HK1178148B - Compositions and methods of reducing side effects and toxicity of methotrexate when given as orotate derivatives - Google Patents

Description

Compositions and methods for reducing side effects and toxicity of methotrexate when administered as orotate derivatives

The present invention is a divisional application of the chinese invention application (entitled "composition and method for reducing side effects and toxicity of methotrexate when administered as orotate derivative"; application No. 200880002637.7; application date 1/18/2008).

Cross reference to other applications

This application is a Continuation-in-part (Continuation-in-part) of U.S. patent application 11/448,703 filed on 7.6.2006, U.S. patent application 11/448,703 being incorporated herein by reference in its entirety.

1. Field of the invention

The present invention relates to orotic acid derivatives of the drugs and in particular to the conversion of methotrexate to their orotate derivatives to enhance bioavailability and drug clearance, thereby reducing toxicity in cancer patients and in non-infectious, non-neoplastic inflammatory conditions such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus and multiple sclerosis.

2. Background of the invention

The field of the present invention relates to the chemical structural modification (chemical structural modification) of methotrexate and related drugs known to have poor bioavailability, causing toxic or adverse drug reaction side effects in non-cancerous tissues by preparing their orotate derivatives. More particularly, the invention relates to derivatives of folic acid (folate) antagonists, methotrexate, trimetrexate and raltitrexed for use as anti-cancer drugs in non-malignant conditions characterized by rapid cell growth.

In the 58 years when Farber first described clinical remission after the folate antagonist aminopterin administration to children with acute leukemia, methotrexate has been used to treat millions of patients with both malignant and autoimmune diseases. Methotrexate is now the most widely used disease-modifying antirheumatic drug (DMARD), which is used by at least 500,000 patients worldwide with rheumatoid arthritis. In current combination medication, methotrexate is used more in patients with rheumatoid arthritis than all biopharmaceuticals. It is the most frequently reported drug used in combination with other DMARDs, where a clear additional therapeutic effect is demonstrated. Oral administration of low dose methotrexate has been used to treat multiple sclerosis with minimal toxicity. However, when administered orally, serum levels of methotrexate are inadequate due to poor oral bioavailability. Concern over severe organ-related toxicity has led to the development of standards for monitoring liver toxicity, which discouraged physicians and limited their early use. Moreover, methotrexate-associated lung disease is the most common subacute syndrome associated with dry cough, often accompanied by dyspnea and fever, recognition of which has led to early recognition and avoidance of permanent lung sequelae in many drug-receiving patients. The recognition that the use of methotrexate was greatly limited by many gastrointestinal, bone marrow and other toxicities before the 80's of the 20 th century, which could be avoided by the use of folate supplements, has also given clinicians and patients safety measures in the use of effective antimetabolites.

However, the overall search for the problems and complexities associated with the use of methotrexate has been relatively rare. Many physicians use folic acid with other physicians using methotrexate with a variety of other drugs, and the patients may have a high risk of adverse effects due to drug interactions. The thoughts and modes of administration associated with maximum weekly doses, use in the elderly, monitoring blood tests, and when to "give up" and add other drugs to be administered with methotrexate are often without particularly rigorous scientific support.

The food and drug administration approved low dose methotrexate for the treatment of psoriasis in 1960 and rheumatoid arthritis in 1988. Methotrexate is now administered worldwide by rheumatologists and has proven to be a very effective, fast-acting, second-line antirheumatic with the best efficacy-toxicity ratio. However, the main reason for stopping methotrexate is not its ineffectiveness but toxicity. Because of their clear and long-lasting efficacy, many efforts are now being made to investigate strategies to reduce or prevent their toxicity. In approximately 30% of rheumatoid arthritis patients, toxicity results in cessation of methotrexate therapy. Thus, the present invention provides 2 approaches to reduce toxicity of methotrexate: 1) by increasing the bioavailability of methotrexate and thereby decreasing the effective dose of methotrexate, and 2) by increasing the clearance of methotrexate.

Side effectsThe side effects of methotrexate are quite common. Severity varies, but most side effects are mild, reversible, and can be treated conservatively. Side effects, such as nausea, changes in transaminases, and stomatitis (stomatitis) occur frequently and are dose-dependent; others, such as pneumonia and hepatocyte changes are not. However, for 30% of patients with rheumatoid arthritis, toxicity led to discontinuation of methotrexate treatment within 1 year. Only a few determinants of toxicity are known, such as increased age and poor renal function. Another important issue is that although the risk of side effects may be somewhat high in the first 6 months, the risk for adverse effects of all kinds is permanent, suggesting the need for long-term monitoring. At least part of the methotrexate side effects appear to be directly related to its folate antagonism and its cytostatic effects, especially in highly renewing tissues. Methotrexate is used at a dose of 5 mg/week to 15 mg/week and a maximum dose of 25-30 mg/week. The low bioavailability of methotrexate explains these results. Thus, the present invention provides a method for increasing the bioavailability of methotrexate by converting it to the orotate form and thereby reducing its toxicity.

