Derivative compounds of N-6-Trimethy-L-Lysine for therapeutic use
The invention provides derivative compounds of N-6-trimethyl-L-lysine (TML) for potential treatment of disorders resulting from deficiencies in the TML-carnitine pathway. The invention also provides a method of purification of TML and TML derivative compounds. The treatment of conditions of the diseases late infantile neuronal ceroid lipofuscinosis (LINCL) and neuronal ceroid lipofuscinosis (NCL) with TML were shown in the original parent application.
This is a division of application Ser. No. 11/105,165, filed Apr. 13, 2005, which claims priority from provisional application No. 60/601,095 filed on Aug. 12, 2004, and incorporates the subject matter identified as Inventions II and IV in the Requirement for Restriction/Election of Apr. 13, 2007, in the parent application. All materials referenced in the prior provisional and non-provisional applications are hereby incorporated by reference. This includes, but is not limited to, all specifications, drawings, and like materials.
Related divisional applications claiming similar priority include “Method of Synthesis and Purification of N-6-Trimethyl-L-Lysine and Derivative Compounds,” Attorney Docket Attorney Docket No. ChG—00111; “Method of Treating Human Being for a Class of Metabolic Defects and Energy Production Disorders,” Attorney Docket No. ChG—00113; and “Method of Treating a Human Being for a Class of Neurological Defects and Seizure Disorders,” Attorney Docket No. ChG—00114.
All books, manuals, articles, and papers that are cited herein are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION1. Field of Invention
This invention relates to the derivative compounds synthesized from N-6-trimethyl-L-lysine (TML), which may be used in the treatment of deficiencies in the TML-carnitine biochemical pathway.
2. Description of Related Art
All material referenced in the prior provisional and non-provisional applications are hereby incorporated by reference.
In the parent application Ser. No. 11/105,165, allowed on Nov. 17, 2010 to issue as a patent, it was shown by carefully compiled experimental results and other scientific demonstration, that therapeutic use of TML may successfully arrest, and in certain respects, reverse the degeneration associated with the group of progressive neurological diseases called neuronal ceroid lipofuscinoses (NCL).
The modified TML derivatives described should have similar or near-similar results and improved biochemical properties. One of ordinary skill in the art would recognize that structural derivatives of TML, such as those mentioned in this application, may participate in the same biological processes and have the same and improved biochemical properties.
Formulations or encapsulations of the compounds shown in Formulas I-VI may be used for efficient intracellular delivery and as a prodrug of TML to proceed to make endogeneous L-carnitine, and to participate in various metabolic activities in the intermediate steps of L-carnitine biosynthesis pathway. These may be used for better adsorption of modified TML into various tissues such as kidney, liver and brain. The R′, R″, and aminoacyl groups are expected to hydrolyze inside the cellular media with one or more intracellular esterases to release free TML. Intracellular esterases are known to hydrolyze esters (Ghosh, M. and Mitra, A. K., Effects of 5′-Ester Modification on the Physicochemical Properties and Plasma Protein Binding of 5-Iodo-2′-Deoxyuridine. Pharm. Res., 8, 771-775, 1991).
The parent application described the symptoms and common as well as distinct characteristics of the spectrum of NCL group, including the following: Batten Disease, Santavuori disease, Late-Infantile Neuronal Ceroid Lipofuscinoses (LINCL), Speilmeyer-Sjogren disease, Kuf disease, Parry disease, Bernheimer-Seitelberger syndrome, Bielschowsky amaurotic idiocy, Bielschowsky disease, Jansky-Bielschowsky disease, Seitelberger disease, late infantile amaurotic idiocy, late infantile Batten disease, subacute late infantile neuronal ceroid-lipofuscinosis, Zeman-Dyken-Lake-Santavuori-Savukoski disease.
At the genetic level, the neuronal ceroid lipofuscinoses (NCL's) result from mutations in at least eight genes, and these mutations are responsible for causing the various expressions of the neurodegenerative diseases collectively identified as NCLs. A summary background of these mutations and a survey of the background reference literature were given in the parent application. See Table A by Gene Locus.
