Hypotonic fluid for endogenous release of liver arginase for cancer treatment

Abstract

The present invention relates to a hypotonic fluid, e.g., water for injection, useful for the treatment of cancer via the endogenous release of liver enzymes, including liver arginase, by infusion of the said fluid into the hepatic vein of a liver segment.

Description

[0001] Hypotonic fluid for endogenous release of liver arginase for cancer treatment

[0002] The present invention relates to a hypotonic fluid, e.g., water for injection, useful for the treatment of cancer via the endogenous release of liver enzymes, including liver arginase, by infusion of the said fluid into the hepatic vein of a liver segment.

[0003] Background

[0004] Depletion of L-arginine (in the following arginine) has been shown to be of utility in treating some cancers such as hepatocellular carcinoma and melanoma, and based on in vitro work, probably many others. The use of arginine-depleting enzymes such as arginase in cancer therapy has been described by, e.g., Shen et al. (Cell Death & Disease 8 (2017), e2720), Zou et al. (Biomedicine & Pharmacotherapy 118 (2019), 109210), Al-Koussa et al. (Cancer Cell International 20 (2020) Article number 150) and Zhang et al. (Cancer Letters 502 (2012), 58-70), the contents of which are herein incorporated by reference. In a recent publication with the inventor as a co-author, Chew HY et al. (Arginase- induced cell death pathways and metabolic changes in cancer cells are not altered by insulin. Sci Rep. 2024 Feb 19;14(1 ):4112. doi: 10.1038 / s41598-024- 54520-z. PMID: 38374190; PMCID: PMC 10876525.), the utility of arginine depletion was shown against 9 cancer lines, 3 of each of the breast, lung, and ovary. Four of these nine cancer lines could be completely eliminated when treated with recombinant human liver arginase at 1 lU / ml; the other five with 10 lll / ml during 9 days. This in vitro study showed that achieving the level of enzymatic activity and duration is of fundamental importance for the successful treatment of cancer.

[0005] The use of arginine-depleting enzymes in vivo is necessary, but in certain cases, it may not be sufficient to cause and maintain systemic, deep depletion of arginine needed to cause rapid, selective killing of cancer cells. The use of an insulin / glucose clamp in parallel with the enzymatic degradation of arginine makes the task of deep arginine depletion much more manageable. Insulin is a growth factor and thus promotes protein synthesis and inhibits protein breakdown. This is of crucial importance when the task is removing an amino acid from circulation, particularly of removing arginine, which is a semi-essential amino acid under tight homeostatic control.

[0006] An increase of vascular permeability by insulin also helps in getting therapeutic enzymes into interstitial fluid space, closer to where most cancerous cells reside. Finally, insulin may also play a role in transporting arginine-degrading enzymes into cancerous cells by stimulating endocytosis.

[0007] As the inventor’s research has shown, with the concomitant use of an insulin / glucose clamp, free arginine levels on the order of 5 to 10 pM could be obtained compared to the normal plasma concentration of about 100 pM. At these free arginine levels, cells, cancerous and healthy, will not proliferate but still can survive for prolonged periods. The target for arginine concentration to result in rapid killing of cancer cells is 1 pM or less.

[0008] The main obstacle to reaching that target of depletion is endogenous production of arginine by the so-called intestinal-renal axis whereby citrulline is produced in the intestines and converted to arginine by mostly kidneys. None of the many approaches tested in experimental dogs to inhibit citrulline production in the intestines have resulted in a satisfactory reduction of plasma citrulline.

[0009] Arterial occlusion of the liver with hepatocellular carcinoma (HCC) has been proposed by the inventor and performed on a small cohort of human patients (Cheng PN, et al. Remission of hepatocellular carcinoma with arginine depletion induced by systemic release of endogenous hepatic arginase due to transhepatic arterial embolization, augmented by high-dose insulin: arginase as a potential drug candidate for hepatocellular carcinoma. Cancer Lett. 2005 Jun 16;224(1 ):67-80. doi: 10.1016 / j.canlet.2004.10.050. Epub 2004 Dec 25. PMID: 15911102.). This conventional palliative intervention for HCC was combined with continuous infusions of insulin / glucose clamp, sodium nitroprusside, vasopressin, and a prostacyclin analog (lloprost), to minimize the side effects and associated risks of low arginine. The clinical results were encouraging but the limitations of this intervention prevented its broader use.