Because of the role of methotrexate in therapy, many researchers have modified the structure of methotrexate in an attempt to synthesize more potent derivatives. U.S. patent 5,698,556 to chan, Carey l.and U.S. patent 5,958,928 to Masahiko Mihara. Methotrexate enters cells via reduced folate (reduced folate) carriers, which also transport naturally occurring reduced folates. Efflux of methotrexate (efflux) occurs by a different mechanism than influx (influx), and efflux is energy-dependent. Multi-drug resistance-associated proteins (Multi-drug resistance-associated proteins) have been identified which transport methotrexate, folate and 5-CHO-FH4 extracellularly. Inhibition of multidrug resistance protein results in significant aggregation of intracellular methotrexate. The present invention provides methods for increasing the efflux and clearance of methotrexate by converting it to the orotate form and increasing its clearance. The basic object of the present invention is to find more potent and less toxic drugs. The relevant subject matter of the above references is specifically incorporated herein by reference.

3. Brief description of the invention

The present invention overcomes the disadvantages inherent in the prior art by providing compositions of orotate derivatives of methotrexate or trimetrexate that exhibit improved bioavailability and renal clearance compared to the non-derivatized form of the drug.

The field of the invention relates to the chemical structural modification of currently used drugs whose known side effect is the cause of tissue toxicity, by preparing their orotate derivatives. More particularly, the present invention relates to orotate derivatives of methotrexate, which are useful as anticancer agents or disease modifying anti-rheumatic drugs (disease modifying anti-rheumatic drugs).

In light of the state of the art, the present inventors have designed orotate derivatives of folic acid antagonists, such as methotrexate orotate and trimetrexate orotate, with chemical organic moieties contained therein that enhance their bioavailability and renal clearance.

The invention also specifically provides a process for the preparation of methotrexate orotate and related derivatives starting from methotrexate, orotic acid and sodium hydroxide (or any other base such as potassium hydroxide, or aluminium hydroxide). The method comprises the following steps: a) reacting sodium hydroxide with orotic acid, extracting the sodium orotate, and reacting the extracted sodium orotate with methotrexate to form sodium methotrexate orotate(sodium methotrexate orotate)。

It is another object of the invention to increase the bioavailability of methotrexate when administered as methotrexate orotate in humans and other mammals.

It is another object of the invention to reduce multidrug resistance when methotrexate is administered as orotate.

The invention can also be used to reduce the toxicity of methotrexate and trimetrexate when administered as orotate salts.

The invention can also be used to enhance the first pass clearance of methotrexate or trimetrexate when administered as orotate salts through the extraction organs.

The invention can also be used to reduce drug interactions and side effects when methotrexate or trimetrexate is administered as orotate salts.

It is another object of the present invention to provide compositions for treating human tumors, and in particular primary or metastatic tumors, hematopoietic cell proliferative disorders (proliferous cell proliferative disorders) and leukemias with sodium methotrexate orotate and reducing the toxic side effects of the drug by reducing the level of the drug in non-cancerous tissues, which are susceptible targets for drug toxicity, by 10% -100% compared to the administration of methotrexate.

A preferred embodiment of the invention includes compositions of methotrexate orotate for use in the treatment of inflammatory diseases including rheumatoid arthritis, psoriasis, multiple sclerosis and other diseases.

4. Brief description of the drawings

FIG. 1 shows the structure of methotrexate orotate

FIG. 2 shows the synthesis of methotrexate orotate

FIG. 3 is a mass spectrum showing methotrexate orotate

FIG. 4 is an NMR chart showing methotrexate orotate

Figure 5 shows the response of SC Du-145 prostate tumors to treatment with methotrexate or methotrexate orotate.

5. Detailed description of the invention

Drug therapy for treating cancer patients can damage a large number of organs and organ systems. The most frequently damaged of these are tissues with rapid cell turnover, such as the hematopoietic system, gastrointestinal tract and urogenital tract. Some drugs are toxic by themselves, but their toxicity is potentiated when they are used in combination with other drugs, the combination possibly being more toxic than the sum of the toxicities of the individual components. Because of the need to achieve optimal drug antitumor effects while keeping end organ toxicity at acceptable levels, the evaluation of patients treated with toxic drugs must be individualized.