No generally effective treatment for many of the diseases mentioned above is currently available and these diseases are generally fatal. The line of treatment proposed in the parent application, is based on administering in the form of a therapeutic agent of very high purity, any of the following to a human being in need thereof: (a) N-6-trimethyl-L-lysine, (b) a prodrug thereof, or (c) a pharmaceutically acceptable salt of said N-6-trimethyl-L-lysine or said prodrug.
The one common characteristics in all these disorders has been found to be the accumulation of autofluorescent storage material in all tissues, particularly pronounced in the central nervous system. This characteristic has been tied to the fundamental role of L-carnitine in metabolism, such as the prevention of hyperammonimia, lipid peroxidation and fatty acid metabolism. It has also been found that availability of TML as the rate limiting step in the regulation of feedback inhibition for L-carnitine biosynthesis is crucial to the biosynthesis of L-carnitine. (Schematic 1). (F. M. Vaz and R. J. A. Wandars, Biochem. J., 361, 417-429, 2002).
A summary of the role of carnitine, which was described in greater detail in the parent application, is outlined below:
(a) From a biochemical standpoint, L-carnitine plays an essential role in energy metabolism. In fatty acid metabolism, it serves as shuttle between the cell sap and the mitochondria inner-workings permitting breakdown of the long carbon fragment. A major part of that role is in maintaining a balance between the concentration of a compound called acyl CoA in the cell compartments and in sugar metabolism.
Optimal ATP production from either dietary or stored fatty acids is dependent on L-carnitine. L-Carnitine has several roles, most of which involve conjugation of acyl residues to the b-hydroxyl group of the L-carnitine with subsequent translocation of this complex from one cellular compartment to another.
(b) Defects in fatty acid oxidation are a source of major morbidity and are potentially rapidly fatal. Fatty acid oxidation defects encompass a spectrum of clinical disorders, including recurrent hypoglycemic, hypoketotic encephalopathy or Reye-like syndrome in infancy with secondary seizures and potential developmental delay, progressive lipid storage myopathy, recurrent myoglobinuria, neuropathy, and progressive cardiomyopathy.
(c) Administration of L-carnitine prevents acute ammonia toxicity and enhances the efficacy of ammonia elimination as urea and glutamine. In addition the cytotoxic effects of ammonia, possibly arising from lipid peroxidation, are ameliorated by L-carnitine. These data indicate the feasibility of utilization of L-carnitine in the therapy of human hyperammonemic syndromes.
(d) L-Carnitine deficiency can be defined as a decrease of intracellular L-carnitine is a factor in inhibition of the mitochondrial oxidation of long-chain fatty acids during fasting causes heart or liver failure (which may cause encephalopathy by hypoketonaemia, hypoglycaemia and hyper-ammonium), lower acetylcholine synthesis in the nervous system.
L-carnitine plays a key and critical role in enhancing fat metabolism. Reports attest to the fact that L-carnitine works by transporting fatty acids to be burned for fuel, increasing both energy supply and lean muscle mass. Most found that unless an individual is deficient in L-carnitine, it is an unnecessary ergogenic aid. This contrasts with an apparent need in case of L-carnitine deficiency (e.g., in the case pursued by the inventors of Late Infantile Neuronal Ceroid Lipofuscinosis—one form of Batten Disease), of the correct operation of the endogenous production of L-carnitine. This need was corroborated in the observations of dogs with Batten Disease given exogenous L-Carnitine (Siakotos A. N., Hutchins G. D., Farlow M. R., Katz M. L., European Journal of Paediatric Neurology 5 Suppl A: 151-6, 2001) and those of the parents of the child who was afflicted with LINCL (discussed below). She was given exogenous L-carnitine for over three years without significant metabolic changes or marked outward observations of her condition. It was only the delivery of exogenous TML to the afflicted LINCL child that yielded significant metabolic and outward, observable changes to her condition.
A discussion of the experimental results presented in the parent application showed effectiveness of TML therapy in the amelioration of several symptoms including: hyperammonemia, a glutamine levels, insomnia, “nervousness” or myoclonus.
