Treating copper disorders

Abstract

The present disclosure relates to compositions containing fixed doses of triethylenetetramine disuccinate and methods for their use in the prevention and treatment of copper-related diseases, disorders and conditions.One object of the present invention is to provide a dose and dosage form of triethylenetetramine disuccinate that is equivalent in human exposure / bioavailability to previously approved forms of triethylenetetramine, such as for the treatment of Wilson's disease.Triethylenetetramine disuccinate is an alternative and superior salt form of trientine.It is more stable, has better distribution and good activity.

Description

[Technical field]

[0001] Field The present invention relates to fixed-dose triethylenetetramine disuccinate formulations and their use in the treatment, prevention or amelioration of diseases, conditions and disorders treatable with copper chelators.

[0002] Incorporation by Reference All U.S. patents, U.S. patent application publications, foreign patents, foreign and PCT published applications, papers and other documents, references and publications noted herein, and all that are listed as citations in any patent(s) issuing therefrom, are hereby incorporated herein by reference in their entireties. The incorporated information is equivalent to a part of this application as if all text and other content were repeated within this application and treated as part of the text and content of this application as filed. [Background technology]

[0003] background The following contains information that may be useful in understanding the present invention. This is not an admission that any of the information is prior art or relevant to the presently described or claimed invention, or that any publication or document specifically or implicitly referenced is prior art or a reference that may be used in assessing the patentability of the described or claimed invention.

[0004] Copper is an essential trace element involved in numerous biological processes in living cells. Analysis of the human proteome has identified 54 copper-binding proteins, of which 12 are copper transporters, roughly half are enzymes, and one (antioxidant 1 copper chaperone, ATOX1) is a transcription factor. Copper-binding proteins include cytochrome oxidase, copper-zinc-superoxide dismutase, lysyl oxidase, tyrosinase, and dopamine-beta-monooxygenase, which are involved in crucial biological processes such as mitochondrial respiration, antioxidant defense, extracellular matrix cross-linking, pigmentation, and neurotransmitter biosynthesis, respectively. A list of copper-requiring enzymes, with special emphasis on enzymes involved in inherited disorders of copper homeostasis, can be found in Horn N., et al. Chelating principles in Menkes and Wilson diseases: Choosing the right compounds in the right combinations at the right time. J. Inorg. Biochem. 2019;190:98-112. The majority of copper in the body is located in organs with high metabolic activity, such as the liver, kidneys, heart and brain. Approximately 5% of the total copper in serum is bound to ceruloplasmin, of which up to 95% is bound to ceruloplasmin.

[0005] Unbound copper behaves as a powerful antioxidant and catalyzes the formation of highly reactive hydroxyl radicals, leading to DNA, protein and lipid damage. Therefore, cellular copper concentrations need to be finely regulated by complex homeostatic mechanisms of absorption, excretion and bioavailability.

[0006] Once absorbed in the gastrointestinal tract, copper reaches the blood where it is mostly bound to ceruloplasmin. Copper transporter 1 (CTR1, SLC31A1), located in the plasma membrane, is the main copper import protein. Within the cell, various metallochaperones receive and deliver copper to specific locations. ATPase copper transport alpha (ATP7A) and ATPase copper transport beta (ATP7B) play central roles in copper homeostasis and are required for copper delivery to the secretory pathway and for the excretion of excess copper from the cell. Dysregulation of copper homeostasis has been implicated in the pathogenesis of several diseases. See Brewer GJ, Copper in medicine. Curr. Opin. Chem. Biol. 2003;7:207-212;Bandmann O., et al. Wilson's disease and other neurological copper disorders. Lancet Neurol. 2015;14:103-113.

[0007] Chelators are chemical compounds that can selectively bind atoms / ions due to their structure, particularly with the formation of complex ring structures that are generally stable. Most copper overload toxicity and clinically significant copper deficiency are associated with genetic defects of copper transporters, such as Wilson's disease (copper overload) and Menkes disease (copper deficiency). However, copper is an essential catalytic cofactor in redox biochemistry, and copper dyshomeostasis leading to its unpaired distribution has been implicated in several disorders, including diabetes, neuropathy, and cancer.

[0008] Wilson's disease is an autosomal recessive disorder caused by mutations in both copies of the ATP7B gene [18,24] that lead to excess copper in the body and is characterized by a series of clinical signs, including liver failure, tremors and other neurological symptoms. Therefore, to manage increased copper levels, Wilson's disease patients have been treated with different copper-reducing agents, including D-penicillamine (DPA), trientine hydrochloride and tetrathiomolybdate. The goal of copper chelation therapy for Wilson's disease is to remove copper that accumulates in tissues (decopperization phase) and prevent reaccumulation (maintenance phase). DPA was introduced in 1956. It is a dimethylated cysteine ​​that mobilizes tissue copper stores and promotes urinary excretion of excess copper. DPA (sold as Curprimine® and Depen®) is the first-line therapy used to treat Wilson's disease (an inherited condition that causes copper to precipitate in the body, resulting in severe symptoms) and cystinuria (an inherited condition that can lead to kidney stones). However, it has a number of severe side effects, including stomach / abdominal pain, nausea, vomiting, loss of appetite, diarrhea, decreased sense of taste, itching or rash, tinnitus (ringing in the ears), mouth sores, poor wound healing, and increased wrinkling of the skin. In addition, this restoration of copper balance is not followed by improvement of neurological symptoms. Instead, DPA treatment may cause the patient's neurological symptoms to worsen, which is thought to be due to a supposed increase in copper levels in the brain. Brewer GJ, et al. Worsening of neurologic syndrome in patients with Wilson's disease with initial penicillamine therapy. Arch. Neurol. 1987;44:490-493. Furthermore, the use of DPA has been limited by hematological and nephrotoxicity (Brewer GJ, Yuzbasiyan-Gurkan V. Wilson disease. Med. (Baltim.) 1992;71:139-164), and in the 1980s alternative anti-copper agents such as trientine came into use.Walshe JM Treatment of Wilson's disease with trientine (triethylene tetramine) dihydrochloride. Lancet. 1982;1:643-647. Triethylenetetramine (TETA), also known as trientine, has been specifically introduced as a hydrochloride salt for the treatment of Wilson's disease patients exhibiting DPA intolerance. Ibid. Trientine dihydrochloride has an improved safety profile compared to DPA, but has a lower copper uremic effect.

[0009] Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive memory loss, language impairment, and disorientation along with recognizable pathological markers including senile plaques and neurofibrillary tangles. From a molecular perspective, AD is characterized by extracellular deposits of β-amyloid protein that accumulate in the brain, eventually leading to neuronal loss. β-amyloid condensation in plaques is associated with high concentrations of Cu(II) and Zn(II) in neocortical tissue, thus suggesting a role for metal imbalance in the development and / or progression of AD. Moreover, β-amyloid binds and reduces Cu(II) to Cu(I), inducing electron transfer to molecular oxygen with the formation of H2O2, leading to apoptotic cell death and other negative outcomes potentially related to oxidative stress. Copper levels in the cerebrospinal fluid of AD patients are 2.2-fold higher than in controls, and increased levels of ceruloplasmin have been observed in the brain and cerebrospinal fluid. Basun H., et al. Metals and trace elements in plasma and cerebrospinal fluid in normal aging and Alzheimer's disease. J. Neural. Transm. Park Dis. Dement. Sect. 1991;3:231-258. Postmortem analyses performed on transgenic mouse models of AD demonstrated that metal chelators can attenuate β-amyloid protein excess.Cherny RA, et al. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001;30:665-676;Dedeoglu A., et al. Preliminary studies of a novel bifunctional metal chelator targeting Alzheimer's amyloidogenesis. Exp. Gerontol. 2004;39:1641-1649.Clinical trials in AD patients have been conducted using D-penicillamine. Squitti R., et al. D-penicillamine reduces serum oxidative stress in Alzheimer's disease patients. Eur. J. Clin. Investig. 2002;32:51-59.

