Reperfusion injury of the liver as a proxy for cancer treatment

Abstract

The present invention relates to a transient intentional ischemia of a part of the liver by occlusion of a selected hepatic vein, subsequently causing reperfusion injury, useful for the treatment of cancer via the endogenous release of liver enzymes, including liver arginase.

Description

[0001] Reperfusion injury of the liver as a proxy for cancer treatment

[0002] The present invention relates to a transient intentional ischemia of a part of the liver by occlusion of a selected hepatic vein, subsequently causing reperfusion injury, useful for the treatment of cancer via the endogenous release of liver enzymes, including liver arginase.

[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: PMC10876525.), 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. can let.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. 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.

[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 endogenous liver enzymes for use in the treatment of cancer, wherein the liver enzymes are released by liver reperfusion injury caused by temporary occlusion of a hepatic vein of a liver segment of the subject to be treated.

[0015] According to the present invention, a temporary occlusion of the venous drainage of a section of the liver is applied to the liver of a subject in need thereof. Removal of the occlusion restores blood flow through the selected section of the liver following ischemia and initiates a cascade of biological responses known as reperfusion injury. A well-documented effect of reperfusion injury of the liver is the release of liver enzymes, including arginase and argininosuccinate synthase, Ikemoto et al. (Liver-type arginase is a highly sensitive marker for hepatocellular damage in rats. Clin Chem. 2001 May;47(5):946-8. PMID: 11325904.). While these responses have attracted much attention related to liver and other organ transplantation, but also to heart attack, stroke, and related conditions with blood clotting causing temporary perfusion occlusion, there is no published reference to using reperfusion injury as an intended therapeutic intervention. The lysis of liver cells caused by reperfusion injury releases endogenous arginine-decomposing enzymes including but not limited to arginase (ARG), and citrulline-converting enzymes including but not limited to argininosuccinate synthase (ASS) causing deep and relatively long-lasting arginine depletion in the subject’s body. In certain embodiments, the level of arginine in the subject’s body is reduced to 1 pM or less for a time period of at least 24 h and up to 72 h.

[0016] The present invention provides treatment for cancer, e.g., a blood cancer such as leukemia and lymphoma, or 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.

[0017] A further aspect of the present invention is a kit comprising medical devices and drugs needed to execute the treatment described herein.

[0018] Still a further aspect of the present invention is a method for the treatment of cancer, comprising temporarily occluding a hepatic vein of a liver segment and causing reperfusion injury of the liver in a subject in need thereof, wherein endogenous liver enzymes are released thereby depleting arginine in the subject’s body.

[0019] 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.

[0020] In particular embodiments, the perfusion of the selected section of the liver is blocked by occlusion of the venous drainage. In all published animal studies, the occlusion is performed by shutting off arterial and portal venous inflows requiring a surgical intervention. This causes ischemia in the whole liver. Occluding only selected arterial vessels as is done for all current treatment interventions on the liver would not induce ischemia, yet it cannot be combined with matched portal vessels. Occlusion of the venous outflow is thus the preferred option and with distinct advantages.

[0021] It can be performed minimally invasively via jugular and / or femoral veins.

[0022] Further, occlusion of the venous outflow will prevent damage to the portal canals of liver lobules. Arterial blood will continue flowing into portal canals and reverse the portal blood flow through the portal canals. Blood flow through the sinusoids will be stopped by occlusion of outflow via central veins of the lobules exposing hepatocytes to ischemia and eventually to reperfusion injury. Arterial capillaries and capillaries of the portal veins will be partially spared during reperfusion.

[0023] In certain embodiments, the occlusion of venous drainage of the liver is performed employing 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. In certain embodiments, the catheter comprises at least one lumen so that it can be threaded over a guide wire and / or for injection of a contrast medium to facilitate positioning at its target site. In certain embodiments, the catheter comprises at its tip an occlusion means, e.g., an inflatable balloon, for occluding the vein into which it is inserted.

[0024] In certain embodiments, the treatment encompasses targeting a single liver lobe or a part of it. In those embodiments, the catheter may remain in its position only during the occlusion period. In further embodiments, the treatment encompasses multiple sessions of occlusion wherein, the balloon is inflated and deflated without removing the catheter. In further embodiments, targeting may involve 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.

