤5X the conventional upper limit of normal.

[0053] - Temperature: 38.3-39.2℃

[0054] - Pulse rate: 90-160 for small breeds; 80-130 for medium breeds

[0055] - Exhale 10-30 / min while resting

[0056] III) Exclusion criteria

[0057] 1. Chemotherapy or radiotherapy within 4 weeks (6 weeks for nitrosoureas or mitomycin C) prior to study entry, or failure to recover from an adverse event due to the agent administered more than 4 weeks ago.

[0058] 2. Prior treatment with any other investigational agent or steroid therapy within two weeks of sampling.

[0059] 3. History of allergic reactions, including enzyme allergies, attributed to compounds with similar chemical or biological composition to the reagents used in this study.

[0060] 4. History of diabetes.

[0061] 5. Uncontrolled intercurrent illness, including but not limited to ongoing or active infection, symptomatic congestive heart failure, cardiac arrhythmia, or anxiety.

[0062] 6. Pregnancy and breast-feeding.

[0063] 7. Esophageal disorders (e.g., esophageal stenosis, esophagitis, and esophageal dilatation (megaesophagus)) or after esophageal foreign body removal or esophageal surgery.

[0064] 8. Presence of intestinal obstruction, significant GI bleeding, hemodynamic instability, uncontrollable vomiting, and decreased level of consciousness.

[0065] IV) Treatment options

[0066] 1. A standard dose of asparaginase (10,000 IU) was administered to test the sensitivity of the tumor and the potential allergy of the dogs to asparaginase.

[0067] 2. Placement of a central venous catheter (CVC) during general anesthesia.

[0068] 3. Monitor recovery from anesthesia.

[0069] 4. On the next day, drug infusions (asparaginase, insulin, and glucose) were started via pre-prepared infusion pumps.

[0070] 5. Monitoring 24 / 7, up to 96 hours.

[0071] 6. Stop the infusion.

[0072] 7. Resume normal life with its owner(s).

[0073] Dogs will be fasted before general anesthesia and during treatment. Parenteral food (glucose, amino acids) and fluids will be given.

[0074] During CVC insertion, dogs were placed under general anesthesia and treated with pain medication (NSAIDs) if necessary.

[0075] Each dog will be treated for 96 hours: 12 hours for preparation, 72 hours for the treatment itself, and 12 hours for the tapering regimen and follow-up.

[0076] There will be 10 dogs in total.

[0077] The dog will be monitored continuously. Any signs of pain, discomfort and symptoms will be monitored, recorded and treated.

[0078] Dog preparation

[0079] I) Lymphoma Diagnosis

[0080] Diagnostic evaluation is performed according to Withrow & MacEwen, (Canine lymphoma and Lymphoid Leukemias, 3rd edition), including signalment, medical history and physical examination. In addition, a complete blood count (CBC) with different cell counts, platelet counts and serum biochemical profiles will be obtained. Whole lymph nodes, such as prescapular or popliteal lymph nodes, will be taken out and histopathological evaluation will be performed by an independent pathologist. In addition, chest and abdominal radiography and ultrasound examinations including ultrasound, guided fine-needle aspirate cytology and / or needle biopsy will be performed.

[0081] II) Lymphoma staging

[0082] After diagnosis, the extent of the disease is determined and correlated with the clinical stage of the disease. According to the WHO's Clinical Staging System for Lymphosarcoma in Domestic Animals, most dogs are in advanced stages (III-IV).

[0083] III) Catheter

[0084] A central venous catheter (CVC) and a standard cephalic vein catheter (eg, Surflo i / v. catheter, 20G, 11 / 4", Terumo) will be inserted.

[0085] IV) Clistir

[0086] A whole bowel irrigation (WBI) was performed 24 hours before the start of treatment. One day before WBI, the dogs were given antibiotics (a combination of vancomycin and gentamicin) for intestinal disinfection.

[0087] drug

[0088] I) L-asparaginase

[0089] L-asparaginase (Kyowa Kirin) will be administered via CVC and Injectomat The syringe pump administered a dose of 4,000 IU / kg / day (or 2 ml / kg / day of 10,000 IU dissolved in 5 ml of 5 mol / L urea).

[0090] II) Insulin

[0091] 2 ml (200 IU) of insulin (Novo Nordisk) was diluted with 48 ml of saline (200 IU / 50 ml = 4 IU / ml) and injected via Injectomat Administer at a dose of 6 to 12 IU / kg / day.