[0010] Firstly, arterial occlusion of the liver causes lysis of only the tumor lesions with minimal damage to healthy liver tissue, which is why this intervention was accepted in the first place. A healthy liver at its smallest anatomical level (classic lobule) comprises three blood flows: (i) arterial inflow, (ii) portal venous inflow, and (iii) venous outflow. Additionally, there are discrete vessels in each of the classic lobules for outflows of the lymph and the bile. All of these fluids move in and out of about a million of lobules in a human liver via branched vessels. A single liver artery and a portal vein bring the blood into the liver which exits via several veins into the vena cava. Arterial inflow accounts for 20 to 30% of the venous blood outflow, while portal venous inflow accounts for the remaining 70 to 80% of the venous outflow. We can refer to the venous outflow as total throughput. If the arterial inflow is cut in the healthy liver, the remaining 70 to 80% of throughput due to portal venous flow can support the basic metabolic needs of hepatocytes. As the liver regenerates / remodels very fast, the arterial flow is reestablished, and near-normal liver function is restored in weeks to months. However, the liver cancer that grows inside the liver fails to develop the portal venous vasculature. Hence, occlusion of the arterial flow causes its necrosis. However, this treatment is palliative because there are always cancer cells at the periphery of the tumor which survive on the support of healthy cells in the surroundings. Moreover, the HCC cells produce on average only about 10% of the arginase of the healthy hepatocytes and in some cases none. The intervention is thus of a hit-or-miss nature and limited only to HCC.

[0011] The other major limitation is the short duration of arginase release caused by arterial occlusion of the liver with HCC - at best 24 to 36 hours. Even then, 5 of the 7 patients treated responded positively and 3 had a durable response, possibly cures. WO 2023 / 066910 discloses that systemic delivery of a medicament comprising an arginase-decomposing enzyme and a citrulline-converting enzyme, e.g., from crude or partially purified liver extracts can lead to a reduction of free plasma arginine to below-detection levels. The production of liver extracts, however, requires specialized facilities and equipment and thus high overall costs. Further, administering an exogenous liver extract might cause an undesired immunological response.

[0012] It was an object of this invention, to provide a means for improving the depletion of free arginine in the blood of a subject, particularly of a human cancer patient or a dog with cancer. More particularly, it was an object of the invention to overcome disadvantages associated with previous treatment schedules involving amino acid depletion with, e.g., administration of PEGylated arginase or PEGylated arginine deiminase (ADI), which are currently investigated in over 30 clinical trials for cancer treatment.

[0013] Summary of the invention

[0014] A first aspect of the present invention relates to a hypotonic fluid for use in a method of treating cancer, wherein the hypotonic fluid is administered to the liver of a subject in need thereof through a hepatic vein thereby causing lysis of liver cells. The lysis of liver cells releases endogenous arginine-decomposing enzymes including but not limited to arginase (ARG), and citrulline-converting enzymes such as argininosuccinate synthase (ASS) causing a deep and long- lasting arginine depletion in the subject’s body.

[0015] The present invention is useful in human and veterinary medicine. Thus, the subject to be treated may be a human patient or a non-human subject, particularly a cat or dog.

[0016] In particular embodiments, the hypotonic fluid is pure water, e.g., water for injection. In certain embodiments, the hypotonic fluid is administered to the liver by means of a catheter inserted into a hepatic vein. The catheter may be inserted into a hepatic vein through the vena cava superior and a jugular vein and / or through the vena cava inferior and a femoral vein. The catheter comprises at least one lumen for the hypotonic fluid and an opening, e.g., at its tip, for administering the hypotonic fluid after being positioned at its target site. According to certain embodiments, the catheter is inserted into the liver vein such that the outflow therefrom is blocked by wedging the end of the catheter to occlude the vein. In certain embodiments, the catheter comprises at its tip an occlusion means, e.g., an inflatable ballon, for occluding the vein into which it is inserted.

