Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/45—Transferases (2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/51—Lyases (4)

Definitions

• the present invention generally relates to a therapeutic composition and a method for the treatment of patients in the state of arginine depletion, and more particularly for the treatment of cancer patients depleted of arginine for therapeutic reasons.
• the composition comprises a nitric oxide donor, a vasoconstricting peptide, and a prostacyclin analog, the combination of which prevents activation of platelets, while maintaining hemodynamic stability.
• Homeostatic response to arginine depletion is counteracted by inhibiting protein breakdown of expandable cellular proteins, mostly in muscles, by known modulators of protein turnover, such as insulin, which promotes protein synthesis, and inhibits lysosomal protein breakdown, and by, for example, a heme-containing compound which inhibits proteasomal protein breakdown.
• ALL acute lymphocytic childhood leukemia
• the sensitivity is due to suppressed asparagine synthesis in ALL cells.
• usefulness of asparaginase remained limited to ALL, which also was found to become resistant to repeated treatments.
• Leukemia cells surviving the initial period of depletion eventually activate their own, normally latent, synthesis of asparagme, a non-essential amino acid.
• Asparaginase is thus usually used for induction in a multi-drug chemotherapy regimen for ALL, which at approximately 75% cure rate is one of the best treatments for any disseminated cancer.
• arginine is a substrate for a number of enzymes, widely distributed in different tissue and cell types (see generally the discussion in Wu G. and Morris S.M. Jr.: "Arginine metabolism: nitric oxide and beyond", Biochem J, 336 (Pt 1): 1-17, 1998).
• Arginine is also a substrate for nitric oxide synthase (NOS), which converts arginine into citrulline and nitric oxide (NO).
• NOS nitric oxide synthase
• NO nitric oxide
• One of these roles is maintenance of platelet inactive state, mostly by NO produced by the vascular endothelium. NO stimulates production of cGMP in platelets in their inactive state. Removal of NO signal leads to depletion of cGMP, and then, through a number of molecular events, to influx of calcium and platelet activation.
• Prostacyclin has a similar, synergistic, role of platelet inactivity maintenance by stimulation of cAMP production (Anfossi G, Russo I, Massucco P, Mattiello L, Balbo A, Cavalot F, Trovati M.:" Studies on inhibition of human platelet function by sodium nitroprusside.
• NO is also a potent vasodilator.
• Blood vessel lumen is under constant control by agonist/antagonist actions of NO and so-called pressor peptides all of which contain arginine (vasopressin, angiotensins).
• pressor peptides all of which contain arginine (vasopressin, angiotensins).
• arginine vapressin, angiotensins.
• hemodynamics Normal function of the heart, and its impact on the circulation of blood, referred to as hemodynamics, depends on the appropriate volume of the blood and the peripheral resistance of the complete vascular system.
• the heart's own feedback control will respond to a potential drop in pressure, due to either reduced blood volume, or reduced peripheral resistance, increasing the pulse rate, even before a measurable pressure drop is registered at the periphery.
• the early workers on essential amino acid depletion for cancer could have not understood multiple dependencies of hemodynamic stability on the substances, which either contain argmine, or are produced from it. NO is a "modern" molecule, perhaps the most intensely studied one in the last decade.
• medical literature remains silent on the problems facing in vivo arginine depletion as a modality for cancer treatment.
• Argmine is also a substrate for arginine decarboxylase (ADC), which converts arginine to agmatine.
• ADC arginine decarboxylase
• ADC is present in the brain and kidneys of mammals, but the metabolic role of agmatine remains rather poorly understood.
• Therapeutic composition of this invention restores systemic levels of NO by continuous delivery of one of the known NO donors, preferably a direct one, such as sodium nitroprusside.
• restoring NO supply in arginine depleted state, leads to excessive vasodilatation for the lack of normal pressor counterbalance (since all of pressor peptides contain arginine and thus cannot be produced at normal rates).
• Therapeutic composition of this invention resolves this difficulty by supplying a pressor peptide, for example vasopressin, or preferably, one of its more stable analogs, such as ornipressin, terlipressin or desmopressin.
• Arginine is a semi-essential amino acid, i.e. the body is capable of producing some, but usually not all of the required arginine from non-essential amino acids as the precursors, namely from proline and glutamine.