[0010] Altered copper homeostasis also plays a role in Parkinson's disease (PD). PD is one of the most common neurodegenerative disorders, affecting approximately 2-3% of the population over a 65-year period. The main hallmark of PD is represented by a typical dopamine-producing neuronal cell loss in the substantia nigra, usually accompanied by α-synuclein aggregates called Lewy bodies, leading to the characteristic symptoms of bradykinesia, muscle rigidity, tremor and other non-motor symptoms. Copper binding to α-synuclein protein is a key event in the pathogenesis of PD, inducing protein fibrillation and increased oxidative stress. Moreover, copper binding to ceruloplasmin is reduced in PD patients, leading to increased levels of free copper associated with oxidative stress and neurodegeneration. Ajsuvakova OP, et al. Assessment of copper, iron, zinc and manganese status and speciation in patients with Parkinson's disease: A pilot study. J. Trace Elem. Med. Biol. 2019:126423.Thus, altered copper homeostasis plays a role in PD. Bjorklund G., et al. Metals and Parkinson's disease: Mechanisms and biochemical processes. Curr. Med. Chem. 2018;25:2198-2214.Copper attenuation therapy in PD was recently reviewed by Tosato, M and Di Marco, V, in Metal chelation therapy and Parkinson's disease: A critical review on the thermodynamics of complex formation between relevant metal ions and promising or established drugs. Biomolecules. 2019;9:269.

[0011] Idiopathic pulmonary fibrosis (IPF) is a form of chronic lung disease that usually affects people between the ages of 50 and 80, in which fibrosis progressively consolidates in the lungs, leading to impaired lung function. Wide heterogeneity in clinical signs and symptoms leads to high variability in therapy course and response. The exact mechanism of IPF pathogenesis remains to be elucidated, but different biological and molecular factors may be involved, including lysyl oxidases, a group of copper-dependent enzymes involved in the covalent cross-linking of type I collagen. In particular, LOXL2 may represent a potential therapeutic target that is profibrotic and highly expressed in IPF lung biopsies. See, for example, Chen L., et al. LOX / LOXL in pulmonary fibrosis: Potential therapeutic targets. J. Drug Target. 2019;27:790-796. A study conducted in 2003 demonstrated that administration of the copper chelator tetrathiomolybdate induced a reduction in serum ceruloplasmin in a mouse model of bleomycin-induced IPF that led to a corresponding reduction in pulmonary fibrosis, paving the way for clinical trials in IPF patients unresponsive to other therapies. Brewer GJ, et al. Tetrathiomolybdate therapy protects against bleomycin-induced pulmonary fibrosis in mice. J. Lab. Clin. Med. 2003;141:210-216.Copper chelation with tetrathiomolybdate has been shown to exert its beneficial effects in IPF by reducing collagen I expression and accumulation and by acting on the expression of copper-dependent lysyl oxidase. Ovet H., Oztay F. The copper chelator tetrathiomolybdate regressed bleomycin-induced pulmonary fibrosis in mice, by reducing lysyl oxidase expressions. Biol. Trace Elem. Res. 2014;162:189-199.

[0012] Diabetes mellitus (DM) is a heterogeneous group of metabolic diseases primarily characterized by a hyperglycemic state accompanied by defects in insulin secretion or action. There are three main types of diabetes: type I, type II and gestational diabetes. DM patients have higher levels of copper in plasma or serum compared to healthy individuals. Qiu Q., et al. Copper in diabetes mellitus: A meta-analysis and systematic review of plasma and serum studies. Biol. Trace Elem. Res. 2017;177:53-63;Li P., et al. Association between plasma concentration of copper and gestational diabetes mellitus. Clin. Nutr. 2019;38:2922-2927.DM pathogenesis and progression have been linked to increased oxidative stress and to imbalance of several metals, including copper. Zheng Y., et al. The role of zinc, copper and iron in the pathogenesis of diabetes and diabetic complications: Therapeutic effects by chelators. Hemoglobin. 2008;32:135-145.Transition between Cu(I) and Cu(II) leads to the generation of reactive oxygen species (ROS) and consequent lipid peroxidation, DNA damage leading to cell death. Copper homeostasis using copper chelators may represent a strategy for diabetes treatment. See Lowe J., et al. Dissecting copper homeostasis in diabetes mellitus. Iubmb Life. 2017;69:255-262.

[0013] Importantly, a series of preclinical and clinical studies using the salt triethylenetetramine hydrochloride demonstrated the potential of the copper chelator trientine to reduce some of the clinical and pathological consequences of diabetes, such as heart failure. Cooper GJ, Phillips AR, Choong SY, Leonard BL, Crossman DJ, Brunton DH, Saafi L., Dissanayake AM, Cowan BR, Young AA, et al. Regeneration of the heart in diabetes by selective copper chelation. Diabetes. 2004;53:2501-2508.In addition, treatment with the copper chelator tetrathiomolybdate was described to promote a significant reduction in insulin resistance in a mouse model of type 2 diabetes. Tanaka A., et al. Role of copper ion in the pathogenesis of type 2 diabetes. Endocr. J. 2009;56:699-706.

[0014] Copper has also been linked to cancer, with studies showing that it is important to maintain and improve the quality of life of patients with laryngeal squamous cell carcinoma (de Jorge FB, et al. Biochemical studies on copper, copper oxidase, magnesium, sulfur, calcium and phosphorus in cancer of the larynx. Acta Otolaryngol. 1966;61:454-458), non-Hodgkin's lymphoma (Shah-Reddy I., et al. Serum copper levels in non-Hodgkin's lymphoma. Cancer. 1980;45:2156-2159), multiple myeloma (Khadem-Ansari MH, et al. Copper and zinc in stage I multiple myeloma: Relation with ceruloplasmin, lipid peroxidation, and superoxide dismutase activity. Horm. Mol. Biol. Clin. Investig. 2018:37), and chronic lymphocytic leukemia (Kaiafa GD, et al. Copper levels in patients with hematological malignancies). malignancies. Eur. J. Intern. Med. 2012;23:738-741), hepatocellular carcinoma (Fang AP, et al. Serum copper and zinc levels at diagnosis and hepatocellular carcinoma survival in the Guangdong Liver Cancer Cohort. Int. J. Cancer. 2019;144:2823-2832), gynecological cancer (Zowczak M., et al. Analysis of serum copper and zinc concentrations in cancer patients. Biol. Trace Elem. Res. 2001;82:1-8), colorectal (Gupta SK, et al.Serum and tissue trace elements in colorectal cancer. J. Surg. Oncol. 1993;52:172-175), lung (Zhang X., Yang Q. Association between serum copper levels and lung cancer risk: A meta-analysis. J. Int. Med. Res. 2018;46:4863-4873), primary brain (Turecky L., et al. Serum ceruloplasmin and copper levels in patients with primary brain tumors. Klin. Wochenschr. 1984;62:187-189) and breast (Dabek JT, et al. Evidence for increased non-ceruloplasmin copper in early-stage human breast cancer serum. Nutr. Cancer. 1992;17:195-201) cancers were analyzed using serum from patients with different types of cancer (Coates RJ, et al. Cancer risk in relation to serum copper levels. Cancer Res. Increased copper content has been determined in tumor tissue samples (Margalioth EJ, et al. Copper and zinc levels in normal and malignant tissues. Cancer. 1989;49:4353-4356) and tissue samples (Margalioth EJ, et al. Copper and zinc levels in normal and malignant tissues. Cancer. 1983;52:868-872). Serum copper levels return to normal upon successful surgical removal or remission of the tumor. In addition, gene expression analysis has demonstrated that copper homeostasis is a marker of tumor remodeling in the colorectum (Barresi V., et al. Transcriptome analysis of copper homeostasis genes reveals coordinated upregulation of SLC31A1,SCO1, and COX11 in colorectal cancer. FEBS Open Bio.2016;6:794-806) and breast cancer (Nagaraja GM, et al. Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: Comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene. 2006;25:2328-2338), revealing multiple alterations of various copper-binding or copper-sensitive proteins, suggesting that dysregulation of copper homeostasis may contribute to the pathogenesis, development and metastasis of cancer.