[0017] In certain embodiments, the treatment encompasses targeting of a single liver lobe or a part of it. In those embodiments, the catheter may remain in its position during the whole treatment procedure. In further embodiments, the treatment encompasses targeting several liver lobes. In those embodiments, a catheter repositioning and / or the positioning of at least one further catheter during the treatment procedure may take place.

[0018] The amount of hypotonic fluid administered to the liver will depend on the amount of liver cells to be lysed which in turn will depend on the type and seventy of the disease to be treated. In certain embodiments, an amount of about 400 to about 800 ml, e.g. about 600 ml to about 750 ml hypotonic fluid per day is administered. In certain embodiments, the hypotonic fluid is administered to the liver in an amount of about 5 ml to about 20 ml, about 7 ml to about 15 ml, and particularly in an amount of about 10 ml per g liver cells to be lysed. In certain embodiments, the hypotonic fluid is administered to the liver for at least one day, and particularly about 2 to 6 days.

[0019] The hypotonic fluid may be co-administered together with at least one further fluid for compensating the side effects of the therapy. In certain embodiments, the hypertonic fluid is co-administered together with a hypertonic fluid, a glucose solution, and / or a mixture of essential amino acids without arginine. In certain embodiments, at least one adjuvant agent is administered which may be selected from an insulin optionally in combination with glucose, a nitric oxide (NO) donor, e.g., sodium nitroprusside (SNP), nitroglycerin and gaseous NO, a pressor peptide, e.g., a vasopressin, and a prostacyclin analog, e.g., Iloprost.

[0020] The present invention relates to the treatment of cancer. In certain embodiments, the cancer is selected from a blood cancer such as leukemia and lymphoma, and a solid cancer such as liver cancer including primary liver cancer and hepatocellular carcinoma, skin cancer such as melanoma, colon carcinoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, gastric cancer, and breast cancer.

[0021] Even though endogenously released liver arginase does not penetrate the brainblood barrier, the intervention is highly likely to have a positive effect even on brain tumors, e.g. glioblastoma multiforme. The brain uses large amounts of arginine (as observed by high concentrations of arginase and nNOS in brain tissue) but it cannot synthesize any. With the blood side of the barrier fully depleted of arginine, cancer cells within the brain will also be depleted. And unlike most other tissues, the brain does not have a large reserve of dispensable proteins to break down.

[0022] A further aspect of the present invention relates to a kit comprising medical devices and drugs for use in the arginine depletion method as described above.

[0023] A further aspect of the present invention relates to a method for the treatment of cancer, comprising administering a hypotonic fluid to a subject in need thereof to the liver of a subject in need thereof through a hepatic vein thereby causing lysis of liver cells.

[0024] Detailed Description of the Invention

[0025] The present invention removes limitations of prior art arginine depletion protocols and makes the endogenous release of liver enzymes for systemic arginine depletion universally applicable to most cancer types. In addition to arginase (ARG), the release of argininosuccinate synthase (ASS) is considered crucially important. Released into vascular circulation, ASS, which combines citrulline and aspartate into argininosuccinate, will inhibit the passage of citrulline from the intestines, the main organ for citrulline synthesis, to kidneys where citrulline is converted to arginine.

[0026] The lysis of hepatocytes will release thousands of different proteins and hundreds of other, smaller molecules. Our research focused on arginine depletion has demonstrated that if combined with protein turnover modulation by, e.g., insulin / glucose clamp, the combined effects of this release can lead to systemic reduction of arginine to the levels required for selective, rapid elimination of cancerous cells. We have also demonstrated that the main side effects of arginine depletion can be mitigated by co-infusion of a nitric oxide (NO) donor, e.g. sodium nitroprusside, and / or a pressor peptide, e.g., vasopressin. Iloprost, a stable analog of prostacyclin has also been useful in preventing platelet aggregation. These interventions prevent blood clotting and hemodynamic instabilities that otherwise could cause serious morbidities.