• the relative need for endogenous sources of arginine varies with age and animal species. For example, milk of many mammals, including humans, is a poor source of arginine and neonates are very much dependent on endogenous production of arginine via so-called intestinal-kidney axis, whereby citrulline is produced by mostly small intestine and then converted to arginine by mostly kidneys.
• Another possibility for inhibition of intestinal-kidney axis is by per os administration of a glycylglycine derivative of delta-N-(phosphonacetyl)-L-ornithine (Gly-Gly-PALO), a powerful and specific inhibitor of ornithine transcarbamylase, an enzyme responsible for intestinal production of citrulline (Hoogenraad N, Totino N, Elmer H, Wraight C, Alewood P, Johns RB, Inhibition of intestinal citrulline synthesis causes severe growth retardation in rats, Am.J.Physiol. 1985 Dec; 249(6 Pt 1): G792-9).
• Gly-Gly-PALO glycylglycine derivative of delta-N-(phosphonacetyl)-L-ornithine
• the present invention generally relates to a therapeutic composition and a method for the treatment of patients in the state of argmine depletion, and more particularly for the treatment of cancer patients depleted of arginine for therapeutic reasons.
• the composition comprises a nitric oxide donor, a vasoconstricting peptide, and a prostacyclin analog, the combination of which prevents activation of platelets, while maintaining hemodynamic stability.
• Homeostatic response to arginine depletion is counteracted by inhibiting protein breakdown of expandable cellular proteins, mostly in muscles, by known modulators of protein turnover, such as insulin, which promotes protein synthesis, and inhibits lysosomal protein breakdown, and by, for example, a heme-containing compound which inhibits proteasomal protein breakdown.
• modulators of protein turnover such as insulin
• a heme-containing compound which inhibits proteasomal protein breakdown.
• a number of more specific inhibitors of the proteasomal pathway have been discovered, and can be beneficially used in combination with arginine depletion.
• Endogenous production of arginine can be inhibited at several of the enzymatic steps required to convert e.g. proline to arginine by the so-called intestinal-kidney axis.
• endogenous production of arginine can be inhibited at several of the enzymatic steps required to convert e.g. proline to arginine by the so-called intestinal-kidney axis.
• proline oxidase by lactate has been shown to allow for very deep reductions of circulating arginine.
• the invention relates to a method for the treatment of cancer by depletion of arginine and the therapeutic compositions useful in such methods.
• the composition enables arginine depletion in the therapeutic window without systemic complications, which otherwise would ensue.
• the composition comprises, in addition to an arginine decomposing enzyme and protein breakdown inhibitor(s): a nitric oxide donor, such as sodium nitroprusside; a pressor peptide, such as vasopressin, or one of its analogs, such as ornipressin, desmopressin, or terlipressin; and prostacyclin, or one of its analogs, such as iloprost.
• a nitric oxide donor such as sodium nitroprusside
• a pressor peptide such as vasopressin, or one of its analogs, such as ornipressin, desmopressin, or terlipressin
• prostacyclin or one of its analogs, such as iloprost.
• Controlled co-infusion of sodium lactate and lactic acid inhibits endogenous production of arginine via intestinal-kidney axis.
• the ratio of sodium lactate-to-lactic acid infusions is adjusted as needed to maintain physiologically normal pH
• the composition may include: an amino acid mixture lacking arginine; an antidote for cyanide, such as hydroxocobalamine, or sodium thiosulfate; blood plasma, or one or more of its derivatives, such as cryoprecipitate rich in clotting factors, or albumin.
• the composition may also include a preparation of argmine, such as a solution of arginine hydrochloride.
• Arginine decomposing enzyme may be arginase, decarboxylase, deiminase, or kinase. The enzyme may be pegylated, or otherwise modified to increase its half-life in circulation.
• Arginase may be type I liver arginase, or type ⁇ .
• It may be human or animal, partially purified, or recombinant. It may also be of bacterial origin, particularly a thermostable type. It may be administered as a drug, or it may be released from the patient's own tissue, particularly from the liver in case of hepatocellular carcinoma. Release may be effected by arterial occlusion of the liver, or by cryo, ultrasonic, or RF ablation of the tumor lesions.