[0015] Collectively, these indications provide support for copper chelation as a strategy for cancer therapy. See Goodman VL, et al. Copper deficiency as an anti-cancer strategy. Endocr. Relat. Cancer. 2004;11:255-263;Lopez J., et al. Copper depletion as a therapeutic strategy in cancer. Met. Ions Life Sci. 2019:19;Garber K. Cancer's copper connections. Science. 2015;349:129. Angiogenesis is essential to support cancer cell growth and tumor metastasis. The mechanism of cancer inhibition by copper chelators is generally attributed to their inhibitory effect on tumor angiogenesis. Goodman VL, et al., supra. Indeed, copper chelators used to treat Wilson's disease, such as trientine, have demonstrated chemotherapeutic properties in experimental preclinical cancer models, leading to several clinical trials. These studies have demonstrated that copper chelation therapy is generally well tolerated because the copper chelators act selectively on cancer cells, increase their copper content, and exert little toxicity on normal cells.Denoyer D., et al. Targeting copper in cancer therapy: 'Copper That Cancer' Metallomics. 2015;7:1459-1476;Gupte A., Mumper RJ Elevated copper and oxidative stress in cancer cells as a target for cancer treatment. Cancer Treat. Rev. 2009;35:32-46.

[0016] It should also be noted that increased efficacy of radiation therapy against primary tumors with reduced side effects can be achieved when combined with antiangiogenic agents, and additive effects of radiation therapy and copper chelation therapy have been observed in a highly metastatic Lewis lung carcinoma mouse tumor model. Khan MK, et al. Combination tetrathiomolybdate and radiation therapy in a mouse model of head and neck squamous cell carcinoma. Arch. Otolaryngol. Head Neck Surg. 2006;132:333-338.

[0017] Copper chelation and immunotherapy combination strategies have also been proposed and evaluated for use with several immunotherapy strategies, including monoclonal antibodies, immune cell activators, immune checkpoint inhibitors, and oncolytic viral vectors. For example, nanoparticle-based copper chelation and immune stimulation strategies have been shown to effectively inhibit breast tumor growth and metastasis in experimental models both in vitro and in vivo. Zhou P., et al. Multifunctional nanoparticles based on a polymeric copper chelator for combination treatment of metastatic breast cancer. Biomaterials. 2019;195:86-99. With regard to immune checkpoint inhibitors, a positive correlation between the copper transport protein CTR1 and programmed cell death protein 1 (PD-1) expression was observed in neuroblastoma and glioblastoma tumor cells. Copper chelation reduces PD-L1 expression and promotes a significant increase in tumor-infiltrating lymphocytes in a syngeneic mouse model of neuroblastoma. Voli F., et al. Copper homeostasis: A new player in anti-tumor immune response. Cancer Res. 2019:79.Thus, copper chelation therapy can also promote the efficacy of programmed cell death protein 1 (PD-1) / programmed cell death ligand 1 (PD-L1)-based immunotherapy. Oncolytic vectors selectively replicate and promote the lysis of cancer cells to attract the patient's immune system against tumor antigens. Changes in the tumor microenvironment in response to induced tumor lysis may limit the efficacy of oncolytic virotherapy. It has therefore been hypothesized that the combination of copper chelation therapy, which affects both the tumor microenvironment and angiogenesis, may promote the efficacy of oncolytic virotherapy. In addition, serum copper levels have a detrimental effect on herpes virus infection. Based on these premises, concomitant copper chelation therapy has been described to increase the antitumor efficacy of oncolytic viruses derived from herpes simplex viruses.Yoo J.Y., et al. Copper chelation enhances antitumor efficacy and systemic delivery of oncolytic HSV. Clin. Cancer Res. 2012;18:4931-4941;Yoo J.Y., et al. ATN-224 [Bis-Choline Tetrathiomolybdate] enhances antitumor efficacy of oncolytic herpes virus against both local and metastatic head and neck squamous cell carcinoma. Mol. Oncolytics. 2015;2:15008。

[0018] Autophagy has a complex role in cancer pathogenesis, progression and response to therapy. Autophagy inhibition has emerged as an effective approach for tumor therapy, especially in cancers with increased levels of basal autophagy. Amaravadi RK, et al. Targeting autophagy in cancer: Recent advances and future directions. Cancer Discov. 2019;9:1167-1181.Different lines of evidence suggest that increased copper content activates a set of autophagy-related genes. Polishchuk EV, et al Activation of autophagy, observed in liver tissues from patients with Wilson disease and from ATP7B-deficient animals, protects hepatocytes from copper-induced apoptosis. Gastroenterology. 2019;156:1173-1189.e1175.Accordingly, copper chelation was shown to inhibit Unc-51-like autophagy activating kinases 1 and 2 (Ulk1 / 2) in lung adenocarcinoma cells. Tsang T., et al. Copper is an essential regulator of the autophagic kinases ULK1 / 2 to drive lung adenocarcinoma. bioRxiv. 2019:816587.Recently, the combination of copper chelation and autophagy inhibition with chloroquine was evaluated to promote pancreatic cancer cell death. Yu Z., et al. Blockage of SLC31A1-dependent copper absorption increases pancreatic cancer cell autophagy to resist cell death. Cell Prolif. 2019;52:e12568.

[0019] In addition to the use of triethylenetetramine dihydrochloride as a therapy for treating individuals with Wilson's disease, it has also been reported to be used to treat individuals with primary biliary cirrhosis. See, e.g., Epstein, O. et al., Gastroenterology 78(6):1442-45 (1980). In addition, trientine has been tested for inhibition of hepatitis and spontaneous development of liver tumors in rats. See, e.g., Sone, H., et al., Hepatology 23:764-70 (1996). Issued U.S. patents describe the use of copper-binding compounds in the treatment of various disorders, including the treatment of diabetes mellitus and its complications, including, for example, diabetic cardiomyopathy. See, U.S. Patent Nos. 6,897,243, 6,610,693, and 6,348,465. Certain copper chelators have also been described for use in treating certain disorders, including cardiovascular, glucose, and vascular disorders.Prior art teachings regarding copper chelators are found in, for example, U.S. Pat. No. 10,543,178 (use of the succinic acid addition salt of triethylenetetramine to treat diabetic neuropathy), U.S. Pat. No. 9,993,443 (use of the succinic acid addition salt of triethylenetetramine to treat tissue damage associated with specific cardiac, glucose-related and vascular disorders), U.S. Pat. No. 8,987,244 (use of various chelators including trientine, 2,2,2 tetramine tetrahydrochloride and 2,3,2 tetramine tetrahydrochloride to reduce copper(II) levels in patients with tissue damage in myocardial, renal, ocular, nervous and vascular tissues), U.S. Pat. No. 8,563,538 (method of treating heart failure in non-diabetic human subjects), and U.S. Pat. No. 8,563,538 (method of treating heart failure in non-diabetic human subjects). No. 8,034,799 (methods of treating heart failure in non-diabetic human subjects with agents capable of reducing copper levels, e.g., copper(II), including trientine and copper chelators such as 2,3,2 tetramine, DPA, N-acetylpenicillamine, trithiomolybdate and tetrathiomolybdate), and U.S. Pat. No. 7,928,094 (use of triethylenetetramine dihydrochloride to treat one or more conditions associated with the long-term complications of diabetes). In summary, an increasing number of in vitro and in vivo studies suggest that copper-involved mechanisms represent potential therapeutic targets for a number of different pathologies. Systemic or tissue-specific states of elevated copper can arise via multiple mechanisms in addition to the genetic defects of copper metabolism observed in Wilson's disease. Dysregulation of copper homeostasis has been observed in a wide range of neurological, fibrotic pulmonary and vascular diseases, as well as in different types of cancer. Copper imbalance in Wilson's disease has been thoroughly investigated, leading to the introduction of copper chelation therapy as a major therapeutic tool that has significantly reduced morbidity, making Wilson's disease a treatable disorder. As noted, treatment with first-line DPA therapy has a number of side effects, including stomach / abdominal pain, nausea, vomiting, loss of appetite, diarrhea, decreased sense of taste, itching or rash, tinnitus (ringing in the ears), stomatitis, poor wound healing, and increased wrinkling of the skin. Trientine hydrochloride (sold as a dihydrochloride salt under the trade names Syprine® and Clovique®) chelates copper and is used to treat Wilson's disease in people who cannot take penicillamine. Syprine® was approved by the FDA in 1985 as a second-line treatment for Wilson's disease. Common side effects of trientine dihydrochloride include skin rash, muscle cramps or contractions, heartburn, stomach pain, loss of appetite, and peeling, cracking or thickening of the skin. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] U.S. Patent No. 6,897,243 [Patent Document 2] U.S. Patent No. 6,610,693 [Patent Document 3] U.S. Patent No. 6,348,465 [Patent Document 4] U.S. Pat. No. 10,543,178 [Patent Document 5] U.S. Pat. No. 9,993,443 [Patent Document 6] U.S. Pat. No. 8,987,244 [Patent Document 7] U.S. Pat. No. 8,563,538 [Patent Document 8] U.S. Pat. No. 8,034,799 [Patent Document 9] U.S. Patent No. 7,928,094 [Non-patent literature]