L-Carnitine may be essential or “conditionally” essential for several groups of people including: normal infants, premature infants, and both children and adults suffering from a variety of genetic, infectious, and injury-related illnesses. For example, some cardiomyopathies which afflict children are due to metabolic errors or deficiencies. There is data that supports treatment of some myocardial dysfunctions with L-carnitine supplementation. (Winter, S., Jue, K., Prochazka J., Francis, P., Hamilton, W., Linn, L., Helton, E. (1995) J. Child Neurol. 10, Supple 2: S45-51.)
For these and other reasons, all of which were described in detail in the parent application, it is believed that TML, or its derivatives which had been proposed in the parent application, can be used for the treatment of a human being diagnosed with one or more of the following: defects in carnitine biosynthesis pathway, inefficiency of endogeneous TML, over-accumulation of TML bound protein at the cellular level, renal failure conditions, hyperammonemic Encepalophathy, over-accumulation of glutamine in the brain, reduced and deficient fatty acid metabolism and shuttling of fatty acid in to mitochondria, insufficient ATP production or subsequent energy production and all the cellular activities associated with this events, defective fatty acid oxidation resulting from carnitine deficiency, hypoglycemia, hypoketotic, encephalopathy, Reye-like syndrome, for recurrent seizures and developmental delay, AIDS or AIDS-like conditions, over-accumulation of lipids causing myopathy, myoglobinuria, neuropathy, cardiomyopathy, ammonia over-production, hyperammonemic syndromes, over accumulation of triacylglycrols, Batten diseases, infantile neuronal lipofuscinoses diseases (Santavvori diseases), Late infantile neuronal lipofuscinoses diseases (Jansky-Bielscowsky), Speilmeyer disease, Sjorgsen disease, Kuf diseases, Parry diseases, Juvenile or adult neuronal lipofuscinoses diseases (“NCL”) disease, lysosomal accumulation of mitochondrial ATP synthase subunit and their by products, ataxia and seizures, various stages of mental impairment, (e.g., learning disability, clumsiness, stumbling, impaired motor skills, and dementia, hyperandrogenism caused by NCL, defective dopamine receptors caused by NCL, epileptic fits, myoclonic epilepsy, Parkinson's disease, and Alzheimer's disease.
In all the conditions as described above, TML derivatives would be equally or more effective after hydrolysis in cellular environment and liberation of free TML.
The experimental results presented during the prosecution of the parent application showed that the progress observed by the medical care-givers to the LINCL child-patient correlated with the administration of high purity TML. See Table B below.
It was also found during the administration of TML to the child that it was essential that high, therapeutic-grade TML be used for treatment purposes.
Therefore, TML derivate compounds were invented and purified by the team of current inventors. These inventions are listed in the present application.
SUMMARY OF THE INVENTIONThe present invention provides derivative compounds of the biologically active TML compound, which may be used to treat various diseases resulting from deficiencies in the TML-carnitine pathway, such as Late Infantile Neuronal Lipofuscinosis (LINCL) or Neuronal Ceroid Lipofuscinosis (NCL).
It is also believed these can be used to treat a human being diagnosed with one or more of the following: defects in carnitine biosynthesis pathway, efficiency of endogeneous TML, over-accumulation of TML bound protein at the cellular level, reduced and deficient fatty acid metabolism and shuttling of fatty acid in to mitochondria, insufficient ATP production or subsequent energy production and all the cellular activities associated with this events, defective fatty acid oxidation resulting from carnitine deficiency, hypoglycemia, hypoketotic, over accumulation of triacylglycrols, lysosomal accumulation of mitochondrial ATP synthase subunit and their by-products. This is described in detail in the allowed parent patent application Ser. No. 11/105,165.
In one embodiment, the invention provides a compound represented by Formula II:
wherein R′ is selected from the group consisting of an alkyl having between 1 and 5 carbon atoms and an aromatic ring.
In one embodiment, the invention provides a compound represented by Formula III:
wherein R″ is an alkyl having 1 to 5 carbon atoms or CH3.
In one embodiment, the invention provides a compound represented by Formula IV:
wherein R″ is an alkyl having between 1 and 5 carbon atoms, or CH3 and R′ is an alkyl having between 1 and 5 carbon atoms or an aromatic ring.