• Protein breakdown inhibitors may be insulin, insulin-like growth factor I, IGF-I, growth hormones, protein breakdown inhibiting peptide aldehydes such as Cbz-Leu-Leu-Leucinal, lactacystin or its analogs, heme or its derivatives, and inhibitors of prostaglandin E 2 production, such as nonsteroidal anti-inflammatory drugs (NSAID's).
• NSAID's nonsteroidal anti-inflammatory drugs
• compositions may be administered separately, or in suitable mixtures, allowing for needed adjustments during the treatment.
• the various embodiments of the therapeutic composition and method for treatment of cancer by depletion of arginine are set forth in the claims and described in detail herein.
• Figure 1 is a graph depicting the change in arginine plasma concentration over time in experimental dogs being treated with arginine depletion with curve 1 showing arginine plasma concentration without an insulin/glucose clamp and curve 2 showing arginine plasma concentration with an insulin/glucose clamp.
• Figure 2 is a graph depicting platelet loss over time in experimental dogs comparing the platelet loss where SNP/terlipressin/iloprost was not administered (curve 1) and where it was administered (curve 2).
• Figure 3 is a graph depicting plasma concentration in experimental dogs being treated with bolus i.v. injections of a crude liver extract alone or in combination with insulin/glucose/SNP/terlipressin/iloprost.
• Figure 4 is a schematic diagram showing plasma concentration of argmine in experimental dogs treated with or without infusion of sodium lactate/lactic acid in addition to pegylated recombinant human liver arginase / insulin / glucose / SNP / terlipressin / iloprost.
• Figure 5 is a chart depicting the dosages derived from the experiments discussed in the present application.
• ALL Acute rymphocytic leukemia (ALL) - a type of childhood leukemia, which responds well to asparaginase treatment.
• Alpha-fetoprotein (AFP) a tumor marker useful for diagnostic and follow-up procedures in hepatocellular carcinoma.
• Arginase (ARG) - converts L-arginine and H 2 O to L-ornithine and urea; found at high concentrations in the liver; other isoforms widely distributed in basically all tissues of all animals, but also in plants and bacteria.
• Arginine - an essential amino acid (L-arginine); by some accounts considered semi- essential, since it can be produced from for example citrulline, which in turn can be produced from praline or glutamine. All amino acids have optical (or stereo) isomers, D and L, and so does arginine. Proteins consist exclusively of L-amino acids, and in this text, if not specified, L-form is implied.
• Arginine decarboxylase ADC
• ADI Arginine deiminase
• Arginine kinase (AK) - converts L-arginine and ATP to N ⁇ -phosphor-arginine and
• Arterial occlusion a procedure frequently used to induce partial tumor necrosis, usually performed by controlled injection of occluding substances, e.g. an oil emulsion and or collagen particles (leading to embolization), but can also be performed by mechanical / surgical means (e.g. ligation).
• occluding substances e.g. an oil emulsion and or collagen particles (leading to embolization)
• mechanical / surgical means e.g. ligation
• Asparaginase - converts L-asparagine and H 2 O to aspartate and NH 3 (drug approved for the treatment of ALL).
• cAMP - cyclic adenosine monophosphate plays an important role in prevention of calcium influx into thrombocytes; its production is stimulated by prostacyclin.
• cGMP - cyclic guanosine monophosphate plays an important role in prevention of calcium influx into thrombocytes; its production is stimulated by NO.
• Desmopressin - an analog of vasopressin (approved drug).
• DIC Disseminated intravascular coagulation
• Hepatocellular carcinoma - a predominant type (about 90% of all cases) of primary liver cancer.
• Hepatitis B and C are considered main causative factors.
• Iloprost - an analog of prostacyclin (approved drug).
• Insulin / glucose-clamp - a therapeutic composition, most frequently used as a diagnostic tool for determining insulin resistance in diabetes; a dose of insulin (usually administered as a primed, fixed-rate continuous infusion) is balanced by a variable dose of glucose required to maintain its normal plasma concentration (by feedback control); hence the name: glucose-clamp (also euglycemic insulin clamp).
• Melanoma a highly invasive cancer, usually of cutaneous origin. Excessive exposure to sunlight is a major risk, and the most important factor in the rapidly rising incidence of melanoma. Early surgical treatment is very effective; overall 5-year survival rate is about 80%. Melanomas are very sensitive to arginine depletion, and generally unable to substitute citrulline for arginine because they fail to produce the required enzymes.