[0092] III) Glucose

[0093] Unless a sugar source is provided, insulin will immediately lower blood glucose levels. Initially at a dose of 14 g / kg / day (for a 25 kg dog => 30 ml / h), then depending on blood sugar, via Large Volume Pump administers a 50% glucose preparation enriched with KCl.

[0094] IV) Essential Amino Acids

[0095] via Large Volume Pump administers amino acid solutions for parenteral nutrition, such as Aminoven (Fresenius Kabi) at a dose of 1 g / kg / day i / v.

[0096] V) Antibiotics

[0097] Give vancomycin by feeding tube ( Eli Lilly) 5 mg / kg / day and gentamicin 2 mg / kg / day are used for intestinal disinfection after intestinal lavage. Cephalosporins such as ceftriaxone ( Aventis) 25 to 50 mg / kg every 12 hours, or equivalent to 1 g of cefazolin sodium ( Eli Lilly), divided into four doses, administered i / v., as a preventive antibiotic for the entire treatment period (six days).

[0098] VI) Lactated Ringer's solution

[0099] An electrolyte source, such as Lactated Ringer's solution, will be used to balance fluid and electrolyte requirements. It will be administered via an infusion pump i / v. at a dose of 1.5 to 3 ml / kg / h. The dose and total volume of Lactated Ringer's solution will be calculated based on the patient's total water and electrolyte requirements (60 ml / kg / d).

[0100] VII) Potassium chloride

[0101] KCl was added to 50% glucose solution at a dose of 20 mmol / L.

[0102] VIII) Diuretics

[0103] If needed, a diuretic such as furosemide Administer at a dose of 2-6 mg / kg i / v. 4 times daily to maintain diuresis.

[0104] IX) Propofol

[0105] Propofol (Fresenius Kabi) will be used to induce general anesthesia, initially at a dose of 1 to 2 mg / kg iv, and maintained at a dose of 0.4 mg / kg / h.

[0106] Assessment of treatment response

[0107] Response to treatment is objectively assessed using the same methods used to diagnose, grade, and stage cancer.

[0108] In human oncology, the criteria for lymphoma treatment evaluation are standardized to the International Working Group Criteria (IWC) for response assessment proposed by Cheson et al. (Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas) and include five designations:

[0109] 1. A complete response (CR) is the complete disappearance of all detectable signs of disease on CT and all disease-related symptoms, and normalization of biochemical abnormalities and a normal bone marrow biopsy (BMB). Nodules previously involved on CT that were greater than 1.5 cm in maximum axial diameter must regress to less than 1.5 cm, and nodules that previously measured 1.1-1.5 cm must decrease to less than 1 cm.

[0110] 2. CRu (uncertain) corresponds to the CR criteria, but the residual mass with a maximum axial diameter greater than 1.5 cm has regressed by more than 75%.

[0111] 3. A partial response (PR) is a reduction of at least 50% in the sum of the products of the largest diameters (SPD) of the six largest nodules, with no increase in size of other nodules and no new sites of disease. Splenic and hepatic nodules must regress by at least 50% in the SPD. BMB is not relevant to the determination of PR.

[0112] 4. Stable disease (SD) is less than PR but not progressive disease.

[0113] 5. Progressive disease (PD) was defined as an increase of more than 50% in the sum of the products of the largest diameters of any previously abnormal nodules or the appearance of any new lesions during or at the end of treatment.

[0114] 6. Recurrent disease (RD) was the appearance of any new lesion or an increase in size of more than 50% in a previously involved site or nodule in a patient who achieved CR or CRu.

[0115] Measurement method

[0116] CT and MRI will be routinely used to measure the target lesions selected for response assessment.Cytology and histology can be used to distinguish PR and CR.All measurable lesions (up to five lesions per organ and a total of ten lesions, representing all organs involved) will be identified as target lesions, and recorded and measured at baseline.Target lesions will be selected based on their size (lesions with the longest diameter) and their suitability for accurate repeated measurements (by imaging technology or clinically).The sum of the longest diameter (LD) of all target lesions will be calculated and reported as the baseline sum LD.Baseline sum LD will be used as a reference for objective characterization of tumors.