It is an object of the present invention to reduce the extent and incidence of initial liver damage caused by a drug by reducing the level of the drug in the liver, preventing the accumulation of the drug in the liver tissue and/or ensuring that the drug leaves the liver tissue faster when chemotherapy is administered thereby reducing the release of free radicals.

In most pharmaceutical companies, many techniques such as combinatorial chemistry, nanotechnology, rapid analog synthesis, automated synthesis open liquid chromatography mass spectrometry (automated synthesis open access chromatography mass spectrometry), and high-speed automated High Performance Liquid Chromatography (HPLC) now affect pharmaceutical chemistry, and their main role is to shorten the cycle time of the synthesis operation. One of the most difficult properties for constructing newly discovered lead molecules (lead molecules) is the required pharmacokinetic profile, especially for orally administered compounds. "most experienced medicinal chemists would prefer to start in a structural series that inherently has good pharmacokinetic properties (albeit with poor efficacy at the target receptor) and then increase efficacy at the target without attempting in another direction", "Organic Chemistry in Drug Discovery, Drug Discovery", Science 303: 1810-.

The use of methotrexate in the form of orotate salts increases the oral bioavailability of methotrexate.

The present invention relates generally to increasing the oral bioavailability of drugs poorly absorbed from the gastrointestinal tract, and to methods of orally administering these drugs to improve patient treatment. In particular, the invention relates to poorly absorbed methotrexate or trimetrexate and converting them to orotate salts to enhance the oral bioavailability of the drug. Thus, the orotate salt of the pharmaceutical agent can be administered at lower doses to provide the efficacy of the higher dose, while reducing the toxic effects of the agent at lower doses. Furthermore, the orotate salts of the drugs have better clearance, i.e., increase the fraction of the drug that is not metabolized by the first pass, thereby reducing the potential for hepatotoxicity. Thus, particularly useful formulations of the orotate salt of the pharmaceutical agent are able to provide rapid action and sustained action using lower doses, and reduce drug interactions and side effects due to continuous administration. The present invention provides a method for synthesizing orotate salts of water-insoluble drugs having ionizable centers for improving the oral bioavailability and efficacy of the drugs.

The absorption of drugs by the oral route is the subject of intense research in the pharmaceutical industry, since good bioavailability means that the drug can reach the systemic circulation through the mouth. Oral absorption is influenced by drug properties and the physiology of the gastrointestinal tract, including dissolution of the drug from the dosage form, the mode of action of the drug with the aqueous environment and membrane, permeation through the membrane, and irreversible removal by first-pass organs (e.g., the intestine, liver, and lung). Some drugs that exhibit low solubility exhibit poor bioavailability or irregular absorption, the degree of irregularity being affected by various factors, such as dose level, dietary status of the patient, and physicochemical properties of the drug.

Due to the large surface area, drug absorption mostly occurs in the small intestine because of the increased absorption area caused by the villi and microvilli. The duodenum and jejunum have the greatest surface area because these regions have the highest concentration of villi and microvilli compared to the ileum. The intestinal circulation is unique in that the intestine is the anterior or posterior tissue that regulates the flow of material to the liver. The intestinal venous blood constitutes about 75% of the blood supplied to the liver. Thus, for drugs that are cleared by the gut, the effect of the liver, kidney or lung on drug metabolism will be reduced. In contrast, for poorly extracted drugs from the intestine, the material is able to reach the next organ, the liver and lungs responsible for removal. Thus, the concentration of drug entering the intestine and intestinal flux alter the rate of drug delivery and affect the proportion in the intestine, and clearance via hepatic first pass metabolism.

"drug bioavailability" is defined herein as the amount of drug available systemically over time. The present invention increases the drug bioavailability of a drug by converting the drug into an orotate salt. This can be achieved by modifying the hydrophilic and lipophilic properties of the drug so that the drug permeates well through the membrane and the blood perfusion rate becomes the overall rate-limiting step in absorption, or by inhibiting the biotransformation of the drug in the gut and/or by inhibiting the active back transpart system (active back transpart system) in the gut which reduces the net transport of the drug through the gut membrane into the bloodstream. In each case, the composition that results in improved bioavailability of the drug is the orotate salt of the drug. For several reasons that are not yet clear, it has been discovered that the conversion of a water-insoluble drug to an orotate salt provides a means of increasing the bioavailability of a sufficient amount of the drug orally administered to a mammal in need of treatment such that the time-integrated systemic concentrations (integrated systems concentrations over time) of the orotate drug is greater than the drug that is not converted to the orotate salt.

Change in integrated systemic concentration over time by area under the curve (AUC) or C_maxBoth parameters are shown as known in the art. AUC is a measure of the area under the curve, which is plotted on the ordinate (Y-axis) against the serum or plasma concentration of the drug and on the abscissa (X-axis) against time. Generally, the values of AUC represent drug concentration over time in units of mass-time/volume. When testing the efficacy of the orotate salt of the pharmaceutical agent, the amount and form of the active agent administered should be the same in both the administration of the orotate salt as the pharmaceutical agent and the administration of the pharmaceutical agent itself.