• Nitric oxide (NO) a ubiquitous signaling molecule with different physiological functions, including vasodilation and inhibition of platelet activation.
• Nitric oxide synthase (NOS) converts L-arginine and oxygen to citrulline and NO (the stoichiometry not clear), found in different forms and in different cell types. Nitric oxide donor - compounds which release NO, either directly, or via metabolic conversions of certain substrates.
• Nonsteroidal Anti-Inflammatory Drugs - NSAID's a class of compounds inhibiting production of inflammatory mediators, including prostaglanding E 2 .
• Ornipressin - an analog of vasopressin with ornithine substituted for arginine, vasoconstrictor (approved drug).
• PEG Polyethyleneglycol
• Prostacyclin - prostaglandin I 2 potent inhibitor of platelet aggregation.
• Prostaglandin E2 - (PGE 2 ) an important inflammatory mediator; promotes protein breakdown Sodium nitroprusside (SNP) - decomposes to NO and cyanide; a direct NO donor; a drug approved for treatment of hypertension.
• SNP Sodium nitroprusside
• composition - a multi-component drug, whereby single components
• active ingredients are either mixed together by the producer, or by the physician prior to application, or kept and delivered separate to the patient during a given treatment session.
• Urea cycle - a set of enzymes, residing in liver, which convert arginine to ornithine to citrulline back to arginine via arginino-succinate; its physiological function is conversion of excess nitrogen to urea, which is then excreted by kidneys.
• the present invention is directed to a therapeutic composition and a method for the treatment of cancer, the composition comprising an arginine-degrading enzyme, which is either released endogenously, particularly in the treatment of hepatocellular carcinoma, by any one of several conventional tumor destruction methods, or infused together with the other composition constituents, namely: a protein breakdown inhibitor, preferably insulin/glucose; a heme derivative; a nonsteroidal anti-inflammatory drug, preferably a cyclo-oxygenase-2 (COX-2) specific; a nitric oxide donor, preferably sodium nitroprusside; a vasoconstricting substance, preferably an analog of vasopressin peptide; and an analog of prostacyclin, preferably iloprost.
• co-infusion of sodium lactate and lactic acid causes a systemic elevation in lactate, which via inhibition of proline oxidase depresses endogenous production of arginine.
• a protein breakdown inhibitor(s) allows reduction of plasma arginine into micromolar range without the influx of other amino acids, which would otherwise result in a deadly ammonia accumulation.
• SNP Sodium nitroprusside
• NO nitric oxide
• B12a hydroxocobalamine, precursor of vitamin B 12
• sodium thiosulfate both of which are approved as antidotes in cyanide poisoning.
• the therapeutic composition is administered to a patient in need of treatment through infusion, an aerosol nasal spray, or in other methods known to one of ordinary skill in the art.
• the therapeutic composition is administered by means of an extracorporeal blood treatment characterized by molecular exchange between the blood and a dialyzing fluid across a molecular sieve membrane, whereby the conventional dialyzing fluid is supplemented by a plurality of low molecular weight organic and inorganic substances at concentrations essentially equal to those found in the normal blood plasma with the exception of at least one essential nutrient, preferably an - In essential amino acid, which is either not present, or is present at a substantially lower concentration.
• the therapeutic composition is administered, either simultaneously or in series with, molecular factors, at normal or at elevated concentrations, involved in the cellular processes of protein synthesis and degradation in order to limit the release of amino acids from non-essential cellular proteins, mainly f ⁇ brillar muscle proteins.
• these factors are branched side chain amino acids (leucine, isoleucine and valine, glutamate), insulin, insulin like growth factors and growth hormones. Insulin was found to be the most effective. Delivery of insulin must be balanced by an appropriate rate of delivery of glucose in order to avoid hypoglycemia. Chemical inhibitors of the protein degradation pathways may also be administered, as well as antibiotics needed to reduce the risk of infection.
• inhibitors may be separately administered by any suitable means such as injection, i.v., aerosol nasal spray, or any other suitable means known to one of ordinary skill in the art, either at the same time as administration of the therapeutic composition of the present invention or sometime before or after administration of the therapeutic composition.
• suitable means such as injection, i.v., aerosol nasal spray, or any other suitable means known to one of ordinary skill in the art, either at the same time as administration of the therapeutic composition of the present invention or sometime before or after administration of the therapeutic composition.
