TECHNICAL FIELD

This disclosure relates to the field of clinical medicine. In particular, the present disclosure relates to the use of pharmaceuticals to treat patient with excessive lactate production and related acidemia.

BACKGROUND

Cells in human body mainly use glucose as fuel substrate to generate energy (ATP, adenosine triphosphate) in a two-step process, i.e., the anaerobic glycolysis and the oxidative phosphorylation or the Krebs cycle (also known as citric acid cycle or tricarboxylic acid cycle). The glycolysis takes place in cytoplasm and does not need oxygen, glucose is broken down to the pyruvate, and then to the lactate. The oxidative phosphorylation takes place in mitochondria and needs oxygen, the pyruvate is converted to acetyl CoA to enter Krebs cycle, intermediates of Krebs cycle are oxidized to water and carbon dioxide.

Excessive lactate production is caused by increased glycolysis during glucose metabolism. Instead converting it to acetyl CoA, more pyruvate is converted to the lactate. Without conversion of pyruvate to acetyl CoA, Krebs cycle can not be initiated. Compared with Krebs cycle, glycolysis is not an efficient way to yield ATP, therefore, a cell with excessive lactate production is associated with ATP scarcity. The ATP is energy current for all living cells in the body. Without sufficient ATP, cells disfunction and eventually die. ATP scarcity is the final common pathway of all diseases before death. Excessive lactate production can be regarded an indicator of cellular ATP scarcity.

In clinic, excessive lactate production is a pathological process of various diseases, particularly critically ill patients. Excessive lactate production from affected cells releases into the blood. It is frequently manifested by hyperlactatemia in clinic. Hyperlactatemia refers to lactate concentration higher than 2 mmol/L in the blood. Severe hyperlactatemia is often accompanied by acidemia, also known as lactate acidemia or lactate acidosis which refers to blood pH less than 7.35. The acidemia further interferes with cellular biochemical reactions of the body aggravating abnormal metabolism. Hyperlactatemia and acidemia can be easily detected by simple measurement of blood. However, if excessive lactate production is only confined to the part of affected organ or if excessive lactate production is not severe enough, the hyperlactatemia and acidemia may not be easily detected by simple the blood testing.

Hyperlactatemia and lactate acidemia are ominous signs in clinic. For examples, patients with lactate level of more than 5 mmol/L have shown a high mortality rate. Patient with sepsis has poor prognosis when lactate level more than 2.5 mmol/L. However, effective treatment of hyperlactatemia is not available so far. Sodium bicarbonate (NaHCO3) has been used to treat lactate acidemia. Although NaHCO3can increase blood pH value, evidences have shown that it has very limited effect or even aggravating outcome for patient with lactate acidemia.

The lactate dehydrogenase (LDH), the enzyme that is responsible for lactate formation, is mainly in cytosol. It catalyzes conversion of the pyruvate to the lactate at a cost of turning NADH (reduced nicotinamide adenine dinucleotide) to NAD+(oxidized nicotinamide adenine dinucleotide) (Pyruvate+NADH+→Lactate+NAD+). The energy from glycolysis is transferred to the NADH through NAD+. The NADH has potential to generate ATP. However, NADH produced in the cytosol cannot directly enter the mitochondrial matrix to generate ATP. Regeneration of NAD+ is critical for continuation of glycolysis. Therefore, enhanced LDH enzymatic reaction wastes NADH energy potential and eventually leads to lactate accumulation. Normal serum LDH may range about 60-450 units/L (Newborn: 160 to 450 Units/L, Infant: 100 to 250 units/L, Child: 60 to 170 units/L, and Adult: 100 to 190 units/L). The LDH is frequently tested and widely used as an indicator for tissue damage in clinic. The LDH activity is often up-regulated in many diseases, both acute and chronic. Increased LDH activity invariably parallel to excessive lactate production. The lactate is generally considered as a glycolytic end product, and most cells can not use it to produce ATP. It can only be converted back to glucose in liver via Cori cycle which consumes ATP.

The oxamate, a salt of oxamic acid, is a non-competitive inhibitor of LDH. Its molecular formula is C2H2NO3−, its molecular weight is 88.042. The oxamate can reduce lactate production by inhibiting LDH. The oxamate has only been proposed for cancer therapy.

The lodoxamide is a Mast cell stabilizer. Ophthalmic lodoxamide Tromethamine (Alomide) is used to treat redness, burning, itching and swelling of the eyes that is caused by allergic reactions.