[0025] In extreme cases, multiple catheters, e.g., 2, 3 or even more catheters can be pre-inserted into different sections of the liver via the right and left jugular and femoral veins and inflated as needed. The duration of the occlusion and the size of the selected section of the liver will depend on the amount of liver cells to be lysed which in turn will depend on the type and severity of the disease to be treated. In certain embodiments, the duration of the occlusion may be from about 10 min to about 100 min, from about 15 min to about 60 min, or from about 30 min to about 45 min.

[0026] The occluded section of the liver may be from about 10% to about 60%, from about 20% to about 40%, or about 30% of the total liver (based on the liver volume).

[0027] In certain embodiments, the occlusion is repeated at least once, e.g., after about 1 to about 3 days.

[0028] The complete process can be repeated many times with weeks to months between treatments. Unlike with all conventional cancer drugs, cancer cells cannot become resistant to arginine depletion.

[0029] In certain embodiments, the method of the present invention further comprises administering exogenous agents, for example, agents providing nutrients to the subject’s body, e.g., at least one of a glucose solution, and a mixture of essential amino acids without arginine.

[0030] 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, isosorbide mono or dinitrate, a pressor peptide, e.g., a vasopressin, and a prostacyclin analog, e.g., Iloprost.

[0031] 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 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.

[0032] Even though most cancer drugs do not penetrate the brain-blood 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 suggested 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. Furthermore, there is published evidence that reperfusion injury of the liver leads to a partial breakdown of the brain-blood-bamer, Zhu L, et al. (Hepatic Ischemia- Reperfusion Impairs Blood-Brain Barrier Partly Due to Release of Arginase From Injured Liver. Front Pharmacol. 2021 Oct 13; 12:724471 . doi: 10.3389 / fphar.2021.724471. PMID: 34721021 ; PMCID: PMC8548691 .). While of some concern in liver transplantation, this unexpected effect of endogenous arginase release caused by liver reperfusion injury may be a real benefit in treating brain cancers.

[0033] A further aspect of the present invention relates to a method, devices, and specific nutritional support for the treatment of cancer, comprising temporary occlusion of the liver venous drainage in a subject in need thereof through a hepatic vein thereby causing lysis of liver cells upon reperfusion.

[0034] Detailed Description of the Invention

[0035] 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.

[0036] The lysis of hepatocytes caused by reperfusion injury 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.

[0037] 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.

[0038] Approach to the liver veins is currently used only for diagnostic procedures, e.g., to measure trans-hepatic venous pressure and thus diagnose portal venous hypertension.

[0039] This approach to the liver veins is now proposed to be used for intervention by temporary, partial blocking of the blood perfusion by, e.g., a Fogarty-type of catheter, to cause controlled ischemia followed by lysis of hepatocytes and release of their proteins, including ARG and ASS, upon reperfusion.

[0040] 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 60% of the total liver volume.

[0041] 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.

[0042] The catheter can be wedged into its target vein, and / or it can include at its tip an occlusion balloon. Both techniques are used for measuring transhepatic venous pressure. The balloon version is preferred because it offers more choices of the size of the vein to be occluded.

[0043] 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.

[0044] Now, if the venous outflow of a section of the liver is occluded, it will prevent perfusion of the core of the lobules occupied by hepatocytes, surrounding hepatic sinusoids. Hepatocytes exposed to low oxygen and blockage of metabolic exchange during occlusion will spill much of their cytosolic contents upon reperfusion by arterial and portal venous blood. The lymphatic flow as well as the venous flow will bring the contents of damaged hepatocytes into the blood circulation which will distribute all the released molecules, including ARG and ASS, throughout the body.

[0045] Liver throughput (venous outflow) in an adult human is 1500 to 1900 ml / minute or about a quarter of cardiac output. About 20 to 30% of that is arterial inflow. 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 IU of arginase per kg body weight per day. Thus, in certain embodiments, approximately 1 g of liver tissue should be exposed to ischemia / reperfusion per kg body weight per day. For a human of 75 kg body weight with about 1500 g of liver, three days of arginine depletion would call for full release of ARG from about 15 to 30% of the liver.

[0046] The amount of liver tissue to be exposed to ischemia / reperfusion injury 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 reperfusion injury of the liver leaves some of its scaffolding (stroma) intact, especially if the portal canals are spared, and an even faster regeneration may be possible.

[0047] 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. List of Figures

[0048] Figure 1 shows flows of fluids in the classic liver lobule (from Krstic RV, Human Microscopic Anatomy, An Atlas for Students of Medicine and Biology, Springer Berlin, Heidelberg, 1991 ).