• these factors and inhibitors may be contained in the dialyzing fluid or may be separately administered by any suitable means such as injection, i.v., aerosol nasal spray, or any other suitable means known to one of ordinary skill in the art.
• the temperature of the patient is controlled during or following administration of the therapeutic composition of the present invention.
• the temperature of the patient is preferably controlled to subnormal levels in order to reduce the muscle protein breakdown in response to removal of the targeted essential amino acid.
• a continuous treatment is carried out over a period of several days, leading to selective killing of the tumor cells.
• This result can be accomplished due to the relaxed cell cycle control mechanisms found in all tumor cells.
• healthy, normally cycling cells exit the cycle and are kept in the rest phase where they can easily survive the harsh conditions of deprivation.
• tumor cells are less restricted and will proceed into the cycle finding themselves vulnerable to conditions of deprivation.
• a majority of cycling tumor cells proceed over the restriction point into the S-phase (DNA synthesis) and are readily killed after, for example, no more than 72 hours of arginine deprivation.
• the few survivors among the cycling tumor cells can again be sent across the restriction point by re-supplying the deprived essential amino acid during a time which is too short for the normal cells to enter the cycle, and then eliminated by repeated deprivation.
• Programming of such cycles is preferably achieved by administration of appropriate concentrations of the therapeutic composition over the period of treatment.
• equilibrating mass transport between the blood and the appropriate dialyzing fluid results in a sufficiently powerful systemic-level control with sustainable extracorporeal blood flow rates and can be achieved by switching between the appropriate concentration formulations of the dialyzing fluid.
• This method of treatment can be readily combined with a suitable protocol of chemotherapy.
• Deprivation of arginine causes most tumor cells to crowd into and to get arrested in the S-phase, while most normal cells manage to complete their cycle and exit into the rest phase (G.sub.O).
• S-phase-specific drugs can thus be used in significantly escalated doses.
• the preferred mode of drug delivery is by loading the drug into (or mixing it with) the therapeutic composition, thus avoiding any risk of overdose. In the case of extracorporeal blood treatment, this allows the drug to be readily removed from circulation by simply switching to a drug-free dialysate at the end of the drug treatment, before the healthy cells are allowed back into the cycle.
• drugs can be infused directly into the blood, taking into account kinetics of removal by the continuous dialysis. As soon as the infusion is stopped, dialysis will quickly reduce the concentration of the remaining drugs. It has been determined, by performing in vitro work on seven different human cancer lines, that arginine is the best target for amino acid deprivation because arginine is used in disproportionate amounts by all cancer cells tested for production of proteins, but also of polyamines.
• the "killing window" is defined by concentrations below 10 micro molar and by deprivation time longer than 72 hours. Normal cells exit the cycle and reemerge from the rest phase apparently undamaged after even ten days of arginine deprivation.
• Arginine has a special role in the physiology of mammals.
• the main pathway for elimination of excess nitrogen is the urea cycle, whereby liver cells use a set of enzymes which turn arginine into ornithine, ornithine into citrulline, and citrulline back into arginine with the net effect of releasing nitrogen from ammonia (which is produced by ultimate degradation of amino acids) as a constituent of urea. Should this process be inhibited by the lack of arginine, the predictable outcome is accumulation of highly toxic ammonia. This phenomenon seems to have escaped the attention of many investigators who have worked on arginine degrading enzymes.
• arginine degrading enzyme Any suitable arginine degrading enzyme can be used in the present invention.
• a preferred choice for an arginine degrading enzyme is co-called biosynthetic arginine decarboxylase (bADC) of E. coli, which in contrast to a similar enzyme, so-called biodegradative arginine decarboxylase (dADC) of E. coli, has very favorable kinetic properties at normal physiological conditions (Wu WH, Morris DR, "Biosynthetic arginine decarboxylase from Escherichia coli.
• the arginine decomposing enzyme is administered by any suitable means including intravenously (i.v.), intraperitoneally (i.p.), intramuscularly (i.m.), nasally, or extracorporeally.
• the arginine decomposing enzyme is administered intravenously.
• the arginine decomposing enzyme is inhaled as an aerosol, which may allow minimization of immunological side effects caused by i.v. or i.m. injections of enzymes. All known arginine decomposing enzymes are large proteins which cannot enter blood circulation through the respiratory membrane.