[0027] Cannulation of the liver vascular system is commonly performed by techniques developed in the field of interventional radiology. In most cases, it is used for therapeutic interventions, and for that, the arterial vessels are cannulated and used to deliver to the liver a high concentration of, e.g., drugs to treat liver cancer or other cancers that have developed metastases to the liver. The aforementioned arterial occlusion of the liver is such an example.

[0028] Approach to the liver veins is currently used mostly for diagnostic procedures, e.g., to measure trans-hepatic venous pressure and thus diagnose portal venous hypertension.

[0029] This approach to the liver veins is now proposed to be used for intervention by infusion of hypotonic fluid, e.g., water for injection, to cause controlled lysis of hepatocytes and release of their proteins, including ARG and ASS. The liver veins can be approached via vena cava cannulated either from the superior direction (cranial in the dog) through a jugular vein (preferably through the right jugular vein) passing through the right atrium to reach the level of the main liver veins and guiding the catheter into one of smaller veins that drain 10% to 20% of the total liver volume.

[0030] Alternatively, and preferably, the vena cava can be entered from the inferior (caudal in the dog) direction via a femoral vein guiding the catheter into one of the smaller liver veins.

[0031] The catheter can be wedged into its target vein, or it can include at its tip an occlusion balloon. Both techniques are used for measuring transhepatic venous pressure. For a prolonged dwelling (several days) of the catheter in the liver vein, the balloon version is preferred.

[0032] In both cases, the venous outflow from the targeted liver section is blocked. Arterial inflow continues and is drained in the retrograde direction by the portal vein but only to the level where the portal vein supplying the occluded venous outflow branches out from the rest of the portal venous system. From that junction, the blood flow from the occluded volume joins the normal portal inflow into the rest of the liver.

[0033] Now, if the hypotonic fluid is infused via a catheter occluding a section of the liver, it will gradually flush out the blood all the way back into the lobules, entering them through the central vein of the lobule and then into hepatic sinusoids. Hepatocytes exposed to low osmotic pressure will swell and then burst spilling their contents into the outflowing mixture of the infused fluid and arterial blood. The low osmotic pressure front will expand from the central lobular vein outwards to the portal canals. The lymphatic flow as well as the portal venous flow will bring the contents of lysed hepatocytes into the blood circulation which will distribute all the released molecules, including ARG and ASS, throughout the body. The preferred hypotonic fluid for flushing out hepatocytes is pure water, the so- called water for injection. In certain embodiments, however, the hypotonic fluid may contain a reduced concentration of electrolytes, e.g., sodium chloride which to normal saline is added to make up 0.9% mass per volume (9 grams per 1 liter which results in an isotonic solution, with an approximate osmolarity of 308 mOsm / l). Thus, in certain embodiments, the hypotonic fluid has an osmolarity of about 250 mOsm / L or less, about 200 mOsm / L or less, about 150 mOsm / L or less, about 100 mOsm / L or less, or about 50 mOsm / L or less.

[0034] To minimize potential systemic disturbance of tonicity, the infusion of hypotonic fluid directly into the liver could be offset by the infusion of hypertonic fluid into a distant vein or into the vena cava, e.g. , via a separate lumen of the same catheter.

[0035] Liver throughput (venous outflow) in an adult human is 1500 to 1900 ml / m inute or about a quarter of cardiac output. About 20 to 30% of that is arterial inflow. The in vitro measurements on pig liver suggest that about 10 ml of water for injection can flush out ARG contained in about 1 g of liver tissue.