Malate dehydrogenase (MDH, EC 1.1.1.37) is the key enzyme in the malate/aspartate shuttle. NADH is oxidized to NAD+by the cytosolic MDH (MDH1), while oxaloacetate is simultaneously reduced to malate. The malate penetrates the mitochondrial inner membrane. Inside the matrix, the malate is converted to oxaloacetate by mitochondrial MDH (MDH2), generating the NADH, which then transfers electrons to the electron transport chain (ETC) in mitochondria. In this way, cytoplasmic reducing power of NADH is transferred to enter the ETC to generate ATP molecules in mitochondria. MDH is highly abundant in vital organs such as liver, heart, muscle and brain etc. It is estimated that the normal serum MDH may range about 79-176 units/L. The MDH activity are reportedly increased mainly in chronic diseases, such as liver cirrhosis. The MDH is not frequently tested in clinic.

Glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) is the key enzyme in the glycerol phosphate shuttle. NADH is oxidized to NAD+by the cytosol GPDH, while dihydroxyacetone phosphate is simultaneously reduced to glycerol-3-phosphate. Glycerol-3-phosphate gets converted back to dihydroxyacetone phosphate by an inner membrane-bound mitochondrial GPDH simultaneously reducing FAD to FADH2. In this way, cytoplasmic reducing power of NADH is transferred to enter the ETC to generate ATP molecules in mitochondria. The GPDH activity are reportedly increased mainly in chronic diseases, such as liver cirrhosis. The GPDH is seldom tested in clinic.

Transaminase, also known as aminotransferases, are enzymes that catalyze a transamination reaction between an amino acid and an α-keto acid. In transamination, the NH2group on one molecule is exchanged with the ═O group on the other molecule. The amino acid becomes a keto acid, and the keto acid becomes an amino acid. The reactions are readily reversible, the direction being determined by which of the reactants are in excess.

Two most important transaminases associated with the malate/aspartate shuttle activity are Aspartate Transaminase (AST, EC 2.6.1.1), also known as glutamic oxaloacetic transaminase (GOT), and Alanine Transaminase (ALT, EC 2.6.1.2), also known as glutamic pyruvate transaminase (GPT). The pyridoxal 5′-phosphate (PLP), also known as active form of Vitamin B6, is the co-enzyme of these two transaminases. The products of enzymatic reaction catalyzed by these two transaminases include aspartate, glutamate, α-ketoglutarate, oxaloacetate and pyruvate.

AST has two isoenzymes, GOT1 or cAST is cytosolic isoenzyme, and GOT2 or mAST is mitochondrial isoenzyme.

AST and ALT are found in various body tissues, including the liver, heart, skeletal muscle, kidneys, brain, and red blood cells etc. In clinic, The AST and ALT are frequently tested in clinic. It is estimated that the normal AST content may range 10-40 units/L, and normal ALT content may range 7-56 units/L. Serum AST and ALT are elevated in many diseases. For examples, there are marked increases of serum AST and ALT in circulatory failure, such as hemorrhagic shock, cardiac arrest, heart failure, and various organ damages, such as hepatitis, kidney disease, brain diseases etc. There are marked increases of serum AST and ALT even in healthy individual with intensive exercise. Although the elevation of AST and ALT can occur in almost all types of organs in response to various damages, they do have some preference to organ. In fact, serum AST elevation is regarded as broader biomarkers of tissue damage, and ALT elevation is more related to liver diseases. It usually takes hours to days for these enzymes to be increased in responding to initial injury. In some patient, transaminase activity simply does not increase despite of elevated mRNA level of transaminase.

Glutamate or glutamine plays a pivotal role in the conversion among amino acids, intermediates of Krebs cycle and malate/aspartate shuttle. Glutamic acid consists of two carboxyl groups (—COOH) and an amine group (—NH2) (molecular formula: C5H9NO4; molecular weight:147.13). It has very limited solubility in water (only 8.6 mg/ml at 25° C.). Glutamine is similar to glutamic acid, except the carboxylic group is replaced by an amide (molecular formula: C5H10N2O3; molecular weight: 146.14). Glutamate is the salt of glutamic acid, such as monosodium glutamate (molecular formula: C5H8NNaO4; molecular weight: 169.11), Magnesium diglutamate (molecular formula: C10H16MgN2O8; molecular weight: 388.62). Glutamic acid, glutamate and glutamine often collectively refer to glutamate as they are easily interchangeable chemically inside body.

Glutamate is used to reduce blood ammonia to treat hepatic encephalopathy in clinic. Ammonia can be detoxified by reacting with glutamate or glutamic acid to form glutamine. For example, the pharmaceutical preparation of sodium glutamate injection usually contains 5.75 g in each 20 ml vial. The pH value of this pharmaceutical preparation concentration is between 7.5-8.5.