• the amino acids specifically arginine
• the enzyme can be encapsulated into a suitable polymer or conjugated with PEG.
• the arginine decomposing enzyme is PEG-ilated (covalently bound to a number of molecules of polyethylene glycol or polyethylene glycol derivatives such as methoxypolyethylene). As the enzyme degrades and loses its activity, it is eliminated from the lungs by a natural process of mucosal excretion.
• this potential toxicity is avoided by the concurrent removal of ammonia by hemodialysis.
• a further advantage of the dialysis of the present invention is the possibility of removal of citrulline and ornithine which are precursors of arginine (these metabolic processes are not confined to liver).
• dialysis can be performed using conventional dialyzing solutions, while some, or all, of these substances, as well as any necessary adjuvants (e.g. glucose with insulin), can be delivered by a controlled infusion into the return line of the extracorporeal circuit.
• This embodiment constitutes a simple controller of the systemic concentration of these substances. The performance of the controller is dependent on the blood flow and the efficiency of the filter, which is predictable, can be monitored essentially on line, and the necessary adjustments of the infusion rate are easily implemented.
• Figure 1 is a schematic depiction of the effect of insulin/glucose on the concentration of plasma arginine in dogs being exposed to depletion of arginine by extracorporeal means (U.S. Patent No. 5,851,985) - if the homeostasis is allowed to develop unperturbed, arginine plasma concentration is maintained at near normal level of 100 micromolar, depicted by curve 1. However, if an insulin/glucose clamp is deployed, arginine can be readily lowered to below 10 micromolar, as shown by curve 2. This effect has been consistently observed in a dozen of experimental sessions lasting up to 6 days.
• liver-type arginase for systemic depletion of arginine was demonstrated by an acute pre-terminal experiment on dogs. Dogs were kept under anesthesia continuously for 24 hours. First, one (of six) liver lobe was surgically removed, and the abdomen closed as for a normal surgical intervention on liver. The approximately 100 g of liver tissue so obtained, was then homogenized, partially purified, and sterile-filtered before being returned to the animal by i.v. infusion during the next 18 to 20 hours.
• Bolus injections (0.4g of liver tissue / kg body weight) at 3 hour intervals resulted in a rapid drop of plasma arginine concentration, followed by an equally rapid recovery, for every injection given, if no insulin was used, Figure 3, curve 1.
• Normal lactate concentration in blood plasma is 1 to 2 millimolar.
• Both, sodium lactate and lactic acid solutions were prepared as 1 molar. In this dog of 12kg body weight the total infusion rate required to maintain the targeted plasma lactate of 10 millimolar was approximately lOOml/h, with the final ratio of lactic acid to lactate of approximately 6. Both solutions were started at infusion rates of 25ml/h, with lactic acid ramping up and sodium lactate ramping down. Plasma pH and lactate were measured in hourly intervals, adjusting the infusion rates as needed. Both ornithine and citrulline were significantly lowered in comparison to all of the experiments performed without elevated lactate.
• Drug dosages presented in this document are based on dog and mice experiments carried out at the School of Veterinary Medicine, University of Zurich, from 1995 until the end of 2000. Rationale for use of a particular drug to control observed side effects of arginine depletion is given in brief- in all cases there is ample scientific literature to support the choices made. Since arginine depletion to the extent and duration needed to effect significant cancer reduction (deduced from in vitro experiments) has never been achieved outside the experimental work of the Zurich group, side effects of the depletion could only be anticipated, but have not been actually observed by any other research team.
• Dosage of most of the drugs used to correct the side effects of arginine depletion was determined by rudimentary search for an effective dose, always respecting the limits of suggested pharmacological dosage for the approved indications.
• Limits for use in animals were calculated from those recommended for humans, using the generally accepted rules of dose adjustment for body surface area. For example, all dosages for humans are multiplied by factor 2 when recalculating for dogs, and by factor 12 when recalculating for mice (DeVita VT, Hellman S, Rosenberg SA, "Cancer - principles & practice of oncology", 4 th ed., pg. 288). Conversely, when recalculating from dogs to humans all dosages were divided by factor 2; from mice to humans by factor 12.
• liver extract was continuously infused i.v. at 4 different rates, delivering approximately 900, 300, 90 or 30 I.E./kg day of arginase.