The present invention provides methods wherein the composition provides an increased bioavailability of the orotate salt of the pharmaceutical agent as measured by AUC of at least 25% relative to dosing of the pharmaceutical agent. The invention also provides methods wherein the composition provides an increased bioavailability of the orotate salt of the pharmaceutical agent as measured by AUC of at least 50% relative to dosing of the pharmaceutical agent. The invention also provides methods wherein the composition provides an increased bioavailability of the orotate salt of the pharmaceutical agent as measured by AUC of at least 100% relative to dosing of the pharmaceutical agent.

The present invention provides compositions that increase bioavailability of the orotate salt of the pharmaceutical agent as measured by Cmax of at least 50% relative to dosing of the pharmaceutical agent. The invention also provides compositions that increase bioavailability of the orotate salt of the pharmaceutical agent as measured by Cmax of at least 100% relative to dosing of the pharmaceutical agent. The invention also provides the composition which provides an increased bioavailability of the orotate salt of the pharmaceutical agent as measured by Cmax of at least 200% relative to dosing of the pharmaceutical agent. Systemic drug concentrations were measured using standard biochemical drug detection techniques (Simmons et al, Anal Lett.39:2009-2021 (1997).

Properties of drugs used as orotate derivatives

The word "drug" as used herein is defined as a chemical used to treat or prevent a disease. Drugs include synthetic and naturally occurring biologically effective substances, as well as recognized drugs, such as those listed in the "the Physician desk Reference," 56 th edition, pages 101-133 (or more recent edition). These references are incorporated herein by reference. The term "drug" also includes compounds having the indicated properties not found or available. The present invention may employ agents that include charged, uncharged, hydrophilic, zwitterionic or hydrophobic species, as well as any combination of these physical properties. Hydrophobic drugs are defined as drugs whose non-ionized form is more soluble in lipids or fats than in water. Preferred types of hydrophobic drugs are those that are more soluble in octanol than in water.

Compounds or drugs from many classes of compounds can be converted to orotate derivatives and administered orally as the orotate derivative. The compound or drug may be, for example, but not limited to, the following species: acetanilide, actinomycin D, doxorubicin, amsacrine, aminoimidazole, aminoquinoline, anilide, anthracyclines, antiestrogens (antiestrogens), benzazepinesClass (I), benzhydryl compounds, benzodiazepines(benzodiazepine), benzofuran, cannabinoid, cephalosporin, cisplatin, colchicine, cyclopeptide, cyclophosphamide, daunorubicin, dibenzazepineDigitoxin, dihydropyridine, doxorubicin, etoposide (epipodophyllotoxin), epirubicin, ergeline, ergot alkaloid, etoposide, 5-fluorouracil, idarubicin, ifosfamide (ifosamide), imidazole, interleukin-2, interferon alpha isoquinoline, macrolide, melphalan, methotrexate, mitomycin-C, mitoxantrone, naphthalene, nitrogen mustard, opioids, oxazines (oxazune), oxazole, paclitaxel, phenothiazine, phenalkylamine, phenylpiperidine, piperazine, piperidine, polycyclicAromatic hydrocarbons, pyridine, pyrimidine, pyrrolidine, pyrrolidone, quinazoline, quinoline, quinine, rauwolfia alkaloids, retinol, salicylates, steroids, stilbene, sulphones, sulphonylureas, tamoxifen, taxol, taxotere, THP-doxorubicin, trastuzumab, triazole, tropane, vinblastine, vincristine or vinca alkaloids.

"side effects" or "toxicity" or "adverse drug reactions" of chemotherapeutic drugs are observed in the acute phase of chemotherapeutic administration and in treated cancer patients with subclinical tissue damage. There is a greater awareness of drug-related tissue side effects that can be very severe, disabling and irreversible. The clinician must be aware of possible tissue/organ complications of chemotherapeutic drugs and where a baseline tissue examination is suitably performed prior to initiating treatment.

"purging" of the drug is performed by perfusing the blood to the extraction organ. "extraction" refers to the irreversible removal (excretion) or change in the ratio of the drug supplied to the organ to a different chemical form (metabolism). Clearance (CL) is therefore calculated as the product of the blood flow through the organ and the proportion of drug extracted by the organ (product).