In one embodiment, the invention provides a compound represented by Formula V:
wherein a, a′, b, b′; c, c′, d, d′, e, and e′ are independently selected from H, deuterium, and an alkyl having between 1 and 5 carbon atoms; R′ is selected from the group consisting of H, an alkyl having between 1 and 5 carbon atoms and an aromatic ring; and, and each N is independently selected from nitrogen and N15 labeled nitrogen.
In one embodiment, the invention provides a compound represented by Formula VI:
wherein the a, b, b′, c, c′, d, d′, e, and e′ are independently selected from H, deuterium, and an alkyl having from 1 to 5 carbon atoms, and each N is independently selected from nitrogen and N15 labeled nitrogen.
In another embodiment, the invention provides a method of purifying the TML compound represented by Formula I
to at least 98% purity.
In another embodiment, the invention provides a method of purifying the TML derivate compounds represented by Formulas II, III, IV, V, and VI above to at least 98% purity.
The method of purifying the TML and TML derivative compounds involves the following steps:
-
- 1. running the TML compound or TML derivative through an ion exchange resin column;
- 2. washing the ion exchange resin column with at least 4 times the volume of water as the amount of present TML compound or TML derivative;
- 3. eluting the washed TML compound or TML derivative from the ion exchange resin column to obtain eluted TML or TML derivative; and
- 4. triturating the eluted TML or TML derivative.
In a preferred embodiment, the method further involves:
-
- 5. dissolving the triturated TML into a polar solvent;
- 6. filtering the dissolved TML through a microglass membrane filter; and
- 7. lyophilizing the filtered TML at room temperature.
Although the process was outlined in the original application and is outlined in preceding paragraphs, a detailed summary of the general process is outlined in the Detailed Description. The synthesis was an improvement of the method reported in the literature, Frederic M. Vaz, Bela Melegh, Judith Bene, Dean Cuebas, Douglas A. Gage, Albert Bootsma, Peter Vreken, Albert H. van Gennip, Loran L. Bieber and Arnold J. A. Wanders; Clin. Chem.: 48:6, 826-834, 2002), and in the application as filed. However, the most critical part of the removal of impurities and careful monitoring of the fractions during ion-exchange column purification is documented. After the synthesis of TML from lysine, a careful collection of fractions was carried out and each fraction was monitored by thin layer chromatography, as reported in the parent application. The elution was done initially with deionized Millipore water, followed by 0.5 M aqueous (aq.) ammonia (NH3), followed by 1 M aq. ammonia. The fractions were visualized by ninhydrin test on the thin layer chromatography (TLC) plate. The early and late eluting fractions were found to be of impure or undesired impurity. The purest fractions thus observed were then combined to yield TML of high purity a was reported with no visible impurities by TLC (greater than 98%) and conform to correct mass spectral data and high-resolution proton NMR analysis.
DETAILED DESCRIPTION OF THE INVENTIONAs used herein, the term “derivative” means any of Formulas II-VI. The invention incorporates both TML and TML derivatives. As such, any mention of TML also encapsulates the TML derivative compounds.
The symbol “—” represents a covalent bond.
Reference to a chain, such as an alkyl, can mean either the branched or unbranched chain unless otherwise noted.
An “alkyl”, as used herein, means either a branched or unbranched alkyl chain, and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and the like.
As used herein, “dilute” means ten percent or less in solution.
As used herein “excess” means a stoicheometry greater than 1:1.
A “mild base,” as used herein, means a dilute base.
The phrase “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient(s), and/or salt must be chemically and/or physically compatible with the other ingredients comprising the Formulation, and physiologically compatible with the recipient thereof.
The invention provides a compound represented by Formula II:
wherein R′ is selected from an alkyl having between 1 and 5 carbon atoms and an aromatic ring.
The invention provides a compound represented by Formula III:
wherein R″ is selected from an alkyl having between 1 and 5 carbon atoms, and an aromatic ring.
The invention provides a compound represented by Formula IV:
wherein R′ is selected from an alkyl having between 1 and 5 carbon atoms, and CH3 and R″ is selected from an alkyl having between 1 and 5 carbon atoms, and an aromatic ring.