[0021] [Non-Patent Document 1] Horn N., et al. Chelating principles in Menkes and Wilson diseases: Choosing the right compounds in the right combinations at the right time. J. Inorg. Biochem. 2019;190:98-112 [Non-Patent Document 2] Brewer GJ, Copper in medicine. Curr. Opin. Chem. Biol. 2003;7:207-212 [Non-Patent Document 3] Bandmann O., et al. Wilson's disease and other neurological copper disorders. Lancet Neurol. 2015;14:103-113 [Non-Patent Document 4] Brewer GJ, et al. Worsening of neurologic syndrome in patients with Wilson's disease with initial penicillamine therapy. Arch. Neurol. 1987;44:490-493 [Non-Patent Document 5] Brewer GJ, Yuzbasiyan-Gurkan V. Wilson disease. Med. (Baltim.) 1992;71:139-164 [Non-Patent Document 6] Walshe JM Treatment of Wilson's disease with trientine (triethylene tetramine) dihydrochloride. Lancet. 1982;1:643-647 [Non-Patent Document 7] Basun H., et al. Metals and trace elements in plasma and cerebrospinal fluid in normal aging and Alzheimer's disease. J. Neural. Transm. Park Dis. Dement. Sect. 1991;3:231-258 [Non-Patent Document 8] Cherny RA, et al. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001;30:665-676 [Non-Patent Document 9] Dedeoglu A., et al. Preliminary studies of a novel bifunctional metal chelator targeting Alzheimer's amyloidogenesis. Exp. Gerontol. 2004;39:1641-1649 [Non-Patent Document 10] Squitti R., et al. D-penicillamine reduces serum oxidative stress in Alzheimer's disease patients. Eur. J. Clin. Investig. 2002;32:51-59 [Non-Patent Document 11] Ajsuvakova OP, et al. Assessment of copper, iron, zinc and manganese status and speciation in patients with Parkinson's disease: A pilot study. J. Trace Elem. Med. Biol. 2019:126423 [Non-Patent Document 12] Bjorklund G., et al. Metals and Parkinson's disease: Mechanisms and biochemical processes. Curr. Med. Chem. 2018;25:2198-2214 [Non-Patent Document 13] Tosato, M and Di Marco, V, in Metal chelation therapy and Parkinson's disease: A critical review on the thermodynamics of complex formation between relevant metal ions and promising or established drugs. Biomolecules. 2019;9:269 [Non-Patent Document 14] Chen L., et al. LOX / LOXL in pulmonary fibrosis: Potential therapeutic targets. J. Drug Target. 2019;27:790-796 [Non-Patent Document 15] Brewer GJ, et al. Tetrathiomolybdate therapy protects against bleomycin-induced pulmonary fibrosis in mice. J. Lab. Clin. Med. 2003;141:210-216 [Non-Patent Document 16] Ovet H., Oztay F. The copper chelator tetrathiomolybdate regressed bleomycin-induced pulmonary fibrosis in mice, by reducing lysyl oxidase expressions.Biol. Trace Elem. Res. 2014;162:189-199 [Non-Patent Document 17] Qiu Q., et al. Copper in diabetes mellitus: A meta-analysis and systematic review of plasma and serum studies. Biol. Trace Elem. Res. 2017;177:53-63 [Non-Patent Document 18] Li P., et al. Association between plasma concentration of copper and gestational diabetes mellitus. Clin. Nutr. 2019;38:2922-2927 [Non-Patent Document 19] Zheng Y., et al. The role of zinc, copper and iron in the pathogenesis of diabetes and diabetic complications: Therapeutic effects by chelators. Hemoglobin. 2008;32:135-145 [Non-Patent Document 20] Lowe J., et al. Dissecting copper homeostasis in diabetes mellitus. Iubmb Life. 2017;69:255-262 [Non-Patent Document 21] Cooper GJ, Phillips AR, Choong SY, Leonard BL, Crossman DJ, Brunton DH, Saafi L., Dissanayake AM, Cowan BR, Young AA, et al. Regeneration of the heart in diabetes by selective copper chelation. Diabetes. 2004;53:2501-2508 [Non-Patent Document 22] Tanaka A., et al. Role of copper ion in the pathogenesis of type 2 diabetes. Endocr. J. 2009;56:699-706

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[0022] Efforts have been focused on identifying and evaluating new chelating compounds and formulations to reduce toxic side effects, enhance ability to cross the blood-brain barrier, and improve patient compliance, and there is a need to develop improved methods of treating subjects with copper disorders. The present disclosure meets these needs, providing methods and compositions for reducing copper and chelating agent side effects, improving drug stability, and avoiding the requirements for refrigeration and cold chain distribution. [Means for solving the problem]

[0023] A brief summary The invention described and claimed herein has many attributes and embodiments, including but not limited to those set forth or described or referenced in this brief summary. It is not intended to be comprehensive, and the invention described and claimed herein is not limited by or to the features or embodiments identified in this introduction, which are included for purposes of illustration only and not limitation.

[0024] The present invention relates to improved fixed dose amounts of triethylenetetramine disuccinate, formulations thereof, and their use for the treatment, prevention or amelioration of diseases, conditions and disorders treatable with copper chelators.

[0025] In one aspect, the invention includes an article of manufacture comprising a single dose capsule or tablet containing a single fixed dose of triethylenetetramine disuccinate, the fixed dose being selected from the group consisting of about 350 mg, 400 mg, about 500 mg, about 600 mg, and about 700 mg of triethylenetetramine disuccinate.

[0026] In another aspect, the article of manufacture further comprises a package insert instructing a user to administer the fixed dose to a patient having a disease, condition, or disorder treatable with a copper chelator. In a further aspect, the disease treatable with a copper chelator is characterized by an excess of copper.

[0027] In another aspect, the disease, condition or disorder treated as described herein is selected from the group consisting of Wilson's disease, heart failure, diabetic cardiomyopathy, left ventricular hypertrophy, diabetes mellitus, Alzheimer's disease, Parkinson's disease, idiopathic pulmonary fibrosis and cancer. In another embodiment, the disease, condition or disorder treated as described herein is selected from the group consisting of frontotemporal dementia (FTD), multiple sclerosis, and amyotrophic lateral sclerosis (Lou Gehrig's disease / motor neuron disease or ALS).

[0028] In another aspect, the disease, condition, or disorder is copper toxicity.

[0029] In another embodiment, the cancer is selected from the group consisting of laryngeal squamous cell carcinoma, non-Hodgkin's lymphoma, multiple myeloma, chronic lymphocytic leukemia, hepatocellular carcinoma, gynecological cancer, colorectal cancer, lung cancer, primary brain cancer, and breast cancer.

[0030] In another embodiment, a fixed dose of triethylenetetramine disuccinate is used to inhibit tumor angiogenesis.

[0031] In another embodiment, a fixed dose of triethylenetetramine disuccinate is used in conjunction with radiation therapy for tumors and cancers, including highly metastatic Lewis lung carcinoma.

[0032] In yet another embodiment, a fixed dose of triethylenetetramine disuccinate is used in conjunction with immune stimulation, including to inhibit breast tumor growth and metastasis.

[0033] In another embodiment, a fixed dose of triethylenetetramine disuccinate is used to reduce programmed cell death protein 1 (PD-1) expression observed, for example, in neuroblastoma and glioblastoma tumor cells.

[0034] In another embodiment, the fixed dose triethylenetetramine disuccinate is used in combination with oncolytic virus therapy, including oncolytic HSV therapy. In yet another embodiment, the fixed dose triethylenetetramine disuccinate is used in combination with oncolytic virus therapy, for example, for localized and metastatic head and neck squamous cell carcinoma.

[0035] In another embodiment, the fixed dose of triethylenetetramine disuccinate is used in combination with an inhibitor of N-acetylaminotransferase. In another embodiment, the fixed dose of triethylenetetramine disuccinate is used in combination with an inhibitor of spermidine-spermine-N(1)-acetyltransferase (SSAT1 and / or SSAT2). In one preferred embodiment, the fixed dose of triethylenetetramine disuccinate is used in combination with an inhibitor of spermidine-spermine-N(1)-acetyltransferase-2 (SSAT2).