Clearance of the drug typically begins from the liver and kidneys, and it is believed that only free and non-protein bound drug can be cleared. For hepatic clearance, passive diffusion of lipophilic drugs through the lipid core of the hepatocyte membrane, particularly ionized molecules (anions and cations) with molecular weights above 400, is increased by sinusoidal carrier systems. Likewise, other transporters at the surface of the tubules transport drugs or their metabolites into the bile. This system has two separate processes, hepatic uptake and biliary excretion (biliary excretion). Hepatic uptake is not a major factor with small molecule lipophilic drugs that readily pass through membranes, but with larger molecular weight compounds (greater than 500) and those containing a large number of H-bonds, hepatic uptake can become the major clearance process even after metabolism occurs.

The present invention provides a method of increasing clearance of an orotate derivative of the pharmaceutical agent from non-cancerous or normal tissues as measured by pharmacological studies of at least 25% relative to dosing of the pharmaceutical agent. The invention also provides a method of increasing clearance of an orotate derivative of the pharmaceutical agent from noncancerous or normal tissues as measured by pharmacokinetic studies of at least 50% relative to dosing of the pharmaceutical agent. The invention also provides a method of increasing clearance of an orotate derivative of the pharmaceutical agent from noncancerous or normal tissues as measured by pharmacological studies of at least 100% relative to dosing of the pharmaceutical agent.

The present invention provides compositions for increasing clearance of an orotate derivative of the pharmaceutical agent from noncancerous or normal tissues as measured by pharmacological studies of at least 50% relative to dosing of the pharmaceutical agent. The invention also provides a composition for increasing clearance of the orotate salt of the pharmaceutical agent from noncancerous or normal tissues as measured by pharmacokinetic studies of at least 100% relative to dosing of the pharmaceutical agent. The invention also provides a composition for increasing clearance of an orotate derivative of the pharmaceutical agent from non-cancerous or normal tissues as measured by pharmacokinetic studies of at least 100% relative to dosing of the pharmaceutical agent.

The absorption or efflux is carried out by one of three methods, passive diffusion, active transport or assisted transport (failed active transport). Passive diffusion is simply the passage of molecules across the mucosal barrier until the concentration of molecules on both sides of the membrane reaches osmotic equilibrium. In active transport, molecules are actively pumped across the mucosa. In assisted transport, a carrier (usually a protein) is required to transport the molecule across the membrane for absorption.

"bioavailability" of an orally administered drug refers to the degree or proportion by which the active portion of the drug or metabolite enters the systemic circulation, thereby reaching the site of action. The physicochemical properties of a drug determine its absorption capacity, but the properties of a dosage form, which depend in part on its design and preparation, can largely influence drug bioavailability. Differences in bioavailability between formulations of a given drug are clinically significant. The concept of equivalence in pharmaceutical products is important in making clinical decisions.

"Chemical equivalence (Chemical equality)" refers to a pharmaceutical product containing the same compound in the same amount and which meets current official standards. However, the inactive ingredients in the pharmaceutical product may vary.

"Bioequivalence" refers to a chemical equivalent that results in the same concentration of drug in blood and tissues when administered to the same person on the same dosage regimen.

"Therapeutic equivalence" refers to a pharmaceutical product that provides substantially the same Therapeutic effect or toxicity when administered to the same person in the same dosage regimen. A bioequivalent product is considered therapeutically equivalent. Sometimes different bioavailability may be therapeutically equivalent, for example when the therapeutic index is broad (ratio of maximum tolerated dose to minimum effective dose).

The rate of "absorption" is important because even when the drug is completely absorbed, it may be absorbed too slowly to produce a therapeutic blood concentration quickly enough, or too quickly to be toxic to achieve a therapeutic level of high drug concentration after each administration.

"Clearance (Clearance)" of the drug is performed by perfusing blood to the extraction (extraction) organ. "extraction" refers to the irreversible removal (excretion) or change in the ratio of the drug supplied to the organ to a different chemical form (metabolism). Clearance (CL) is therefore calculated as the product of the blood flow through the organ and the proportion of drug extracted by the organ (product). Figure 2 illustrates the interplay of hepatic and renal clearance.

Clearance of the drug typically begins from the liver and kidneys, and it is believed that only free and non-protein bound drug can be cleared. For hepatic clearance, passive diffusion of lipophilic drugs through the lipid core of the hepatocyte membrane, particularly ionized molecules (anions and cations) with molecular weights above 400, is increased by the sinusoidal carrier system. Likewise, other transporters at the surface of the tubules transport drugs or their metabolites into the bile. This system has two separate processes, hepatic uptake and biliary excretion. Hepatic uptake is not a major factor with small molecule lipophilic drugs that readily pass through membranes, but with larger molecular weight compounds (greater than 500) and those containing a large number of H-bonds, hepatic uptake can become the major clearance process even after metabolism occurs.