Normal serum glutamic acid and glutamine range about 18-140 μmol/L and 390-730 μmol/L respectively. Normal serum aspartic acid and asparagine range about 0-26 μmol/L and 15-130 μmol/L respectively.

In clinic, serum glutamine and glutamate concentration are often reduced or even be depleted very quickly in many patients, particularly critically ill patients. Contrary to the decrease of glutamate, alanine concentration is often increased in patients with excessive lactate production. The increased ratio of Alanine/Glutamate was reportedly used as an index of reversibility of tissue damage. The alanine is shuttled to the liver where the nitrogen enters the urea cycle or consume α-ketogiutarate to generate pyruvate to make glucose which is known as alanine cycle, glucose-alanine cycle or Cahill cycle. This cycle consumes ATP. Therefore, alanine is not beneficial to or may even aggravate the outcome. However, glutamate is often mistakenly combined with alanine, for examples, almost all amino acid nutrition infusion products contain L-alanine. Dipeptiven® (Fresenius Kabi), a dipeptide containing L-alanine and -glutamine, is available in clinic to treat critically ill patients. The results of Dipeptiven® treatment have been controversial. For examples, Wischmeyer PE (Glutamine: Mode of action in critical illness. Crit Care Med 2007 Vol. 35, No. 9 (Suppl.), 5541-5544) reported beneficial effect; Heyland (A Randomized Trial of Glutamine and Antioxidants in Critically Ill Patients N Engl J Med. 2013; 368(16):1489-97) and Chen (The effect of glutamine therapy on outcomes in critically ill patients: a meta-analysis of randomized controlled trials. Crit Care. 2014; 18(1): R8.) reported negative effects.

Many amino acids can be converted to glutamate or glutamine, and can be used as fuel to generate ATP as well. Amino acids in human body can be categorized into essential (including Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenyalanine, Threonine, Tryptohan and Valine), non-essential amino acid (Alanine, Aspartic acid, Asparagine, Glutamic acid, Serine, Selenocysteine and Pyrrolysine), and conditionally essential (including Arginine, Cysteine, Glutamine, Glycine, Proline and Tyrosine). Conditionally essential amino acid cannot be synthesized under pathophysiological conditions such as trauma, sepsis, surgery, wound, severe catabolic distress.

Leucine, Isoleucine and Valine belong to branched-chain amino acid (BCAA) which has an aliphatic side chain with a branch. The final catabolic products of BCAA are acetyl-CoA and succinyl-CoA, which can enter Krebs cycle for ATP production. In clinic, serum BCAA is often decreased or depleted in patients associated with hyperlactatemia.

Currently amino acid injections are mainly for intravenous nutritional supplement. Pharmaceutical companies manufacturing amino acid injections include Baxter, B Braun, Clintec Nutrtion, Hospira, ICU Medical, Fresenius Kai etc. Their products include Glycine, Prosol, Aminosyn, Clinimix, FreAmine, HepatAmine, Hepatasol, Kabiven, Novamine, Nutrinea, Olimel, Perikabiven, Plenamine, Smofkabiven, Travasol, Trophamine, Vamin etc. These amino acid formulations usually contain large amount of alanine, D-glucose or lipid. Mounting evidence have also shown that D-glucose and lipid can aggravate hyperlactatemia and lactate acidemia.

In central nervous system (CNS), glutamate and aspartate are excitatory amino acid neurotransmitters. Current dogmatic theory considers glutamate and aspartate as excitotoxin that can destroy CNS tissue by excessive activation of glutamate receptors such as the N-methyl-D-aspartate receptor (also known as the NMDA receptor). The excessive NMDA receptor activity causes calcium ions (Ca2+) to influx into cells. High Ca2+concentrations activate a cascade of cell degradation processes such as proteases, lipases, nitric oxide synthase, free radicals leading to cell death. Overwhelming studies have reported glutamate and aspartate release after various injuries to brain and spinal cord. Therefore, persons skilled in the art believe that glutamate damages brain tissue and mediate a wide range of neurologic diseases, such as brain hypoxia-ischemia, trauma etc. In spite of glutamate usage in peripheral organ systems, persons skilled in the art have been trying to avoid the glutamate in neurological diseases because of the concern of prevailing excitotoxicity theory and reports that glutamate administration may induce brain damage due to its excitotoxic and edema-aggravating potential.