[0036] The human liver contains about 500 lll / g of ARG. In the dog, this is several times higher. Endogenously released human liver arginase has a half-life of several hours. Interstitial and plasma volume add up to about 20% of body weight (BW). Maintaining about 1 lll / ml of ARG activity in this amount of fluid calls for the release of about 200 III of arginase per kg body weight in about 12 hours or 400 III of arginase per kg body weight per day. Thus, in certain embodiments, approximately 1 g of liver tissue should be flushed out per kg body weight per day. For a human of 75 kg body weight with about 1500 g of liver, three days of continuous flushing would call for full release of ARG from about 15% of the liver. The water infusion of about 10 ml / g of liver to be flushed amounts to 750 ml / day, which is about a third of fluid maintenance volume. Another third of the maintenance volume could be delivered as hypertonic fluid (e.g. as 1.8% saline) and the final third as a glucose infusion to balance the effects of insulin. A mixture of essential amino acids but without arginine could also be delivered with these additional infusions. The amount of liver tissue to be lysed by administration of hypotonic fluid according to the present invention does not cause critical medical issues. For comparison, two-thirds or even three-quarters of the liver can be safely surgically resected to remove cancerous lesions. The same amount of liver can be removed from a donor for a partial liver transplant. About 80% of the resected liver volume will grow back in less than 2 weeks. The flushing of the liver by hypotonic fluid will leave some of its scaffolding (stroma) intact and an even faster regeneration may be possible.

[0037] In the aforementioned in vitro study by Chew HY, et al., the cells were kept in the arginine-depleted medium for 9 days. For many cancer types, e.g., blood cancers, HCC, and melanoma, even two days can result in the total elimination of cancerous cells. Canine blood cancers needed no more than 3 days, while some sarcomas took up to 6 days, as reported in Wells JW, et al. ((2013) Arginase Treatment Prevents the Recovery of Canine Lymphoma and Osteosarcoma Cells Resistant to the Toxic Effects of Prolonged Arginine Deprivation. PLoS ONE 8(1 ): e54464. https: / / doi.org / 10.1371 / journal.pone. 0054464), co-authored by the inventor. Six days of continuous treatment by infusion of liver extracts or selective hemodialysis has been shown to be safe in dogs. The human HCC patients in the aforementioned study with arterial occlusion of the liver were treated for up to 6 days with all adjuvant medications without any adverse side effects.

[0038] List of Figures

[0039] Figure 1 shows flows of fluids in the classic liver globule (from Krstic RV, Human Microscopic Anatomy, An Atlas for Students of Medicine and Biology, Springer Berlin, Heidelberg, 1991 ).

[0040] Figure 2 shows a venous flow reversal in globules by infusion of hypotonic fluid according to the present invention. Figure 3 is a schematic representation of fluid flows in the liver.

[0041] Figure 4 shows the reversal of flows in a section of the liver infused by hypotonic fluid.

[0042] Figure 5 shows a kit with medical devices and drugs needed for the intervention.

[0043] Detailed Description of the figures

[0044] The classic liver lobule illustration in Figure 1 shows arterial blood inflow 10 via the intralobular artery (IIA), portal venous blood inflow 11 via the interlobular portal vein (IIV), and venous blood outflow 12. Venous outflow 12 through the central lobular vein (CV) and sub-lobular vein (SV) combines flows 13 from portal canals (PC) that flow through liver sinusoids (LS). The intralobular bile duct (BD) collects bile outflow 14.

[0045] Figure 2 illustrates flows in a classic liver lobule with the hypotonic fluid (e.g., water for injection) flow 20, entering the venous outflow vessel (SV) in the retrograde direction. The flows 21 through the liver sinusoids (SV) towards portal canals (PC) are now reversed. Arterial inflow 10 is maintained and it combines with the hypotonic fluid flow 20 into the portal flow 22, now reversed. Bile outflow 14 is diminished as hepatocytes (HP) are lysed by the flow of hypotonic fluid through the sinusoids.

[0046] Figure 3 is a schematic representation of the fluid flows in the liver. The main liver artery 100 branches into the arteries 101 of the liver lobes (Li). In the human liver, there are 8 lobes; in the dog, there are 6. Arterial vessels branch out into smaller and smaller vessels down to the level of about one million classic liver lobules shown in detail in Figures 1 and 2, and here schematically as CLn. Portal vein 102 also branches out to the individual lobules.

[0047] In contrast to the single artery and portal vein, several liver veins take venous outflow directly into the vena cava. For simplicity, only a single liver vein 103 is shown in Figure 3. Bile and lymph outflows are schematically represented by the respective vessels 104 and 105.