[0036] In another aspect, a fixed dose of triethylenetetramine disuccinate is used to prevent, treat or manage autophagy.

[0037] In another embodiment, the article of manufacture includes a number of capsules equal to a daily dose of triethylenetetramine disuccinate, the daily dose being selected from the group consisting of about 2400 mg per day to about 3000 mg per day of triethylenetetramine disuccinate. In another embodiment, the dose and dosing is between about 2.336-2.337 mg of triethylenetetramine disuccinate for every milligram of triethylenetetramine dihydrochloride or triethylenetetramine tetrahydrochloride.

[0038] In another embodiment, the triethylenetetramine disuccinate in the article of manufacture has a purity of at least about 95%. In a further embodiment, the purity is at least about 99%.

[0039] In another embodiment, the triethylenetetramine disuccinate in the article of manufacture is a crystalline form of triethylenetetramine disuccinate.

[0040] In another embodiment, the triethylenetetramine disuccinate in the article of manufacture is triethylenetetramine disuccinate anhydrate.

[0041] In another aspect, the triethylenetetramine disuccinate in the article of manufacture is non-hygroscopic and has good stability under normal room temperature storage conditions.Importantly, the anhydrous crystalline form of the triethylenetetramine disuccinate article of manufacture described herein has a shelf life of at least about 12 months (and up to 5 years) at room temperature without significant degradation of triethylenetetramine disuccinate API, and remains within the impurity specifications for triethylenetetramine disuccinate drug substance.In one embodiment, the term "without significant degradation" means that the purity of triethylenetetramine disuccinate is at least about 98.5%, does not exceed about 0.5% of degradation products, and does not exceed about 0.1% of new unidentified impurities for at least about 12 months.

[0042] In another embodiment, the article of manufacture with a fixed dose of triethylenetetramine disuccinate is in the form of a capsule. In another embodiment, the article of manufacture with a fixed dose of triethylenetetramine disuccinate is in the form of a tablet. In a further embodiment, the capsule or tablet of triethylenetetramine disuccinate is formulated in such a way as to provide delayed or sustained release, thereby resulting in a modified pharmacokinetic profile from the associated immediate release form.

[0043] In yet a further aspect, the present invention also includes a method of managing or treating a subject having a copper chelator-treatable disease, comprising administering triethylenetetramine disuccinate to said subject in an amount ranging from about 2400 mg per day to about 3000 mg per day of triethylenetetramine disuccinate. In one aspect of the method, the copper chelator-treatable disease is characterized by excess copper. In another aspect, the triethylenetetramine disuccinate used in the method is at least about 95% pure, at least about 99% pure, or 100% pure. In another aspect, the triethylenetetramine disuccinate used in the method is a crystalline form of triethylenetetramine disuccinate. In yet another aspect of the method, the triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydrate. In yet another aspect of the method, the triethylenetetramine disuccinate is in the form of a fixed dose tablet or capsule. In one preferred embodiment, the fixed dose of triethylenetetramine disuccinate is about 400 mg, about 500 mg, about 600 mg or about 700 mg. In another preferred embodiment of the method, the subject is a human.

[0044] In another embodiment, three fixed dose tablets or capsules of 400 mg fixed dose triethylenetetramine disuccinate are given twice per day (2400 mg per day).

[0045] In another embodiment, three fixed dose tablets or capsules of 500 mg fixed dose triethylenetetramine disuccinate are given twice per day (3000 mg per day).

[0046] In another embodiment, the triethylenetetramine disuccinate fixed dose tablet or capsule is 350 mg.

[0047] In another embodiment, the total amount given per day is 2800 mg as four 350 mg tablets or capsules BID.

[0048] In another aspect, the fixed dose of triethylenetetramine disuccinate is used to lower or normalize copper(II) content in a subject.In one embodiment, the fixed dose of triethylenetetramine disuccinate reduces total copper in a subject.In another embodiment, the fixed dose of triethylenetetramine disuccinate is used to treat a subject for a disease, disorder or condition that would benefit from a copper(II) chelator.

[0049] In one preferred embodiment of the method of the present invention, a fixed dose of triethylenetetramine disuccinate is delivered orally.

[0050] In another embodiment, the fixed dose of triethylenetetramine disuccinate maintains total copper in the subject within the normal human serum or plasma range of about 0.8-1.2 milligrams / L or about 10-25 micromoles / L. In another embodiment, the fixed dose of triethylenetetramine disuccinate maintains total copper in the subject within at least about 70%, such as at least about 75%, of the normal range of about 0.8-1.2 milligrams / L or about 10-25 micromoles / L. In another embodiment, the fixed dose of triethylenetetramine disuccinate maintains total copper in the subject within about 75% to about 85%, or about 85% to about 95% of the normal range of copper in human plasma or serum. In one aspect of the method of the invention, the copper status of the subject given a fixed dose of triethylenetetramine disuccinate is determined by evaluating copper in the subject's urine.

[0051] In one aspect of the invention, the method employs a pharmaceutical composition comprising a fixed dose of substantially pure triethylenetetramine disuccinate. In another aspect, the method employs a pharmaceutical composition comprising substantially pure triethylenetetramine disuccinate and a pharma- ceutically acceptable excipient.

[0052] In one aspect of the invention, the method employs a fixed dose of a crystalline form of triethylenetetramine disuccinate.

[0053] In another aspect of the invention, the method employs a fixed dose of triethylenetetramine disuccinate anhydrous.

[0054] In certain embodiments, the fixed dose triethylenetetramine succinate is a triethylenetetramine disuccinate polymorph.

[0055] A preferred pharmaceutical composition for use in the method of the present invention comprises, consists essentially of, or consists of a fixed dose of substantially pure triethylenetetramine disuccinate. Another preferred composition is a fixed dose of substantially pure triethylenetetramine disuccinate anhydrate. Another preferred composition is a composition comprising, consists essentially of, or consists of a fixed dose of substantially pure triethylenetetramine disuccinate crystals having alternating layers of triethylenetetramine molecules and succinate molecules.

[0056] In another embodiment of the invention, the method maintains copper levels in a subject at about 70% to about 100% of normal, thereby inducing a decrease in copper titers and / or reducing copper levels in a mammalian patient.

[0057] The total dosage of triethylenetetramine disuccinate may be given in single or divided dosage units (e.g., BID, TID) and preferably maintains normal urinary and / or plasma copper levels in the subject, or levels not below about 70%-75% of normal. Fixed doses of triethylenetetramine disuccinate are typically administered BID.

[0058] In some embodiments, the method comprises, consists essentially of, or consists of administering to the subject a tablet or capsule containing a fixed dose of triethylenetetramine disuccinate. Preferably, the fixed dose of triethylenetetramine disuccinate is administered orally in the form of a capsule.

[0059] In any of the procedures described and / or claimed herein, the fixed triethylenetetramine disuccinate dosage regimen administered to a subject will not reduce physiological levels of copper in the subject to a depleted or otherwise dangerously low level.

[0060] The present invention also includes articles of manufacture, e.g., kits of parts, that include or consist essentially of one or more of the fixed dose triethylenetetramine disuccinates described herein, e.g., oral fixed dose triethylenetetramine disuccinates, and a printed set of instructions (e.g., package inserts) that describe their use in therapy, e.g., in the treatment of heart failure, diabetic cardiomyopathy, left ventricular hypertrophy, Wilson's disease, cancer, etc. In one embodiment, the kit does not include a physical set of instructions, but mentions or describes their availability online, in the cloud, in a flash drive, or in another storage mechanism. In one embodiment, the instructions describe that triethylenetetramine disuccinate is administered to a patient with Wilson's disease who has previously received triethylenetetramine dihydrochloride or DPA.

[0061] Both the foregoing general description and the following detailed description are exemplary and explanatory. They are intended to provide further details of the invention and are not intended to be construed as limiting. Other objects, advantages and novel features will become apparent to those skilled in the art from the following detailed description of the invention. [Brief description of the drawings]

[0062] [Figure 1] Figure 1 is a schematic diagram of the final PK / PD model used to describe TETA, MAT and DAT plasma concentrations and urinary copper excretion versus time in Example 3. Symbols are defined in the List of Abbreviations and Table 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] Detailed Description Various formulation methods are practiced to achieve the desired outcome of maximizing the pharmacokinetic profile of absorption, distribution, metabolism, and elimination of drugs. The molecule triethylenetetramine is basic due to the four nitrogen atoms, each of which carries a lone pair of electrons. It is a colorless oily liquid, but like many amines, it is yellowish in color due to impurities resulting from air oxidation. It is soluble in polar solvents.