[0049] Figure 2 shows a blood flow reversal in the portal canals of the liver lobules when venous outflow is blocked causing stasis in the sinusoids.

[0050] Figure 3 is a schematic representation of fluid flows in the liver.

[0051] Figure 4 shows the flows in a section of the liver with venous drainage occluded.

[0052] Figure 5 shows a kit with medical devices, medications, and nutritional support needed for the intervention.

[0053] Figure 6 shows the enzymatic activity of AST (alanine amino transferase) and AST (aspartate amino transferase) after inducing liver reperfusion injury during the cancer treatment of the dog Koki.

[0054] Figure 7 shows the plasma concentration of arginine and citrulline after inducing liver reperfusion injury during the cancer treatment of the dog Koki.

[0055] Figure 8 shows an x-ray picture of the skull of the dog Koki suffering from an osteosarcoma before treatment (a) and 3 months after treatment (b).

[0056] Detailed Description of the figures

[0057] 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.

[0058] Figure 2 illustrates flows in a classic liver lobule with the venous outflow 20 via the venous outflow vessel (SV) blocked. The flows 13 (Figure 1 ) through the liver sinusoids (SV) are now interrupted. Arterial inflow 10 is maintained and it reverses the portal flow 21 . Bile outflow 14 is diminished as hepatocytes (HP) are lysed by interruption of the flow through the sinusoids and eventual reperfusion injury.

[0059] 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 CLi. Portal vein 102 also branches out to the individual lobules.

[0060] 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.

[0061] 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 exposed to partial blockage of perfusion by closing off the venous drainage 110. The outflow of venous blood from that segment is now blocked 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 -- arterial blood enters lobular portal canals and exits via the intralobular vein. In most cases, only a part of a liver lobe must be exposed to ischemia. That depends on the type of cancer being treated, and consequently on the combined effects of the enzymatic activity released as a consequence of reperfusion injury and the duration of arginine depletion needed. If necessary, two or even three liver lobes could be used.

[0062] Figure 5 shows kit 200 comprising the devices 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, with an insertion set 203, and a gastric feeding tube 204.

[0063] Medications for treating side effects of arginine depletion are packaged separately, 210. They include an NO donor 205 (e.g., SNP for infusion), an antidote for cyanide 206, nitroglycerin or isosorbide patches 207 (an alternative to SNP), vasopressin 208, or an analog thereof, for infusion or oral delivery.

[0064] 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.

[0065] For dogs, s.c. injection of slow (long) acting insulin is the preferred mode of delivery.

[0066] Nutritional preparation for gastric feeding 220, and a connection line 221 to the gastric feeding tube 204 are packaged separately.

[0067] The nutritional preparation comprises:

[0068] 1 . Glucose, dextrose, or saccharose

[0069] 2. Beta-hydroxybutyrates of calcium, sodium, potassium, and magnesium

[0070] 3. Vegetable or fish oil

[0071] 4. Nine essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) plus aspartate 5. Water

[0072] 6. Lecithin

[0073] 7. Fiber

[0074] 8. Vitamins and antioxidants

[0075] 9. Stabilizers

[0076] Nutritional preparation 220 can be continuously delivered via line 221 into gastric feeding tube 204 by a conventional pump.

[0077] 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.

[0078] 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 of recombinant enzymes 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.

[0079] Another important advantage - on top of avoiding the rapid clearance of foreign proteins by the innate immune system - is the reduction of the risk 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. Autoimmune hepatitis is a known but rare condition. 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.

[0080] 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), nitroglycerin or isosorbide, e.g. isosorbide mononitrate or dinitrate. 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 / injections of sodium thiosulfate or B12a, known antidotes to cyanide, can be used to minimize the toxic effects of cyanide.

[0081] 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 damaged by reperfusion injury. 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.

[0082] Additionally, the activity of ASS requires aspartic acid, which may be added to the above-listed essential amino acids.