[0048] Figure 4 shows the flows during intervention according to the invention. While the global flows 100, 101 , 102, 103, 104, and 105 move in the same direction, a segment of the liver, here represented as the lobe Ls, is flushed out by infusion 110 of a hypotonic fluid into its outflow vein. Note that the outflow of venous blood from that segment is now blocked (X) by either a wedged catheter or a catheter with an occlusion balloon. The flow 111 through the portal vein of segment Ls is now reversed. In most cases, only a part of a liver lobe must be flushed out. That depends on the type of cancer being treated, and consequently on the combined effects of the enzymatic activity released and the duration of arginine depletion. If necessary, two or even three liver lobes could be used.

[0049] Figure 5 shows kit 200 comprising devices and medications for the treatment according to the invention. It preferably includes a catheter 201 of, e.g., Fogarty type (with an occlusion balloon 201 a), a guide wire 202, and an insertion set 203. An NO donor 204 (e.g., SNP for infusion), an antidote for cyanide 205, or nitroglycerin patches 206 (an alternative to SNP), vasopressin 207, or an analog thereof, for infusion or oral delivery, and an infusion line 208. A hypotonic fluid (e.g., water for injection) 211 , a solution of glucose (20 or 50%) 212, and a solution of amino acids 213 can also be packaged as a subset kit 210. Insulin must be kept at a low temperature and is typically unsuitable for inclusion in a kit. For human use, the ideal mode of insulin delivery is by an insulin pump with feedback provided by a glucose monitoring device. This system is now generally available to diabetic patients, and it greatly simplifies the administration of the insulin / glucose clamp - glucose can be delivered at a fixed rate, with the insulin pump maintaining the desired normal glycemia.

[0050] Using an endogenously released enzyme(s) has distinct advantages compared to the administration of exogenous enzymes, e.g., recombinant enzymes such as recombinant human liver arginase (or any modification of it) or recombinant arginine deiminase. The immune system is continuously dealing with liver enzymes released into circulation by the normal turnover of the liver. Thus, there is no overt response that could come about with the infusion of a foreign protein. The solution that all pharmaceutic companies have taken by PEGylation is counterproductive. These large molecules remain trapped within the vascular system and hence do not result in total systemic arginine depletion. The breakdown of dispensable proteins, mostly in the muscle tissue, releases amino acids into interstitial fluid. Enzymes within the vascular system can cause only a moderate reduction of arginine in most of the interstitial fluid.

[0051] Another important advantage - on top of the rapid clearance of foreign proteins by the innate immune system - is the avoidance of the development of a specific immune response that develops within weeks from the initial exposure. The specific immune response can make the use of foreign enzymes ineffective and may limit their use to only a short time, e.g., to a single session of several days. Because the development of a specific immune response to self-proteins is much less likely, the treatments according to the invention can be used multiple times. An important fact in this context is that there is no acquired resistance to arginine depletion - it works with the same efficiency through multiple cycles of attack on cancerous cells as shown in vitro. All known cytostatic drugs are prone to the acquirement of drug resistance.

[0052] From the in vitro research on arginine depletion with animal blood, to research on experimental dogs and clinical applications in dogs and human patients, we have identified the major systemic risks of arginine depletion to be related to platelet “activation”. Activation is a misnomer - in their normal, quiet physiological state platelets actively produce cGMP and cAMP stimulated by external signals of NO and prostacyclin, respectively. Lack of either of those signals interrupts the production of cGMP and / or cAMP, which in turn causes an influx of calcium, rupture of the membrane, and release of thrombogenic molecules. The only substrate for NO synthesis is arginine. It is thus critically important - in arginine deprived state - to provide NO externally which can be done by NO donors, e.g., sodium nitroprusside (SNP) or nitroglycerin. In addition to keeping platelets quiet, NO is also a potent vasodilator. The vasodilation is countered by pressor peptides, e.g., vasopressin, angiotensin I, and II, all of which contain arginine and are short-lived in circulation. Maintaining normal hemodynamics as represented by, e.g., heart rate, blood pressure, tissue perfusion, external delivery of an NO donor must be balanced by external delivery of a pressor. In all our research we have successfully used vasopressin with minor adjustments of the rate of infusion based on heart rate -- increased heart rate can be readily reduced by an increase in the infusion rate of vasopressin. SNP breakdown releases NO and cyanide. Infusions of sodium thiosulfate or B12a can easily control the toxic effects of cyanide.