The invention provides a compound represented by Formula V:
wherein a, a′, b, b′, c, c′, d, d′, e, and e′ are independently selected from H, deuterium, and an alkyl having between 1 and 5 carbon atoms and R′ is selected from H, an alkyl having between 1 and 5 carbon atoms, or an aromatic ring.
In a preferred embodiment, each N is independently selected from nitrogen or N15 labeled nitrogen.
Improved Method of Synthesis and Purification of TML and TML Derivatives
Starting Raw Material: L-lysine HCl (Sigma-Aldrich, St. Louis, Mo.), dimethylsulfate (99.99%) Sigma-Aldrich, St. Louis, Mo.), alkaline copper carbonate (Sigma-Aldrich, St. Louis, Mo.), double distilled water (highly purified), Whatman 3MM Chromatography blotting paper (Whatman Inc., Florham Park, N.J.), sodium hydroxide (NaOH, 99%+) (Sigma-Aldrich, St. Louis, Mo.), Dowex SOWX8 ion exchange column (The Dow Chemical Company, Midland, Mich.).
L-lysine HCL (50 gm; 0.274 mol)) was dissolved in distilled water (500 mL) and copper carbonate basic (72 gm; 0.326 mol) was added. The mixture was boiled at 85 centigrade for 10 minutes.
The reaction mixture was cooled to room temperature and filtered with Whatman 3MM paper. The clear filtrate was mixed with dimethylsulfate, 100 mL (1.055 mol) at room temperature, after which 325 mL of aq. sodiumhydroxide solution (10% aq.; 1.055 mol; w/v, in dd water) was added drop-wise during 30 minutes, then stirred at room temperature for 60 min.
A 17″height×2″dia Dowex5OWx8 ion exchange column (H+ form) was washed with de-ionized water prior to the addition of the TML solution.
The solution containing the TML was then added to Dowex5OWx8 column.
The column was copiously washed with 500 mL of distilled water. This process was repeated with 1700 mL water and 1000 mL and 700 mL fractions were collected. The collection and monitoring of these fractions were accomplished by thing layer chromatography (TLC) and ninhydrin color tests.
Subsequently, 2M ammonium hydroxide solution was run and 8 fractions (each fraction 50 mL, followed by 100 mL) were collected. TLC analysis was performed on all the fractions (tic system:MeOH:water:Aceticacid::80:10:10). These fractions are detailed in Table C below.
The eight fractions were combined and evaporated to yield an oil. The oil was subsequently lyophilized at room temperature to yield a solid.
The solid was triturated in acetonitrile and filtered and washed with acetonitrile again.
The solid was dissolved in methanol/water (95:5::Methanol:ddWater) and filtered with glass micro filter paper and the filtrate was evaporated and lyophilized.
Large-Scale Purification of TML
A larger scale synthesis has been achieved, which is further amenable to large-scale production of TML. The larger scale synthesis of TML incorporating step of final clean up to achieve purity of at least 98% or greater, and free of foreign materials, has been demonstrated. The literature procedure does not teach synthesis of high purity TML or TML derivative, which could be applicable to pharmaceutical-grade product. Thus, the purification steps that allow larger scale synthesis can be described as follows:
-
- 1. Running crude TML or TML derivative through an ion exchange resin column;
- 2. Washing the ion exchange resin column with at least 4 times the volume of water as the amount of present crude TML or TML derivative;
- 3. Eluting the washed TML or TML derivative from the ion exchange resin column;
- 4. Freezing the eluted solution and then lyophilize at room temperature to prevent or minimize any decomposition of obtained TML or TML derivative; and
- 5. Triturating the lyophilized solid TML or TML derivative.
In another embodiment, the following steps may include:
-
- 5. Dissolving the triturated TML or TML derivative into a polar solvent;
- 6. Filtering the dissolved TML or TML derivative through a microglass membrane filter; and
- 7. Lyophilizing the filtered TML or TML derivative at room temperature.