The present invention provides a method of increasing the clearance of the orotate salt of methotrexate as measured by pharmacokinetic studies of at least 25% relative to dosing of the pharmaceutical agent. The invention also provides a method of increasing the clearance of the orotate salt of methotrexate as measured by pharmacokinetic studies of at least 50% relative to dosing of the pharmaceutical agent. The invention also provides a method of increasing clearance of an orotate derivative of methotrexate as measured by pharmacokinetic studies of at least 100% relative to dosing of the pharmaceutical agent.

The present invention provides a composition that increases clearance of the orotate salt of the methotrexate as measured by pharmacokinetic studies of at least 50% relative to dosing of the pharmaceutical agent. The invention also provides a composition for increasing clearance of the orotate salt of methotrexate as measured by pharmacokinetic studies of at least 100% relative to dosing of the pharmaceutical agent. The invention also provides a composition for increasing clearance of the orotate salt of the pharmaceutical agent as measured by pharmacokinetic studies of at least 200% relative to dosing of the pharmaceutical agent.

Reasons for the low bioavailability

When the drug rapidly dissolves and passes through the intestinal membrane rapidly, absorption tends to be complete, but absorption of orally administered drugs is not always complete. Before reaching the vena cava, the drug must travel down the gastrointestinal tract and pass through the intestinal wall and liver (the common site of drug metabolism). The drug is therefore metabolized during first pass metabolism before it is detected in the systemic circulation. Many drugs have low oral bioavailability due to extensive first pass metabolism.

Low bioavailability is most common in oral dosage forms of poorly water soluble, slowly absorbed drugs. Slow or incomplete absorption may be affected by more factors than fast and complete absorption. I.e., slow or incomplete absorption results in different therapeutic responses. Slow absorption in the gastrointestinal tract also leads to increased acute and delayed phase chemotherapy-induced nausea and vomiting.

Insufficient time in the gastrointestinal tract is a common cause of low bioavailability. Absorbed drug is exposed throughout the gastrointestinal tract for no more than 1-2 days and to the small intestine for only 2-4 hours. If the drug is not able to dissolve rapidly or is unable to penetrate through the epithelial membrane (e.g., if the drug is highly ionized and polar), time at the site of absorption may be insufficient. In these cases, bioavailability varies greatly and is low. Age, sex, activity, genetic phenotype, stress, disease or previous gastrointestinal surgery may affect the bioavailability of a drug.

Reactions competing with absorption can reduce bioavailability. These reactions include complex formation, hydrolysis by gastric acid or digestive enzymes, binding in the intestinal wall, absorption of other drugs, and metabolism by the luminal microflora.

Assessment of bioavailability from plasma concentration-time data typically involves determining the maximum peak concentration, the time to reach the maximum peak concentration of the plasma drug, and the area under the plasma concentration-time curve (AUC). Plasma drug concentrations increase with the extent of absorption. The peak is reached when the drug elimination rate equals the absorption rate. AUC is the most reliable measure for bioavailability. It is directly proportional to the total amount of unchanged drug that reaches the systemic circulation.

If the plasma level profiles of the drug products are substantially identical (invasive super-compounds), they can be considered bioequivalent in degree (extent) and rate of absorption. Drug products with similar AUC, but different shapes of plasma level curves are to the extent equivalent, but their absorption rate-time profiles are different.

Absorption is carried out by one of three methods, passive diffusion, active transport or assisted transport. Passive diffusion is simply the passage of molecules across the mucosal barrier until the concentration of molecules on both sides of the membrane reaches osmotic equilibrium. In active transport, molecules are actively pumped through the mucosal layer. In assisted transport, a carrier (usually a protein) is required to transport the molecule across the membrane for absorption.

Methotrexate (MTX)

Methotrexate is known under the generic name methotrexate, NSC-740, and is available under the trade name: MEXATE, FOLEX, RHEUMATREX. It can be in the form of tablet, powder and solution. The sodium methotrexate tablets contained 2.5mg methotrexate in 100 vials. Lyophilized preservative-free methotrexate sodium for injection, available in powder form in 20mg,50mg and 1g vials. It can be reconstituted with any sterile free fluid (e.g., water or 0.9% saline) without preservatives. Preservative protected methotrexate sodium for injection, available as 25mg/mL in 2mL (50mg) and 10mL (250mg) vials.

The invention provides equivalent doses of methotrexate sodium in the form of orotate for each formulation and further doses as required. Methotrexate orotate can be administered orally, intravenously, intra-arterially or intrathecally.

A method for reducing side effects of a drug by conversion to an orotate derivative.