SUMMARY

Accordingly, the instant invention discloses that the oxamate, a non-competitive inhibitor of LDH, can be used as pharmaceutical composition to treat a patient with excessive lactate production and lactate acidemia. The lodoxamide has two similar chemical groups as the oxamate, it can also inhibit LDH, and can be used as in a pharmaceutical composition to treat patient with excessive lactate production and lactate acidemia.

The dosage of the oxamate and lodoxamide is generally in the range of about 0.00001-1,000 mg/kg body weight, specifically in the range of about 1-100 mg/kg body weight. The route of administration can be oral, intravenous or intramuscular injection, the preferred route is intravenous or intramuscular injection.

The inventor has also surprisingly discovered that amino acids (except alanine), particularly, glutamate, aspartate, BCAA can reduce lactate production. The malate, oxaloacetate, pyruvate and α-ketoglutarate can also reduce lactate production.

One side effect of sodium glutamate injection is alkalemia in clinic. The sodium and potassium salt of malate, oxaloacetate, aspartate, glutamate, pyruvate and α-ketoglutarate have higher pH value, can lead to alkalosis in blood. This is because sodium and potassium salts of these substrates are salt of strong base and weak acid. Therefore, sodium and potassium salts in this invention are especially useful to formulate pharmaceutical compositions, because they not only reduce lactate but also correct acidemia. For example, pharmaceutical preparation of sodium glutamate 5.75 g in each 20 ml water, the pH value is between 7.5-8.5. Unexpectedly, the inventor found that this preparation can be used for increasing blood pH value to correct acidemia.

The inventor has also surprisingly discovered that in contrary to current excitotoxic theory, glutamate, glutamine and aspartate can be used to reduce lactate production and ameliorate lactate acidemia in all neurological diseases too, particularly cerebral ischemia, brain trauma.

The dose of amino acids or malate, oxaloacetate, pyruvate and α-ketoglutarate can range between 0.001 to 10 g/kg, specifically in the range of about 0.1-3 g/kg.

The inventor has also surprisingly discovered that the MDH, GPDH, AST, ALT, PLP (or Vitamin B6) and their substrates all can be used as pharmaceutical composition to treat patients with excessive lactate production and lactate acidemia.

To reduce the amount of NADH in cytosol, two pathways are recruited, i.e. the shuttles and the LDH catalyzed reaction. The shuttles include malate/aspartate shuttle and the glycerol phosphate shuttle. These shuttles can bring cytosolic reducing equivalents into the mitochondrial matrix, without NADH itself actually entering the matrix. The malate/aspartate shuttle activity involves coupled transaminase reaction with cytosolic and mitochondrial transamination, and shares substrates including aspartate, glutamate, oxaloacetate, α-ketoglutarate and pyruvate. Unlike the LDH, the MDH and GPDH activity are not increased quickly enough in many acute pathological conditions. The AST and the ALT are not increased quickly enough either in many acute pathological conditions.

MDH, GPDH, AST, ALT, PLP (or Vitamin B6) and their substrates can compete with the LDH by enhancing malate/aspartate shuttle activity. Enhanced transamination reaction catalyzed by the AST and ALT orchestrates enhanced malate/aspartate shuttle. This is the new mechanism to reduce lactate production and ameliorate lactate acidemia. NADH is oxidized to NAD+by the cytosolic MDH, and the oxaloacetate is needed to produce malate. Malate enters mitochondria. By this way, the cytosolic MDH compete with LDH for consuming NADH, the more NADH is used to form malate, the less NADH is consumed to form lactate, hence lactate production and lactate acidemia can be reduced by enhanced malate/aspartate shuttle activity.

Accordingly, the instant invention discloses MDH, GPDH, AST, ALT, PLP as a pharmaceutical component to treat patients with excessive lactate production and lactate acidemia.