• the two highest rates lowered plasma arginine into sub-micromolar range.
• lymphatic system remained after 18 hours above therapeutic level at 15 micromolar (normal plasma concentration of arginine is about 150 micromolar).
• Circulation between the vascular and lymphatic systems would have been much higher if the animals were not under anesthesia, but this result points to a crucially important issue: plasma concentration is only a crude measure of arginine depletion, which for the full effect on a disseminated cancer should be effected in all of extracellular fluid. It should thus be anticipated that truly effective in vivo doses would be much higher than could be calculated based on enzymatic activity and say the total volume of extracellular compartment. In case of asparaginase, for example, the most similar approved and practiced treatment, the effective doses ultimately determined were 100 times higher than the original estimates.
• the minimal effective dose in acute dog experiments of 300 I.E./kg/day would translate into 150 I.E./kg/day for a human.
• the activity is about 18001.E./mg, so the activity-based dose would translate into 0.08 mg/kg/day, or approximately 6 mg/day for a 70 kg patient.
• bolus injections of lO'OOO I.E./m2 are given at 3-day intervals, typically four times.
• the body surface area is 2 m2, i.e., a typical bolus injection is 20O00 I.E., which corresponds to 100 to 200 mg of protein.
• Higher doses of asparaginase of 25 '000 I.E./m2 are also not uncommon, resulting in bolus injections of up to 500 mg of protein.
• therapeutic proteins may cause serious side effects just due to their un- physiological presence in circulation, such as provoking a non-specific (in early use) immune response.
• Insulin selected must be suitable for i.v. infusion. In all animal experiments at the University of Zurich the product used was Insulin, ACTRAPID ® HM (human, monocomponent), 100 I.E./ml, from Novo Nordisk. The purpose of insulin is to inhibit the normal physiological response the body would otherwise mount to depletion of arginine - initiation of a (potentially) massive protein breakdown (mostly in muscles) to normalize concentration of free arginine. Animal experiments have clearly demonstrated the essential need for inhibition of this response in arginine depletion. Insulin, which is a very potent natural anabolic hormone, was selected for its widespread use, familiarity, availability and safety record in treatment of diabetes, but also for its well demonstrated, if less appreciated, effectiveness in controlling protein wasting in cancer patients.
• Euglycemic diagnostic clamps are usually administered with insulin doses of 0.5 mI.E./kg/min (low physiologic); 1.0 ml.E./kg/min (high physiologic); 4.0 ml.E./kg/min (supraphysiologic), during 2 hours.
• the dose used in acute dog experiments corresponds to supraphysiologic dose in diagnostic euglycemic clamps.
• Glucose (dextrose) infusion is deployed in order to avoid hypoglycemia which otherwise would be induced by infusion of insulin.
• High concentration (50%) glucose solution suitable for i.v. infusion is recommended in order to avoid over-hydration of the patient.
• relatively high rates of glucose infusion should be anticipated in order to balance out the effects of insulin.
• glucose requirement was 8.7 mg/kg/min;, or 12.5 g/kg/day.
• glucose rates were lower (10.9 and 5.3 mg/kg/min, respectively), so that an average glucose infusion rate of 7 g/kg/day could be anticipated at the suggested insulin infusion of 1.5 I.E./kg/day. It is believed that a maximum rate of 16 g/kg/day at 6 I.E./kg/day of insulin should be more than sufficient. In the event that this high rate of glucose infusion fails to normalize plasma glucose concentration, insulin infusion should be appropriately reduced.
• nitroprusside is a direct donor of nitric oxide (NO). Its use in arginine depleted patients is essential in order to avoid loss of thrombocytes -NO stimulates production of cGMP within platelets, the lack of which leads to their activation. Depletion of NO is an eminently predictable consequence of arginine depletion - arginine is the only known substrate for NO production.
• SNP used was purchased from Sigma Co., St. Louis, since the only approved SNP on the Swiss market, NLPREDE ® from Roche, was discontinued several years ago (in the United States there are three FDA-approved SNP products from: Abbott, Elkins Sinn and Gensia Sicor Phar s).
• Generally recommended dose for long term (days to weeks) application to treat hypertension in humans is 2.5 microgram/kg/min.