[0083] Dosages of 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. For human patients:

[0084] SNP 0.7 mg / kg / day up to a maximum of 1 .5 mg / kg / day

[0085] Vasopressin 0.3 U / kg / day, up to a max of 3.0 U / kg / day (Ornipressin) lloprost 0.7 pg / kg / day, up to a maximum of 3.0 pg / kg / day

[0086] Insulin 1.5 U / kg / day, up to a maximum of 6.0 U / kg / day

[0087] Glucose 6.0 g / kg / day, up to a maximum of 16.0 g / kg / day

[0088] Amino acids 1 .0 g / kg / day (essential w / o arginine)

[0089] Nitroglycerin 0.6 mg / kg / day up to a maximum of 1.3 mg / kg / day

[0090] Sodium thiosulfate (pentahydrate) 3.3 mg / kg / day up to 7.0 mg / kg / day

[0091] B12a 3.7 mg / kg / day up to a maximum of 8.0 mg / kg / day

[0092] For dogs, these dosages are doubled.

[0093] To verify the position of the infusion catheter and occlusion of the vein, a contrast can be injected before inflating the balloon.

[0094] Combining arginine depletion with different chemotherapeutics has been studied in search of synergies on the molecular level. Radiation is the most interesting to be combined with arginine depletion according to this invention. Its duration is very short and cells at rest can tolerate orders of magnitude higher doses than cycling cells. For example, a two to three-day session of arginine depletion can be combined with radiation to commence on the second or third 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 / third day, even in the whole-body radiation mode. When the endogenous release of liver enzymes stops, 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.

[0095] Example 1

[0096] Food Composition for enteral nutrition of cancer patients

[0097] The indicated weight amounts of ingredients refer to a total volume of 1300 ml for the composition.

[0098] Amino acids:

[0099] L-Leucine 8.6 g

[0100] L-lsoleucine 3.9 g

[0101] L-Valine 5.2 g

[0102] L-Lysine HCI 5.6 g

[0103] L-Methionine 3.5 g

[0104] L-Phenylalanine 1.6 g

[0105] L-Threonine 4.8 g

[0106] L-Tryptophan 0.9 g

[0107] L-Histidine 1.6 g

[0108] Mix of essential amino acids 35.7 Q

[0109] Magnesium L-aspartate** 2.5 g

[0110] Amino acids total 38.2 Q

[0111] Fat:

[0112] Sunflower Oil 2.5 g

[0113] Polysorbate 80 1 g Carbohydrates:

[0114] Maltodextrin DE 6 395 g

[0115] Pectin 5 g

[0116] Acacia gum 5 g

[0117] Carbohydrates total 405 o

[0118] Other:

[0119] Trisodium citrate 0.46 g

[0120] Calcium lactate pentahydrate 3.00 g

[0121] Calcium glycerophosphate 1.35 g

[0122] Calcium pantothenate 1.55 g

[0123] Potassium citrate 0.820 g

[0124] Potassium chloride 0.600 g

[0125] Potassium sorbate 1 .210 g

[0126] Ferrous sulfate - Iron 0.0207 g

[0127] Disodium phosphate 0.622 g

[0128] Zinc sulfate heptahydrate 0.0367 g

[0129] Copper gluconate 0.006 g

[0130] Manganese sulfate monohydrate 0.002 g Potassium Iodide 160 pg

[0131] Sodium Selenite 110 pg

[0132] Vitamin D 2 pg

[0133] Vitamin A 210 pg

[0134] Vitamin C 500 mg

[0135] Vitamin E 4.44 mg

[0136] Vitamin K1 0.23 mg

[0137] Thiamine - B1 0.31 mg

[0138] Riboflavin - B2 0.72 mg

[0139] Niacin - B3 2.22 mg

[0140] Vitamin B6 0.22 mg

[0141] Folic Acid - B9 38 pg

[0142] Vitamin B12 5 pg

[0143] Biotin 120 pg Pantothenic Acid 2.22 mg

[0144] Choline Bitartrate 36 mg

[0145] L-Carnitine 600 pg

[0146] Taurine 600 pg

[0147] Water about 1000 ml

[0148] ** Magnesium aspartate (or potassium aspartate) is preferred to aspartic acid to avoid shift to low pH.

[0149] A composition of the above ingredients (but without vitamins and other conventional micronutrients) was used in dogs treated for cancer by arginine depletion.

[0150] Example 2

[0151] Case Report

[0152] Koki, a 5-year old male 40 kg Rottweiler mix with an osteosarcoma on the skull was treated with arginine depletion induced by reperfusion injury of a section of the left liver.

[0153] The tumor was located above the right temple, protruding by 3 to 4 cm above the surrounding bone. The whole right side of the head was extremely painful. The eye was red, pushed out to the side. The dog could not eat solid food and did not allow the owner to open his mouth for delivering any tablets.