Accordingly, the instant invention also discloses the combination of MDH, GPDH, AST, ALT, PLP, their substrates and amino acids as pharmaceutical compositions to treat patients with excessive lactate production and lactate acidemia. The combination is advantageous because it can promote the substrate enzymatic reaction and enhance malate/aspartate shuttle. The AST and ALT catalyzed enzymatic reactions produces oxaloacetate, aspartate, glutamate and α-ketoglutarate. These enzymatic reactions are tightly linked with malate formation and malate/aspartate shuttle. Exogenous oxaloacetate, malate, aspartate, glutamate and α-ketoglutarate and amino acids can replenish substrate consumption. Therefore, their combination can further promote malate/aspartate shuttle, lead to synergetic effect, and maintain long efficacy. Any combination may be chosen. Examples of these combination are: MDH+AST; MDH+ALT; MDH+AST+ALT; MDH+AST+glutamate; MDH+ALT+glutamate; MDH+AST+PLP+glutamate; MDH+ALT+PLP+glutamate; MDH+oxaloacetate, AST+glutamate; ALT+glutamate; AST+glutamate+Aspartate; ALT+glutamate+pyruvate; AST+PLP+glutamate; ALT+PLP+glutamate; MDH+malate oxaloacetate+glutamate+aspartate+α-ketoglutarate; GPDH+AST; GPDH+ALT; GPDH+AST+ALT; GPDH+AST+glutamate; GPDH+ALT+glutamate; GPDH+AST+PLP+glutamate; GPDH+ALT+PLP+glutamate; AST+oxaloacetate+glutamate+aspartate+α-ketoglutarate; ALT+oxaloacetate+glutamate+aspartate+ketogiutarate pyruvate; Malate+oxaloacetate+glutamate+aspartate+α-ketoglutarate+pyruvate BCAA etc. The preferred combinations are MDH+AST, MDH+AST+glutamate, MDH+oxaloacetate, AST+glutamate, AST+glutamate+aspartate, Glutamate+aspartate etc.

The MDH, GPDH, AST, ALT can come from animal extract or can be manufactured by bioengineering recombinant protein technology.

The dosage of the MDH, GPDH, AST, and ALT is generally in the range of about 1-100,000 units/kg body weight or 0.0001 ng to 1 g/kg body weight, specifically in the range of about 1-1,000 units/kg body weight or 0.01 ng to 0.01 g/kg body weight. The dose of PLP can be in a range about 1-1000 mg/kg, specifically in the range of about 1-100 mg/kg.

The preferred route of the MDH, GPDH, AST, ALT, substrates and amino acids is intravenous or intramuscular injection.

The magnesium is cytoprotective. It has synergetic effect and can be used to formulate pharmaceutical compositions too. For example, magnesium oxide, magnesium sulfate, magnesium chloride can be used to form magnesium salt of malate, oxaloacetate, aspartate, glutamate, pyruvate and α-ketoglutarate. The Mg2+can be used to formulate the pharmaceutical compositions of MDH, GPDH, AST, ALT and PLP.

One feature of the instant invention is a method for treating a patient having excessive lactate production or lactate academia. The method includes administering to the patient in need thereof an effective amount of at least one substance selected from the group consisting of glutamate, aspartate, BCAA, malate, pyruvate, oxaloacetate, α-ketoglutarate, AST, ALT, PLP, MDH, GPDH, Oxamate, Lodoxamite and salts thereof.

In some embodiments, the substance can be administered at a dosage of from at least 10 ng (e.g., at least 100 ng, at least 1 μg, at least 10 μg, at least 100 μg, at least 1 mg, at least 0.005 g, at least 0.01 g, at least 0.05 g, at least 0.1 g, at least 0.5 g, or at least 1 g) per kilogram body weight to at most 10 g (e.g., at most 8 g, at most 6 g, at most 5 g, at most 4 g, at most 2 g, at most 1 g, at most 0.5 g, at least 0.1 g, at least 0.05 g, or at least 0.01 g) per kilogram body weight.

Another feature of the instant invention is that MDH, GPDH, AST, ALT, PLP, their substrates and their combination can be used to treat patients with excessive lactate production and lactate acidemia caused by ischemia/reperfusion injury. The treatment can begin prior to blood reperfusion. The treatment can last for a suitable period, for example, 5 minutes to 3 hours to allow ischemia issue to generate enough ATP, then restore blood flow to the ischemic region. This is particularly useful when treating patient with thrombolytic therapy (such as tissue plasminogen activator, tPA), percutaneous aspiration thrombectomy or angioplasty etc. In some embodiments, the MDH, GPDH, AST, ALT, PLP, their substrates or their combination can be administered at a dosage of from at least 0.0001 ng (e.g., at least 0.001 ng, at least 0.01 ng, at least 0.1 ng, at least 1 ng, at least 10 ng, at least 100 ng, at least 1 μg, at least 10 μg, at least 100 μg, or at least 1 mg) per kilogram body weight to at most 1 g (e.g., at most 0.8 g, at most 0.6 g, at most 0.5 g, at most 0.4 g, at most 0.2 g, at most 0.1 g, at most 0.05 g, or at most 0.01 g) per kilogram body weight.

Another feature of the instant invention is that glutamate, glutamine, aspartate and their combination with MDH, GPDH, AST, ALT and PLP (or vitamin B6) can be used to treat patients with neurological diseases having excessive lactate production and lactate acidemia, although this is contrary to current excitotoxic theory that glutamine, glutamate and aspartate are excitotoxin to neuronal tissue.