Methotrexate enters cells at conventional concentrations through the facilitated transport of folate transporters. At higher concentrations, it enters the cell by passive diffusion. Oral absorption of methotrexate is rapid but poor and unpredictable and decreases with increasing dose and the presence of food. Methotrexate is widespread in body tissues and is approximately 50% bound to plasma proteins (protens). The elimination of methotrexate from plasma has been shown to be age and dose dependent with a half-life of 0.75-2.0 hours, a beta half-life of 3.5-10.0 hours, and a gamma half-life of 27 hours. Most methotrexate (50% -80%) was eliminated unchanged in urine during the first 12 hours. Methotrexate clearance is similar to that of creatinine and therefore should be used with care for patients with renal impairment. Some of the disadvantages and difficulties associated with the use of methotrexate have been addressed by the present invention, which addresses the above problems by structurally modifying sodium methotrexate to sodium methotrexate orotate.

The present invention describes methotrexate orotate and methods for increasing the oral bioavailability of drugs that are poorly absorbed from the gastrointestinal tract by converting the drug into the orotate salt. The present invention describes an increase in the clearance of methotrexate when administered as an orotate derivative compared to the form of the drug itself, thereby reducing the toxic potential of methotrexate at the time of administration and for long periods after healing of the primary cancer or disease. Thus, particularly useful formulations of orotate derivatives of the drugs can provide rapid onset and sustained action with lower doses and reduce drug interactions and side effects. All cited references are incorporated herein in their entirety.

Orotic acid, a free pyrimidine, is important in the synthesis of uridylic acid (UPP), the major pyrimidine nucleic acid. Pyrimidines play an important role in cellular regulation and metabolism. They are substrates for DNA/RNA biosynthesis, regulators of the biosynthesis of some amino acids, and cofactors in the biosynthesis of phospholipids, glycolipids, sugars, and polysaccharides. The classical pathway of nascent pyrimidine biosynthesis ends with the synthesis of UMP. Biochemistry, compiled by Lubert Stryer, W.H.Freeman & Co NY, 4 th edition, 739-762 (1995). 5-fluorouracil has also been reported to be toxic to the liver, as measured by binding in the acid-soluble fraction, RNA and DNA in normal liver tissue in rats. Administration of orotic acid reduced binding in liver and intestinal RNA, thus suggesting that administration of orotic acid reduced 5-FU induced hepatotoxicity. El Hag IA et al, In vivo 1: 309-. The present invention provides drug orotate derivatives that undergo dissociation (dissolution) to release the drug as a charged molecule and free orotate, which reduces drug-induced liver, heart or other tissue toxicity.

The present invention provides methods and compositions for increasing the efficacy of orotate derivatives of the pharmaceutical agents as measured by improvement in bioavailability and clearance of methotrexate where said agents are known to cause toxicity or may cause long term toxicity due to accumulation of the agent in tissues.

6. Examples of the embodiments

Example 1.

Chemical synthesis of sodium methotrexate orotate

Figure 2 shows the synthesis of sodium methotrexate orotate. Orotic acid (1.74g) was treated with a solution of sodium hydroxide (0.45g) in water (100 mL). The mixture was warmed, stirred for 1 hour, and then stored in a refrigerator overnight. Ethanol (etanol) (30mL) was added to the solution and the precipitate was collected by filtration to give sodium orotate as a colourless solid, which was dried in vacuo overnight and used in the next step (1.51 g).

The sodium orotate (0.43G) and methotrexate (1.000G,1 eq.) obtained in the above procedure were suspended in water (60mL) and stirred for 4 hours at 50 ℃ under argon. The solution was cooled in a refrigerator overnight and then the precipitate was collected by filtration. The solid was dried in vacuo for 24 h to give sodium methotrexate orotate (J-1220-10-I,1.27g) as a colorless solid. Mass spectrometry (FIG. 3) and nuclear magnetic resonance (FIG. 4) indicated a structure of methotrexate sodium orotate.

Example 2

Effect of SC DU-145 prostate tumors on treatment with methotrexate and methotrexate orotate Should be taken

The purpose of the experiment was to evaluate the antitumor efficacy of Methotrexate (MTX) and its orotate derivatives (MTX orotate) against Subcutaneous (SC) implantation of DU =145 human prostate tumor xenografts in male athymic NCr-nw/nu mice.

Pharmaceutical formulation-2.7 mg/mL MTX solution (Sigma-Aldrich, catalog No. M9929, lot No. 114Kl 572 on days 15 and 19 and catalog No. a6770, lot No. 21H0324 on days 19,23 and 35) freshly prepared on each day of treatment with a 2% sodium bicarbonate in water for injection (WFI) solution. The 2.7mg/mL solution was diluted to 1.8 and 1.2mg/mL with sodium bicarbonate in water for injection.