[0053] Lysis of hepatocytes will release arginase and thus rapidly deplete arginine, especially locally. In addition, NO is synthesized by endothelial cells, which will also be lysed by infusion of hypotonic fluid. Platelets enter liver portal canals with arterial blood and could be activated (deprived of NO) on their retrograde passage into portal venous vessels. In the treatment according to this invention, SNP and vasopressin as well as lloprost could be delivered directly into the liver to minimize the risks of platelet activation and blood clotting.

[0054] As an alternative to the infusion of SNP, NO could be delivered as gas injected into the hypotonic fluid, thus avoiding the delay due to the decomposition of SNP, but also eliminating the toxicity of cyanide.

[0055] Our in vivo work with human patients and dogs showed the benefit of infusing essential amino acids but without arginine. These nine essential amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In particular embodiments, these nine essential amino acids may be administered together with L-aspartate.

[0056] Preferred adjuvant drugs to mitigate side effects of arginine depletion developed in experimental dogs and applied to clinical cases in human and canine cancer patients are listed below: - SNP

[0057] - Vasopressin

[0058] - Iloprost

[0059] - Insulin

[0060] - Glucose

[0061] - Amino acids

[0062] - Nitroglycerin

[0063] - NO (gas)

[0064] - Sodium thiosulfate (as an antidote to cyanide)

[0065] - Vitamin B12a

[0066] Arginine depletion according to the present invention may be combined with other tumor treatment protocols including administration of chemotherapeutics and / or immunotherapeutics and / or radiation therapy. Radiation is the most interesting to be combined with arginine depletion. Its duration is very short and cells at rest can tolerate orders of magnitude higher doses than cycling cells. For example, a two-day session of arginine depletion can be combined with radiation to commence on the second day, whereby most healthy, cycling cells are in the rest phase (Go) and cancer cells are not. Several high-intensity sessions can be delivered during the second day, even in the whole-body radiation mode. When the endogenous release of liver enzymes is stopped, normal cells will slowly reinter the cycle not earlier than 12 to 18 hours after arginine concentration returns close to normal. This combination is of particular interest for non-operable brain tumors.

[0067] Example 1

[0068] Release of arginase from pig liver cells

[0069] Two pig livers were flushed in parallel by pure water and Ringer solution during two periods of 2 hours each by wedging catheters (2 mm outer diameter) into a random vein. Both fluids were pumped at 100 ml / h. About a half of fluid pumped into the liver was collected in the tray in which the livers were placed during flushing. Fluid collected from the water flushed liver was much darker red than the fluid collected from the Ringer flushed liver. With time of flushing the surface area of flushed liver increased (hence the volume, since most of the thickness was flushed by the end), easily recognized by change of the color on the surface.

[0070] Water flushed liver swelled more and got much harder than the Ringer flushed liver, which remained of about the same stiffness as the surrounding, unflushed liver. The volume of liver flushed was estimated by cutting it through the thickness and measuring the surface affected which turned pale in comparison to red liver surrounding it.

[0071] Activity of arginase measured in the fluid collected in the tray in each of the two hour periods corresponded to average activity of arginase in the pig liver tissue, i.e. water was removing essentially all of arginase contained in the volume of the liver flushed out.

[0072] Example 2

[0073] Treatment protocol for cancer patients

[0074] The patient to be treated may be human patient or a non-human mammal, e.g. a dog, suffering from cancer, e.g., HCC (hepatocellular carcinoma) optionally with occult systemic metastasis.

[0075] Water for injection is passed at a rate of 15 to 45 ml / h preferably at 25 to 35 ml / h for an average human patient or 10 to 20 ml / h for a 20 kg dog by means of a catheter inserted into a hepatic vein of the patient and occluding a section of the liver for a time of 24 to 72 hours.