This process is an improvement of what is disclosed in Frederic M. Vaz, Bela Melegh, Judit Bene, Dean Cuebas, Douglas A. Gage, Albert Bootsma, Peter Vreken, Albert H. Van Gennip, Loran L. Bieber And Ronald J. A. Wanders, Clin. Chem. 48:6, 826-834, 2002.
Quality Control
The following quality control parameters were obtained:
-
- A. TLC' Samples 1 and 2 were purified TML made according to the invention. Sample 3 was a reference TML purchased from Sigma-Aldrich. The tic plates were Baker-flex silica gel 1B-F. TLC Purity' was greater than 99%, and the spots were observed after staining the spot with ninhydrin (10% in methnol) (FIG. 1).
- B. 1H NMR: The 1H NMR (Proton in D20) was run on 300 MHz; 1.3932 ppm (methylene at C-2; 2H, broad singlet), 1.6643 ppm (methylene at C-3 2H, broad, multiplet); 2.13 ppm (methylene at C-4; 2H, broad singlet), 3.2928 ppm (methylene proton at C-5, 2H; triplet); 3.2167 ppm (alpha H; 1H; triplet); 3.0798 ppm (trimethyl H's; 9H) (FIGS. 2a and 2b).
- C. Mass Spectrum: Chemical Formula C9H20N 202, Molecular weight; 189.28. Four major fragmentation peaks were observed in positive mode; m/e 189.3, m/e 211.2 (+Na ion), m/e 377.5 (possibly dimer formation) and m/e 399.5 (possibly Na+ ion addition on dimmer) (FIG. 3).
Salt Formation
The TML synthesized (as described above) had no external salt. The carboxylic group (which is negatively charged) and the trimethyl group (which is positively charged) form an internal salt. The alpha amino group picks up the proton from the ionized carboxylic group. The molecular weight of this TML is 188.3 From our Mass Spectral analysis (positive ion) we get the molecular ion peak at 189.28 (One extra mass in positive ion is proton adding from matrix). This data confirms MW of 188.3.
The skilled artisan will understand that TML can exist as an external salt as well, such as a potassium salt.
Claims
1. A compound represented by Formula I
- with a pharmaceutically acceptable purity level.
2. The compound of claim 1, wherein said purity level is at least 98%.
3. A compound represented by Formula II
- wherein R′ is selected from the group consisting of (an alkyl having between 1 and 5 carbon atoms, and an aromatic ring).
4. The compound of claim 3, wherein the compound is at least 98% pure.
5. A compound represented by Formula III
- wherein R″ is selected from the group consisting of (an alkyl having between 1 and 5 carbon atoms, CH3, and an aromatic ring).
6. The compound of claim 5, wherein the compound is at least 98% pure.
7. A compound represented by Formula IV
- wherein R′ is an alkyl having between 1 and 5 carbon atoms, or CH3, and wherein R″ is an alkyl having between 1 and 5 carbon atoms or an aromatic ring.
8. The compound of claim 7, wherein the compound is at least 98% pure.
9. A compound represented by Formula V
- wherein a, a′, b, b′; c, c′, d, d′, e, and e′ are independently selected from the group consisting of (H, deuterium, and an alkyl having between 1 and 5 carbon atoms); R′ is selected from the group consisting of (H, an alkyl having between 1 and 5 carbon atoms and an aromatic ring); and each N is independently selected from nitrogen and N15 labeled nitrogen.
10. The compound of claim 9, wherein the compound is at least 98% pure.
11. A compound represented by Formula VI
- wherein the a, b, b′, c, c′, d, d′, e, and e′ are independently selected from the group consisting of (H, deuterium, and an alkyl having from 1 to 5 carbon atoms), and each N is independently selected from nitrogen and N15 labeled nitrogen.
12. The compound of claim 11, wherein the compound is at least 98% pure.
Type: Application
Filed: Mar 9, 2011
Publication Date: Jul 7, 2011
Inventors: Suresh C. Srivastava (Burlington, MA), Sant K. Srivastay (Burlington, MA), Stanley J. Szymanski, JR. (Sewickley, PA)
Application Number: 12/932,940
International Classification: C07C 229/26 (20060101);