[0117] Response Standard

[0118] I) Evaluation of target lesions

[0119] *Complete response (CR): All target lesions disappear

[0120] * Partial response (PR): The sum of LD of target lesions decreased by at least 30% with the baseline sum of LD as reference

[0121] *Progressive disease (PD): The sum of LD of target lesions is taken as reference, with the minimum sum LD recorded since the start of treatment as reference.

[0122] An increase of at least 20% or the appearance of one or more new lesions

[0123] *Stable disease (SD): The disease has not been sufficiently reduced to

[0124] Complies with PR, but has not increased sufficiently to comply with PD

[0125] II) Evaluation of non-target lesions

[0126] * Complete response (CR): Disappearance of all non-target lesions and normalization of tumor marker levels

[0127] * Incomplete response / stable One or more non-target lesions persist and / or tumor marker levels remain within normal limits

[0128] Sexually Transmitted Disease (SD): above

[0129] *Progressive disease (PD): Appearance of one or more new lesions and / or unequivocal progression of existing non-target lesions

[0130] Confirmation

[0131] In order to designate the status of PR or CR, changes in tumor measurements will be confirmed by repeated assessments, which should be performed no less than 4 weeks after the criteria for response are first met. Longer intervals as determined by the study protocol may also be appropriate. In the case of SD, after study entry, follow-up measurements must meet the SD criteria at least once at the minimum interval defined in the study protocol (usually no less than 6-8 weeks).

[0132] Total response duration

[0133] Duration of overall response was measured from the time that CR or PR (whichever status was recorded first) met the measurement criteria until the first date of objectively documented relapse or PD, using the smallest measurement recorded since the start of treatment as reference for PD.

[0134] Duration of stable disease

[0135] The SD was measured from the start of treatment until the criteria for disease progression were met, using the smallest measurement recorded since the start of treatment as the reference.

[0136] Response Review

[0137] All responses will be reviewed by independent experts at the completion of the study.

[0138] Results Report

[0139] All patients included in the study must be assessed for response to treatment, even if there are major deviations from the protocol or they are ineligible. Each patient will be assigned to one of the following categories:

[0140] 1) Complete response

[0141] 2) Partial response

[0142] 3) Stable disease

[0143] 4) Progressive disease

[0144] 5) Premature death due to malignant disease

[0145] 6) Premature death due to toxicity

[0146] 7) premature death due to other causes, or

[0147] 8) Unknown (cannot be evaluated, insufficient data)

[0148] All patients meeting the eligibility criteria should be included in the primary analysis of response rate. Patients in response categories 4 to 8 should be considered unresponsive to treatment (progressive disease). Therefore, incorrect treatment regimen or drug administration will not lead to exclusion from the analysis of response rate.

[0149] All conclusions will be based on all eligible patients. Sub-analyses can then be performed based on subsets of patients, excluding those patients who have been identified as major protocol deviations (e.g., premature death due to other causes, early discontinuation of treatment, major protocol violations, etc.). 95% confidence intervals will be provided.

Claims

1. Use of an asparaginase preparation in the preparation of a medicament for treating leukemia, lymphoma, colon cancer, breast cancer or pancreatic cancer, wherein the asparaginase preparation comprises asparaginase in monomeric form; wherein the asparaginase preparation is an aqueous preparation of asparaginase in a chaotropic agent; The chaotropic agent is urea with a concentration of 3 mol / l to 8 mol / l.

2. The use according to claim 1, The chaotropic agent is urea with a concentration of 4 mol / l to 6 mol / l.

3. The use according to claim 1, The chaotropic agent is urea with a concentration of 5 mol / l.

4. The use according to any one of claims 1 to 3, The asparaginase is asparaginase produced in bacterial cells, including recombinant asparaginase.

5. The use according to any one of claims 1 to 3, The asparaginase is an asparaginase produced in Escherichia coli or Dietrichia davidianalis, including recombinant asparaginase.

6. The use according to any one of claims 1 to 3, wherein the asparaginase preparation is prepared for administration by intravenous infusion.

7. The use according to claim 4, wherein the asparaginase preparation is prepared for administration by intravenous infusion.

8. The use according to claim 5, wherein the asparaginase preparation is prepared for administration by intravenous infusion.

9. The use according to any one of claims 1-3 and 7-8, wherein the asparaginase preparation is prepared for administration to a subject under conditions that promote protein transport into interstitial fluid.

10. The use according to claim 4, wherein the asparaginase preparation is prepared for administration to a subject under conditions that promote protein transport into interstitial fluid.