[0064] The dihydrochloride salt of triethylenetetramine is classified as a BCS Class III drug (high solubility, low permeability). It is sold under the trade names Syprine® (250 mg), Clovique® (250 mg) and Cufence (300 mg). Triethylenetetramine is also available in the tetrahydrochloride salt form, marketed as Cuprior® (150 mg). Other than the high variability in the oral bioavailability of the salt forms of triethylenetetramine dihydrochloride, little was previously known about the relative bioavailability of these different salt forms (see, e.g., Lu, Jun, Triethylenetetramine Pharmacology and its Clinical Applications, Molecular Cancer Therapeutics Volume 9, Issue 9, pp. 2458-2467 (September 2007)), and nothing was known about the bioavailability of the salts of triethylenetetramine disuccinate, which the inventors have determined to be a BCS Class I drug (high solubility, high permeability) rather than a BCS Class III drug.

[0065] Triethylenetetramine dihydrochloride salts sold as Syprine® and Clovique® are recommended to be taken on an empty stomach at a dose of 750-1250 mg / day given to adults in divided doses between 2 to 4 times per day, with a maximum dose of 2000 mg / day. The product is provided in capsule form at a dose of 250 mg / capsule (containing the equivalent of 200 mg of triethylenetetramine). The Cufence® triethylenetetramine dihydrochloride salt product is sold as 200 mg capsules, with a recommended dose of 800-1600 mg (4-8 capsules) per day in 2-4 divided doses.

[0066] Syprine® was approved for medical use in the United States in November 1985 for the treatment of Wilson's disease. Cloviquie® was approved for the treatment of Wilson's disease in the United States in October 2019. Cufence® was approved for medical use in Wilson's disease patients by the European Union in July 2019. Cloviquie® is the first FDA-approved trientine product in a portable blister pack that provides room temperature stability for up to 30 days, potentially providing additional convenience to patients. Cuprior™ is a tetrahydrochloride salt of triethylenetetramine. This product is available in 150 mg strength tablets, each tablet containing the equivalent of 75 mg of triethylenetetramine. It was approved for the treatment of Wilson's disease by the European Union in September 2017. Both of these products are formulated for immediate release.

[0067] One object of the present invention is to provide a dosage and formulation of triethylenetetramine disuccinate that is equivalent in human exposure / bioavailability to previously approved forms of triethylenetetramine, e.g., for the treatment of Wilson's disease.

[0068] Triethylenetetramine disuccinate is an alternative and superior salt form of trientine. It is more stable, has better distribution and good activity. It preferably significantly exceeds the triethylenetetramine dihydrochloride compound currently used to treat Wilson's disease in terms of its resistance to light, temperature and moisture. It does not require cold chain storage or special packaging like the dihydrochloride salt. However, the appropriate dosage is unknown.

[0069] Assessment of dosing is complex, based on a number of factors including drug molecular weight, drug stability and half-life, drug solubility, drug permeability across mucosal barriers, bioavailability (availability of drug to systemic circulation or site of pharmacological action), tissue distribution and clearance, drug tissue:blood ratio, plasma protein binding, activity by dose, breakdown to active metabolites, mean plasma concentrations, and route of excretion, as well as the need for a bolus or loading dose and / or sustained or basal drug levels. Aside from molecular weight, most of these variables are unknown for the copper chelator triethylenetetramine disuccinate.

[0070] Optimal fixed dosing of triethylenetetramine disuccinate for the treatment of disease has been discovered and is provided herein based on the studies described in the Examples below, including the intestinal absorption study in Example 1 and the in vivo distribution study in Example 2, as well as the human clinical study results described, interpreted and evaluated in Examples 3 and 4, which revealed unexpected findings including, among others, about the copper chelating activity of triethylenetetramine disuccinate and its breakdown to major metabolites, mean plasma concentrations, whole body tissue distribution, tissue:blood ratios after oral administration, and triethylenetetramine disuccinate bioavailability.

[0071] Example 1 describes an in vitro intestinal absorption study that shows that triethylenetetramine disuccinate will have good absorption in humans (estimated to be approximately 70%).

[0072] Example 2 is a quantitative in vivo study of tissue distribution of the labeled copper-depleting compound triethylenetetramine disuccinate after oral administration to male albino and male pigmented rats. In both species, significant tissue penetration was seen throughout 42 different body tissues, including brain, heart, lung, and liver. In male pigmented rats, maximum tissue concentrations of radioactivity were evenly distributed between the 1- and 8-hour time points. The highest levels of radioactivity were seen in various tissues, including lung, at 1 hour post-dosing, with lung penetration continuing for the entire 8 hours. At 24 hours post-dosing, elimination was ongoing in male pigmented rats, with approximately half of the tissues measured having levels of radioactivity below the limit of quantification. At 72 hours post-dosing, elimination of radioactivity in male pigmented rats was nearly complete, with approximately 65% ​​of the tissues below the limit of quantification.

[0073] Example 3 describes human population pharmacokinetic and pharmacodynamic modeling of triethylenetetramine, its two major metabolites, and copper excretion following oral two-way crossover administration of triethylenetetramine disuccinate and triethylenetetramine dihydrochloride in a clinical study in healthy adult volunteers that, among other things, characterizes the bioavailability of triethylenetetramine disuccinate in humans. The population PK analysis included samples from this study in which each subject received triethylenetetramine disuccinate and triethylenetetramine dihydrochloride (Syprine®) in a double-blind, dose-escalation, two-way crossover design.

[0074] Example 4 describes further analysis of the data obtained in the study of Example 2 comparing triethylenetetramine disuccinate and triethylenetetramine dihydrochloride (Syprine®). The study of Example 2 led to the discovery that administration of triethylenetetramine as a disuccinate salt resulted in lower exposure indices (Cmax and AUC) of triethylenetetramine and its metabolites. The modeling in Example 3 compared absorption kinetics and provided a more comprehensive assessment of the relative bioavailability of the two salt forms in the context of the study design of Example 3. The analysis in Example 4 applied model-based population analysis to the data to obtain an overall assessment of the pharmacokinetics of triethylenetetramine and its two major metabolites (monoacetylated (MAT) and diacetylated (DAT) forms), as well as to further assess the pharmacodynamics of urinary copper excretion, to consider potential covariates with PK / PD parameters such as gender, age and dose, and in comparing the PK / PD of Syprine® to triethylenetetramine disuccinate from the bioequivalence study in Example 2, particularly with respect to bioavailability.

[0075] Triethylenetetramine dihydrochloride is a copper chelator approved by the FDA for second-line treatment of Wilson's disease. It is available in 300 mg capsules in Europe, with two capsules typically administered BID to treat Wilson's disease (total of 1200 mg per day). Triethylenetetramine dihydrochloride (Syprine®) is available in 250 mg capsules in the United States, with two capsules typically administered BID to treat Wilson's disease (total of 1000 mg per day). There has been no systematic evaluation of Syprine® doses and / or dose intervals. However, from limited clinical experience, the recommended initial doses of Syprine® in the United States are 500-750 mg / day for pediatric patients and 750-1250 mg / day for adults (maximum of 2000 mg / day), given in divided doses two, three or four times a day.

[0076] Triethylenetetramine disuccinate is an alternative and superior salt form of triethylenetetramine, but its target dosage is unknown and cannot be known from the prior art. The inventors have found that to reproduce the bioavailability of triethylenetetramine at 300 mg of triethylenetetramine dihydrochloride, about 701 mg of triethylenetetramine disuccinate is required. To reproduce the bioavailability of triethylenetetramine at 250 mg of triethylenetetramine dihydrochloride, the inventors have found that about 584 mg of triethylenetetramine disuccinate is required. To reproduce the bioavailability of triethylenetetramine at 250 mg of triethylenetetramine tetrahydrochloride (bioequivalent to the dihydrochloride salt), the inventors have found that about 350 mg of triethylenetetramine disuccinate is required.