MTX orotate (batch J1220-B-I) was synthesized as described in example 1 from MTX (batch 114K 1572). 4.25mg/mL MTX orotate solution was freshly made up to 2.83 and 1.89mg/mL on each day of treatment with 2% bicarbonate in water for injection. The control group was treated with 2% sodium bicarbonate in water for injection. The two compounds and the vehicle were administered to mice, the dose being calculated by accurately weighing the body weight of the individual animals on each day of treatment, and the injection volume was 0.1mL/10g body weight.

7 mice of 10 mice per group were injected intraperitoneally (ip), 1 every 4 days, 3 times in total (q4d x 3, days 15,19,23 and 35) as follows: group 1 used 2% sodium bicarbonate solution and groups 2, 3 and 4 used 27, 18 and 12 mg/kg/dose (dose) of MTX, respectively. Groups 5,6 and 7 were treated with MTX orotate at doses of 42.5, 28.3 and 18.9 mg/kg/dose, respectively (MW =454.4 based on MTX, MW =714.7 for MTX orotate).

Tumor measurement and use of formula LxW²/2=mm³Determining a volume; and assume 1mm³=1mg to calculate the weight. The study was terminated on day 47 after tumor implantation.

As a result:

tumor weight-MTX administered at doses of 27, 18 and 12 mg/kg/dose had no effect on tumor inhibition. Intraperitoneal administration of MTX orotate at equivalent doses of 42.5, 28.3 and 18.9 mg/kg/dose had no effect at 2 low doses, but at the highest dose was less inhibitory of the growth of DU-145 prostate tumor xenografts implanted subcutaneously in male NCr-nu/nu mice. There was no significant difference in tumor growth when MTX orotate was administered intraperitoneally compared to MTX administered intraperitoneally. However the highest dose of MTX orotate showed minor benefit relative to MTX (figure 1).

Example 3

Pharmacokinetic analysis of methotrexate and methotrexate orotate

Pharmacokinetic studies were performed in rats to determine the PK profile and oral bioavailability of methotrexate and compare it to methotrexate orotate. The experiment was performed in male Spagrue-Dawley rats. The compound is administered intravenously (IV; methotrexate 10 mg/kg; methotrexate orotate 15.7 mg/kg; vehicle 1% NaHCO)₃0.9% saline solution: 1mL/kg) or by intragastric administration (PO; methotrexate: 100 mg/kg; methotrexate orotate: 157 mg/kg; the solvent is 1% NaHCO₃0.9% saline solution) and the plasma level of the compound in the plasma is determined at a specific time. The percent oral bioavailability can be determined by calculating the ratio of the area under the curve for the concentration of compound PO to IV (estimated to infinity as is common practice). Of course normalization to different doses of IV versus PO dosing was considered to determine oral bioavailability.

Results

The pharmacokinetic properties of methotrexate and methotrexate orotate for IV and PO treatment are summarized in tables 1 and 2. Images of plasma levels (mean + SEM) versus time are shown for IV and PO methotrexate salts and methotrexate.

The IV pharmacokinetic profiles of methotrexate and methotrexate salts are very similar, with almost identical elimination half-lives. Methotrexate orotate does have a higher apparent volume of distribution and plasma clearance, giving it a slightly lower area under the curve (AUC). Methotrexate administered at high doses (100mg/kg) exhibits poor (6%) oral bioavailability when administered orally, with multiple peaks and troughs, and a multimodal distribution (dispersion) (Goodman and Gilman, the pharmaceutical Basis of Therapeutics, Ed 10,2001). Methotrexate has a prolonged residual time in plasma and a widely varying elimination half-life. The oral bioavailability of methotrexate orotate was 2 times that of methotrexate.

Methotrexate orotate appears to enter the plasma somewhat slower than methotrexate, but its elimination half-life is shorter. Overall, its oral pharmacokinetic profile is very similar to that of methotrexate.

TABLE 1

TABLE 2

These results indicate that methotrexate administration at high doses (100mg/kg) showed poor oral bioavailability (6%) when administered orally, compared to 2-fold bioavailability (14%) with methotrexate orotate. Methotrexate orotate appears to enter the plasma slower than methotrexate, but its elimination half-life is shorter than that of methotrexate. These observations suggest that methotrexate orotate is an improved derivative of methotrexate.

The present invention is not to be limited in scope by the embodiments disclosed in the examples which are intended as illustrations of one aspect of the invention and any method which is functionally equivalent is within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (2)

1. Use of sodium methotrexate orotate for the manufacture of a pharmaceutical composition as an anti-cancer agent or a disease modifying anti-rheumatic agent, said sodium methotrexate orotate having a compound of the formula:

2. a pharmaceutical composition comprising sodium methotrexate orotate.