• mice experiments delivery of 10 microgram/kg/min, s.c, is being used apparently avoiding any risk of platelet loss. This would translate into human dose of 0.9 microgram/kg/min. Since the planned delivery in human patients will also be i.v., the recommended dose is 0.5 microgram/kg/min. Should any loss of platelets be observed after a period of 12 hours of arginine depletion, this dose could be escalated to the maximum recommended dose of 1.0 microgram/kg/min (in increments of 0.25 microgram/kg/min for every 12 h).
• hydroxocoba ⁇ amin B12a, or precursor to vitamin B 12
• an antidote for cyanide which is the product of SNP decomposition
• No signs of cyanide poisoning were seen in any of more than 20 dogs which have been given SNP infusions at comparable rates, for between 1 and 6 days, with or without hydroxocobalamin.
• vasopressin replacement became apparent in first dogs depleted of arginine (in those cases by selective dialysis; later be enzymatic decomposition) and given SNP in order to maintain platelets. Since all of the pressor peptides contain arginine and are short lived molecules, in arginine depleted state there will be a general suppression of vasoconstricting signals. Replenishing NO (with SNP), which is also a strong vasodilator, leads to excessive vasodilatation and hemodynamic disturbance (an increase in pulse rate and eventually a drop in blood pressure). It is thus essential to balance NO effects with an appropriate pressor molecule.
• vasopressin There are several approved analogs of the natural vasopressin peptide. In most of the experiments on dogs, glypressin from Ferring was used; for the last dog and now for mice, ornipressin (POR 8 ® ), also from Ferring, was selected for its generally more effective pressor function in comparison to glypressin (which has a relatively stronger anti diuretic effect).
• glypressin was used at typically 0.13 mg/kg/day, which for humans would translate into 0.6 mg/kg/day. Since l g of glypressin corresponds to approximately 5 I.E. of ornipressin, this would be equivalent to the dose used in mice.
• ornipressin for i.v. infusion in esophageal bleeding, 20 I.E. of ornipressin are diluted in 100 ml of saline and infused over 20 minutes. This can be repeated several times, as needed. The suggested dose of 201.E./day for a 70 kg patient seems reasonable.
• administration of ornipressin is to be adjusted as needed to balance vasodilatory effects of SNP and is best adjusted by keeping the pulse rate normal: SNP will raise the pulse; ornipressin will lower it. It is recommended to increase the SNP delivery rate in several steps, adjusting ornipressin infusion at each step to lower the pulse back to normal.
• Prostacyclin is another molecule required by platelets to stay inactive. Prostacyclin stimulates production of cAMP which, like cGMP, is essential in preventing activation. In contrast to NO, prostacyclin synthesis does not directly depend on arginine. It appears though from both the dog and mice experiments, that after 2 to 3 days of arginine depletion, production of prostacyclin is also suppressed leading to activation of platelets, with increased risk of DIC. When dialysis was used as means of argmine depletion, replacement of prostacyclin from the very beginning was essential to avert the risk of platelet loss. Iloprost is a more stable analog of prostacyclin and is available from Schering, Berlin, under the trade name Uomedin ® . Recommended dose for humans is 0.5 to 2 ng/kg/min. hi mice the dose currently used is 12 ng/kg/min, which appears sufficient to prevent DIC. This would correspond to human dose of 1 ng/kg/min.
• Prostacyclin and NO have a synergistic action on platelets, so should any loss of platelets be observed within the first 12 hours of depleted argmine, both SNP and iloprost delivery rates should be increased proportionally. This will in turn require a measured increase in vasopressin dose in order to prevent vasodilation (prostacyclin is also a vasodilatator), i.e. an increase in the pulse rate and a drop in blood pressure.
• lipid infusion of 100 g/day, or 1.5 g/kg/day should help maintenance of the patient nutritional status during the several days of arginine depletion.
• Pisters P.W. et al. Protein and amino acid metabolism in cancer cachexia: investigative techniques and therapeutic interventions, Crit Rev Clin Lab Sci, 1993;30(3):223-72; from Dept. of Surgery, Memorial Sloan-Kettering Cancer Center.
• Pisters P.W. et al. Insulin action on glucose and branched-chain amino acid metabolism in cancer cachexia: differential effects of insulin, Surgery, 1992, Mar;l 11(3):301-10; from Dept. of Surgery, Memorial Sloan-Kettering Cancer Center.

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