[0077] Thus, the inventors have discovered not only that fixed doses comprising or consisting essentially of about 701 mg triethylenetetramine disuccinate and about 584 mg triethylenetetramine disuccinate and about 350 mg triethylenetetramine disuccinate are optimal for dosing this salt form, but also that daily doses of about 2336 mg and 2804 mg triethylenetetramine disuccinate, on a 1000 mg / day and 1200 mg / day dosing basis, respectively, are optimal for the treatment of Wilson's disease and other copper disorders.

[0078] Using the above pediatric and adult dosing ranges of triethylenetetramine dihydrochloride per day of 500 to 750 mg for children and 750 to 1250 mg for adults (with a maximum of 2000 mg / day), the dose ranges of triethylenetetramine disuccinate per day are from about 1168 mg to about 1752 mg for children and from about 1752 mg to about 2920 mg for adults (with a maximum of 4672 mg / day). Doses are increased if clinical response is inadequate or free serum copper is persistently greater than 20 mcg / dL, with long-term maintenance doses reassessed every 6 to 12 months.

[0079] Another approved daily dose for trientine dihydrochloride is 1200-2400 mg / day in 2-4 divided doses for adults, and lower doses, typically 600-1500 mg / day for children, depending on age and weight, also typically given in divided doses. Based on the findings herein, a superior salt of triethylenetetramine disuccinate would be dosed at about 2803 mg / day to about 5606 mg / day for adults, and about 1402 mg / day to about 3504 mg / day for children, depending on age and weight, both typically given in divided doses.

[0080] Cuprior® (triethylenetetramine tetrahydrochloride) is also indicated for the treatment of Wilson's disease in adults, adolescents and children aged 5 years and older who are intolerant to D-penicillamine therapy and is sold as a 150 mg tablet. The approved and recommended Cuprior® dosing regimen for adults is between 450 mg and 975 mg (3 to 6 1 / 2 tablets) per day in 2 to 4 divided doses. The triethylenetetramine disuccinate dosing regimen for adults will be between about 1051 mg and about 2278 mg per day (typically using a fixed dose of 350 to 350.4 mg, which corresponds to a 150 mg triethylenetetramine tetrahydrochloride tablet). The starting dose in children is lower than for adults and depends on age and weight. In general, Cuprior® doses for children are usually between 225 mg and 600 mg (1 1 / 2 to 4 tablets) per day, in 2 to 4 divided doses. Triethylenetetramine disuccinate dosing regimens for children will be between about 525 mg and about 1400 mg per day.

[0081] The inventors have further discovered that other fixed doses of triethylenetetramine disuccinate for optimal dosing and bioavailability are about 350 mg, about 400 mg, about 500 mg, about 600 mg and about 700 mg of triethylenetetramine disuccinate, including about 350.4 mg, 584 mg and about 701 mg of fixed doses of triethylenetetramine disuccinate. Exemplary effective amounts are described herein, including doses in the range of about 2300 mg per day to about 2800 mg per day, for example, given as multiple fixed doses of triethylenetetramine disuccinate comprising or consisting essentially of about 350 mg, 400 mg, about 500 mg, about 600 mg and / or about 700 mg. Other fixed doses of triethylenetetramine disuccinate are given equivalently from about 1050 mg / day to about 2300 mg / day, from about 1400 mg / day to about 3500 mg / day, from about 2400 mg / day to about 3000 mg / day, and from about 2800 mg / day to about 5600 mg / day.

[0082] For example, four 350 mg triethylenetetramine disuccinate capsules given BID would be equivalent to 2800 mg per day (roughly equivalent to 2804 mg per day, which is the dose of triethylenetetramine disuccinate that we have found to be bioequivalent to the 1200 mg per day triethylenetetramine dihydrochloride administered in Europe to treat Wilson's disease). For example, three 400 mg triethylenetetramine disuccinate capsules given BID would be equivalent to 2400 mg per day (roughly equivalent to 2337 mg per day, which is the dose of triethylenetetramine disuccinate that we have found to be bioequivalent to the 1000 mg per day triethylenetetramine dihydrochloride administered in the United States to treat Wilson's disease).

[0083] For example, three 500 mg triethylenetetramine disuccinate fixed dose tablets / capsules etc. given BID would be equivalent to 3000 mg per day, roughly equivalent to the bioequivalent dose of 2804 mg triethylenetetramine disuccinate per day that we have found. For example, four 600 mg triethylenetetramine disuccinate fixed dose tablets / capsules etc. given BID would be equivalent to 2400 mg per day, roughly equivalent to the bioequivalent dose of 2337 mg triethylenetetramine disuccinate per day that we have found. For example, two 700 mg triethylenetetramine disuccinate fixed dose tablets / capsules etc. given BID would be equivalent to 2800 mg per day, roughly equivalent to the bioequivalent dose of 2804 mg triethylenetetramine disuccinate per day.

[0084] Other convenient fixed dose amounts of triethylenetetramine disuccinate can be calculated and manufactured to provide a daily bioequivalent dose, such as about 2804 mg per day and about 2337 mg per day. For example, five doses of 280 mg triethylenetetramine disuccinate given BID can be used to provide 2800 mg per day. Also, by way of example, four doses of 290 mg triethylenetetramine disuccinate given BID can be used to provide 2320 mg per day.

[0085] Triethylenetetramine disuccinate may be given in fixed doses of about 350 mg, about 584 mg, and about 701 mg as two doses BID, which are equivalent to daily doses of about 1400 mg, about 2336 mg, and about 2804 mg of triethylenetetramine disuccinate, respectively. These and other fixed doses and total daily dose amounts described herein may be used to treat Wilson's disease and other copper disorders, including those described or referenced herein.

[0086] Generally, dosages are between about 2.336 to 2.337 mg of triethylenetetramine disuccinate for every milligram of triethylenetetramine dihydrochloride or triethylenetetramine tetrahydrochloride.

[0087] By way of example, the inventors have also found that 2804 mg of triethylenetetramine disuccinate per day, given as, for example, two 700 mg capsules administered twice daily, would be expected to produce a significant cupuric effect with minimal side effects and negligible adverse effects throughout the dosing interval on serum copper levels or other laboratory test parameters for the treatment of cardiac disease, including, for example, cardiac disease in type 2 diabetic patients whose cardiomyopathy (e.g., elevated left ventricular mass) may be treated at these dose amounts. Six months of treatment of elevated left ventricular mass with triethylenetetramine disuccinate dosed as described to provide approximately 2800 mg per day would significantly lower the elevated left ventricular mass toward normal.

[0088] The inventors have further discovered that fixed doses of triethylenetetramine disuccinate for optimal dosing and bioavailability given as multiple fixed doses comprising or consisting essentially of about 350 mg, 400 mg, about 500 mg, about 584, about 600 mg and / or about 700 or 701 mg of triethylenetetramine disuccinate will be useful for treating a copper-related disease, disorder or condition as described herein.

[0089] In one aspect, the present invention relates to newly discovered fixed dose amounts of triethylenetetramine disuccinate, its formulations, and their uses for the treatment, prevention, or amelioration of diseases, conditions, and disorders treatable with copper chelators.

[0090] In certain embodiments, triethylenetetramine disuccinate is administered in an initial dose (or loading dose), followed by a maintenance dose, where the loading dose is about or at least 1.5 times more than the maintenance dose, about or at least 2 times more than the maintenance dose, about or at least 2.5 times more than the maintenance dose, or about or at least 3 times more than the maintenance dose. The maintenance dose may be, for example, about 350 mg, 400 mg, about 500 mg, about 584 mg, about 600 mg, and / or about 700 or 701 mg, up to 1 to 4 times per day. In one embodiment, the loading dose is administered 1, 2, 3, 4, or 5 times before the first maintenance dose, and may be given 1, 2, 3, or 4 times per day.

[0091] Thus, by way of example, in one embodiment, in a triethylenetetramine disuccinate loading dose regimen of 2337 mg per day, triethylenetetramine disuccinate is administered in a daily loading dose (which may be provided in one or several dosages throughout the day) of at least about 3505 mg (1.5x), at least about 4674 mg (2x), at least about 5842 mg (2.5x), or at least about 7001 mg (3x). In one embodiment, the triethylenetetramine disuccinate loading dose is administered in two doses per day, over 1, 2, 3, 4, or 5 days or more as needed. Other triethylenetetramine disuccinate loading doses are calculated accordingly based on triethylenetetramine disuccinate maintenance doses given daily or at other frequencies, such as 2804 or other maintenance doses given daily.