[0154] In full anesthesia, guided by a C-arm fluoroscope, a guide wire was inserted through the right jugular vein, through the vena cava to the level of a left liver vein. A Fogarty-type FR 8 catheter with a balloon at the tip was passed over the guide wire into the liver vein. The balloon was inflated, and occlusion was verified by injection of a contrast medium. Occlusion was re-verified at 30 and 60 minutes. After deflating the balloon at 60 minutes, blood flow was reestablished, confirmed by a contrast medium.

[0155] A central venous catheter was inserted to facilitate blood sampling. A long lasting insulin was injected s.c. at 6 lll / kg daily, on three consecutive days.

[0156] An enteral formulation, free of arginine and of high carbohydrate content (c.f. Example 1 ) was fed continuously with a pump via a nasogastric tube delivering sufficient amount of glucose (on average about 1 g / kg / h) to maintain normal blood glucose. From the first day on, a 20% glucose solution was delivered i.v. for the remaining 2 days.

[0157] Liver enzymes AST and ALT rose rapidly after the occlusion and remained high (with AST above 1000 I U / l, indicating liver damage) for about a day, Figure 6. Not measured, but known to be released with AST / ALT were arginase (ARG) and argininosuccinate synthase (ASS). This resulted in a significant reduction of plasma arginine and citrulline, Figure 7. With reduced levels of AST and ALT, the treatment was terminated at 72 hours.

[0158] The clinical outcome was very positive. The tumor size was significantly reduced by several weeks and pain has resolved in a matter of days allowing the dog to be fed his regular food. By three months, he was able to eat bones.

[0159] X-rays taken pre and at 3 months post the treatment, Figures 8a and b, showed remarkable reduction of the osteosarcoma, with only small bumps of mineralized tissue remaining at the site of the tumor. We expect the bone to remodel to its original shape in several months.

Claims

Claims1 . Endogenous liver enzymes for use in a method for the treatment of cancer, wherein the liver enzymes are released by liver reperfusion injury caused by temporary occlusion of a hepatic vein of a liver segment of the subject to be treated.

2. The endogenous liver enzymes of claim 1 for the use of claim 1 , which comprise arginine-decomposing enzymes including but not limited to arginase (ARG).

3. The endogenous liver enzymes of claim 1 or 2 for the use of claim 1 or 2, which further comprise citrulline-converting enzymes including but not limited to argininosuccinate synthase (ASS).

4. The endogenous liver enzymes of any one of claims 1 -3 for the use of any one of claims 1-3, wherein the treatment reduces the level of arginine in the subject’s body to 1 pM or less for a time period of at least 24 h, and preferably 48 to 72 h.

5. The endogenous liver enzymes of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the duration of the occlusion is from about 10 min to about 120 min, from about 15 min to about 60 min, or from about 30 min to about 45 min.

6. The endogenous liver enzymes of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the occluded section of the liver is from about 10% to about 60%, from about 20% to about 40%, or about 30% of the total liver volume.

7. The endogenous liver enzymes 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.

8. The endogenous liver enzymes of any one of claims 1 -3 for the use of any one of the preceding claims, wherein the temporary occlusion is caused by inserting a catheter into the hepatic vein, particularly by inserting a catheter through the vena cava superior and a jugular vein and / or through the vena cava inferior and a femoral vein.

9. The endogenous liver enzymes 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 glucose solution, and a mixture of essential amino acids without arginine.

10. The endogenous liver enzymes 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 isosorbide, e.g. isosorbide mononitrate or dinitrate, a pressor peptide, e.g., a vasopressin, and a prostacyclin analog, e.g., Iloprost.11 . The endogenous liver enzymes 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.

12. The endogenous liver enzymes 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.

13. A kit comprising medical devices needed to execute any one of the preceding claims, including at least one catheter, e.g., a Fogarty type catheter with an occlusion balloon, at least one guide wire, an insertion set, and at least one gastric feeding tube.

14. The kit of claim 13 further comprising medications for treating side effects of arginine depletion, including an NO donor, e.g., SNP for infusion, nitroglycerin or isosorbide, an antidote for cyanide, vasopressin or an analog thereof, for infusion or oral delivery, and optionally insulin.

15. A method for the treatment of cancer, comprising temporarily occluding a hepatic vein of a liver segment and causing reperfusion injury of the liver in a subject in need thereof, wherein endogenous liver enzymes are released thereby depleting arginine in the subject’s body.

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