[0076] The water gradually flushes out the blood all the way back into the lobules, entering them through the central vein of the lobule and then into hepatic sinusoids. Hepatocytes exposed to low osmotic pressure swell and then burst thereby releasing the amino acid depleting enzymes ARG and ASS into the outflowing mixture of the infused fluid and arterial blood.

[0077] The procedure is monitored by an analysis of (i) liver enzymes AST and ALT; (ii) blood clotting parameters; (iii) hematology (thrombocytes); (iv) amino acids, (v) the amino acid depleting enzymes ARG and optionally ASS, and (vi) a tumor marker, e.g., alfa-fetoprotein (AFP).

[0078] The intervention is augmented by pharmacological interventions to enable and sustain deep, systemic arginine depletion by liver enzymes incidentally released by hepatocytes.

[0079] The pharmacological interventions are:

[0080] 1 . Insulin / glucose clamp, wherein i.v. infusion of glucose would preferably by replaced by special enteral food, e.g. an enteral composition comprising the nine essential amino acids optionally plus L-aspartate, a carbohydrate source and / or ketone bodies.

[0081] 2. A nitric oxide donor, e.g., i.v. sodium nitroprusside, or, preferably, daily replaced nitroglycerin patches.

[0082] 3. A vasopressin (preferably as oral tablets).

[0083] All of these interventions may be given continuously during the period of time where a significant release of liver enzymes, established by monitoring ARG and optionally ASS.

[0084] By means of this protocol, HCC can be eliminated not only in the liver but in potential, occult systemic metastases.

Claims

Claims1 . A hypotonic fluid for use in a method of treating cancer, wherein the hypotonic fluid is administered to the liver of a subject in need thereof through a hepatic vein thereby causing lysis of liver cells, wherein the lysis of liver cells causes the release of arginine-decomposing enzymes including but not limited to arginase, and citrulline-converting enzymes such as argininosuccinate synthase.

2. The hypotonic fluid of claim 1 for the use of claim 1 , which has an osmolarity of about 250 mOsm / L or less, about 200 mOsm / L or less, about 150 mOsm / L or less, about 100 mOsm / L or less, or about 50 mOsm / L or less.

3. The hypotonic fluid of claim 1 or 2 for the use of claim 1 or 2, which is pure water, particularly water for injection.

4. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the hypotonic fluid is administered to the liver by means of a catheter inserted into a hepatic vein.

5. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the hepatic vein is the vein of a liver lobe or a part of a lobe.

6. The hypotonic fluid of any one of claims 1 -3 for the use of claim 4 or 5, wherein the catheter is inserted into the hepatic vein through the vena cava superior and a jugular vein and / or through the vena cava inferior and a femoral vein.

7. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, wherein an amount of about 400 to about 800 ml hypotonic fluid per day is administered.

8. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the hypotonic fluid is administered to the liver for at least one day, and particularly about 2 to 3 days.

9. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the hypotonic fluid is administered to the liver in an amount of about 5 ml to about 20 ml, particularly in an amount of about 10 ml per g liver cells to be lysed.

10. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, further comprising administering at least one of a hypertonic fluid, a glucose solution, and a mixture of essential amino acids without arginine.11 . The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, further comprising administering at least one adjuvant agent selected from an insulin optionally in combination with glucose, a nitric oxide (NO) donor, e.g., sodium nitroprusside (SNP), nitroglycerin and gaseous NO, a pressor peptide, e.g., a vasopressin, and a prostacyclin analog, e.g., Iloprost.

12. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, for the treatment of a cancer selected from a blood cancer such as leukemia and lymphoma, and a solid cancer such as liver cancer, including hepatocellular carcinoma, skin cancer such as melanoma, colon carcinoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, gastric cancer, brain cancer, and breast cancer.

13. The hypotonic fluid of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the subject is a human or a non-human mammal, particularly a cat or dog.

14. A kit comprising medical devices and drugs for the use in a method of any one of the preceding claims.

15. A method for the treatment of cancer, comprising administering a hypotonic fluid to a subject in need thereof to the liver of a subject in need thereof through a hepatic vein thereby causing lysis of liver cells.

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