11. The use according to claim 5, wherein the asparaginase preparation is prepared for administration to a subject under conditions that promote protein transport into interstitial fluid.

12. The use according to claim 6, wherein the asparaginase preparation is prepared for administration to a subject under conditions that promote protein transport into interstitial fluid.

13. The use according to any one of claims 1-3, 7-8 and 10-12, wherein the asparaginase preparation is prepared for administration to the subject under conditions wherein an insulin-glucose clamp is provided to the subject.

14. The use according to claim 4, wherein the asparaginase preparation is prepared for administration to the subject under conditions wherein an insulin-glucose clamp is provided to the subject.

15. The use according to claim 5, wherein the asparaginase preparation is prepared for administration to the subject under conditions wherein an insulin-glucose clamp is provided to the subject.

16. The use according to claim 6, wherein the asparaginase preparation is prepared for administration to the subject under conditions wherein an insulin-glucose clamp is provided to the subject.

17. The use according to claim 9, wherein the asparaginase preparation is prepared for administration to the subject under conditions wherein an insulin-glucose clamp is provided to the subject.

18. The use according to any one of claims 1-3, 7-8, 10-12 and 14-17, wherein the medicament is for treating leukemia or lymphoma.

19. The use according to claim 4, wherein the medicament is used to treat leukemia or lymphoma.

20. The use according to claim 5, wherein the medicament is for treating leukemia or lymphoma.

21. The use according to claim 6, wherein the medicament is used to treat leukemia or lymphoma.

22. The use according to claim 9, wherein the medicament is for treating leukemia or lymphoma.

23. The use according to claim 13, wherein the medicament is for treating leukemia or lymphoma.

24. The use according to any one of claims 1-3, 7-8, 10-12, 14-17 and 19-23, wherein the medicament is for human pharmaceutical use.

25. The use according to claim 4, wherein the medicament is for human pharmaceutical use.

26. The use according to claim 5, wherein the medicament is for human pharmaceutical use.

27. The use according to claim 6, wherein the medicament is for human pharmaceutical use.

28. The use according to claim 9, wherein the medicament is for human pharmaceutical use.

29. The use according to claim 13, wherein the medicament is for human pharmaceutical use.

30. The use according to claim 18, wherein the medicament is for human pharmaceutical use.

31. The use according to claim 24, wherein the medicament is for treating a human patient suffering from leukemia or lymphoma.

32. The use according to any one of claims 25-30, wherein the medicament is for treating a human patient suffering from leukemia or lymphoma.

33. The use according to claim 24, wherein the medicament is for treating a human patient suffering from acute lymphoblastic leukemia, acute myeloid leukemia or non-Hodgkin's lymphoma.

34. The use according to any one of claims 25-30, wherein the medicament is for treating a human patient suffering from acute lymphoblastic leukemia, acute myeloid leukemia or non-Hodgkin's lymphoma.

35. The use according to any one of claims 1-3, 7-8, 10-12, 14-17 and 19-23, wherein the medicament is for veterinary use.

36. The use according to claim 4, wherein the medicament is for veterinary use.

37. The use according to claim 5, wherein the medicament is for veterinary use.

38. The use according to claim 6, wherein the medicament is for veterinary use.

39. The use according to claim 9, wherein the medicament is for veterinary use.

40. The use according to claim 13, wherein the medicament is for veterinary use.

41. The use according to claim 18, wherein the medicament is for veterinary use.

42. The use according to any one of claims 1-3, 7-8, 10-12, 14-17 and 19-23, wherein the medicament is for treating a cat or a dog.

43. The use according to claim 4, wherein the medicament is for treating a cat or a dog.

44. The use according to claim 5, wherein the medicament is for treating a cat or a dog.

45. The use according to claim 6, wherein the medicament is for treating a cat or a dog.

46. ​​The use according to claim 9, wherein the medicament is for treating a cat or a dog.

47. The use according to claim 13, wherein the medicament is for treating a cat or a dog.

48. The use according to claim 18, wherein the medicament is for treating a cat or a dog.

49. The use according to claim 35, wherein the medicament is for treating an animal patient suffering from leukemia or lymphoma.

50. The use according to claim 42, wherein the medicament is for treating an animal patient suffering from leukemia or lymphoma.

51. The use according to any one of claims 36-41 and 43-48, wherein the medicament is for treating an animal patient suffering from leukemia or lymphoma.