[0092] In one embodiment, the fixed dose of triethylenetetramine disuccinate described herein is administered twice a day (BID) to provide the desired daily dosage. In another embodiment, the fixed dose of triethylenetetramine disuccinate is administered three times a day (TID) to provide the desired daily dosage. In yet a further embodiment, the fixed dose of triethylenetetramine disuccinate is administered four times a day (QID) to provide the desired daily dosage.

[0093] Importantly, the anhydrous crystalline form of the triethylenetetramine disuccinate article of manufacture described herein has a shelf life of at least about 12 months (and up to 5 years) at room temperature without significant degradation of the triethylenetetramine disuccinate API and remains within the impurity specifications for the triethylenetetramine disuccinate drug substance. In one embodiment, the term "without significant degradation" means that the purity of triethylenetetramine disuccinate is at least about 98.5%, has no more than about 0.5% degradation products, and has no more than about 0.1% new unidentified impurities over a period of at least about 12 months.

[0094] definition Copper(II) referred to herein is Cu +2 Or copper +2 or "copper(II)" +2 These are also known as cations.

[0095] The term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or ingredients from the medicament (or step in the case of a method). The phrase "consisting of" excludes any element, step, or ingredient not specified in the medicament (or step in the case of a method). The phrase "consisting essentially of" refers to the specified materials and that does not materially affect the basic and novel characteristics of the medicament (or step in the case of a method). Basic and novel features of the invention are described throughout the specification and include the ability of the compounds, compositions, and methods of the invention to reduce copper levels, reduce total copper, reduce copper value, reduce copper(II), and / or chelate copper(II). Basic and novel features of the invention also include the ability of the compounds, compositions, and methods of the invention to provide a clinically significant change in a copper-related disease, disorder, or condition, or a symptom thereof. In another embodiment of the method of the present invention, the basic and novel features of other compositions and methods of the present invention include the ability to reduce inflammation and / or combat vascular leakage. This concept incorporates the ability to reduce or eliminate the adverse effects of, for example, excess fluid movement through the walls of the vasculature or into the extracellular matrix, as well as diseases initiated or exacerbated by such processes. In addition to the use of the terms "comprising" and "essentially consisting of" in the appended claims, the term "comprising" may be substituted for "essentially consisting of" when used in the specification, and vice versa. Thus, by way of example, the phrase "composition comprising X" when used in the specification may be written in the claims as either "composition comprising X" or "composition essentially consisting of X".

[0096] As used herein, the terms "subject" or the like, including "individual" and "patient," all of which may be used interchangeably herein, refer to any mammal, including humans. Preferred mammals herein are humans, including, by way of example, adults, children, and including people with Wilson's disease, heart failure, cardiomyopathy, left ventricular hypertrophy, diabetes, or cancer. In certain embodiments, the subject, individual, or patient is a human.

[0097] As used herein, "mammal" has its ordinary meaning and includes primates (e.g., humans and non-human primates), laboratory animals (e.g., rodents such as mice and rats), farm animals (such as cows, pigs, mink, sheep and horses), and domestic animals (such as dogs and cats). In one aspect of the invention, a fixed dose of triethylenetetramine disuccinate is added to the animal's feed or water. The invention includes an article of manufacture that includes a fixed dose of triethylenetetramine disuccinate in the animal's feed or water.

[0098] As used herein, the term "subjected to a patient" or "administered to" includes any active or passive mode that ensures the in vivo presence of triethylenetetramine disuccinate. Preferably, the administration mode is oral. However, all other administration modes (particularly parenteral, e.g., intravenous, intramuscular, etc.) are contemplated.

[0099] The term "treating a copper-related disease, disorder or condition" or the like refers to preventing, slowing, reducing, diminishing, arresting and / or reversing a disease, disorder or condition characterized by pathological, excess or unwanted copper, or a disease, disorder or condition treatable with a copper(II) chelator, including, for example, Wilson's disease, heart failure, diabetic cardiomyopathy, left ventricular hypertrophy, diabetes mellitus, Alzheimer's disease, Parkinson's disease, idiopathic pulmonary fibrosis and cancer, or one or more symptoms thereof. The triethylenetetramine disuccinate dosages and treatment methods described herein may be used to treat copper-related diseases, disorders or conditions.

[0100] "Treating copper overload" refers to preventing, slowing, reducing, decreasing, stopping and / or reversing pathological, excess or unwanted copper in a subject, in whole or in part, and / or treating one or more symptoms of excess or unwanted copper.Triethylenetetramine disuccinate dosages and treatment methods described herein may be used to treat copper overload.

[0101] The term "preventing" means to prevent, in whole or in part, or to ameliorate or control. Thus, preventing a disease, disorder or condition means to prevent, in whole or in part, or to ameliorate or control the disease, disorder or condition. The triethylenetetramine disuccinate dosages and treatment methods described herein may be used to prevent copper overload and copper-related diseases, disorders or conditions.

[0102] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of triethylenetetramine disuccinate described herein. Exemplary effective amounts are described herein and include doses in the range of about 2400 mg per day to about 3000 mg per day, comprising or consisting essentially of a fixed dose of triethylenetetramine disuccinate. In one embodiment, the effective amount of triethylenetetramine disuccinate is at least about 95% pure, at least about 99% pure, or 100% pure. In another embodiment, the effective amount of triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate. In yet another embodiment of the method, the effective amount of triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydrous. In yet another embodiment of the method, the effective amount of triethylenetetramine disuccinate is in the form of a fixed dose tablet or capsule. In one preferred embodiment, the effective fixed dose of triethylenetetramine disuccinate is about 400 mg, about 500 mg, about 600 mg or about 700 mg. A fixed dose of 350 mg has also been described.

[0103] Thus, in one aspect, "effective amount" refers to an amount effective at the required dosage and for the required period of time to achieve the desired therapeutic or protective result.For example, but not by way of limitation, "effective amount" can refer to the amount of triethylenetetramine disuccinate disclosed herein that can treat the signs and / or symptoms of copper-related disease, disorder or condition, or otherwise treat copper overload.In one embodiment, the utility of the amount is evaluated by determining the subject's response and / or the copper amount in the subject's urine or plasma after administration of triethylenetetramine disuccinate as disclosed herein.Preferably, the effective amount maintains normal copper levels, or maintains the subject's copper levels within at least about 70% of normal or other levels described herein.

[0104] As used herein, "protective effective amount" refers to an amount that is effective at the required dosage and for the required period of time to achieve the desired protective result.Typically, a protective fixed dose of triethylenetetramine disuccinate is used before or at the early stage of copper-related disease, disorder or condition in a subject, so that the protective effective amount may, but is not necessarily, less than a therapeutically effective amount.A protective dose can also serve as a maintenance dose once copper-related disease, disorder or condition is under control, for example, with an initial, bolus or loading dose (single or multiple), all as described herein.

[0105] By "pharmaceutically acceptable" is meant, for example, a carrier, diluent or excipient that is compatible with other ingredients of the formulation and generally safe for administration to the recipient or does not cause undesired adverse physical reactions upon administration. "Pharmaceutically acceptable carrier" as used herein refers to an ingredient other than the active ingredient in a pharmaceutical formulation that can be safely administered to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives. Pharmaceutically acceptable diluents, carriers and / or excipients are useful in preparing pharmaceutical compositions and include substances that may be co-administered with the compounds described herein while still allowing them to perform their intended function, and that are generally safe, non-toxic, and not biologically or otherwise undesirable. Pharmaceutically acceptable diluents, carriers and / or excipients include those suitable for use in veterinary medicine and for use in human medicine.

[0106] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of triethylenetetramine disuccinate contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to whom the formulation will be administered. The pharmaceutical formulation of the present invention comprises a fixed dose of triethylenetetramine disuccinate as disclosed herein and a pharmaceutical acceptable carrier.

[0107] "Copper chelators" include those that bind or modify copper, selectively bind or modify copper(I) or copper(II) values, and are used to normalize blood and / or tissue copper levels and prevent unwanted copper accumulation. Copper chelators include prodrugs thereof. Other agents that normalize copper values ​​and other agents that selectively bind or modify copper(II), whether now known or later developed, are included within this definition.

[0108] "Copper sequestering agents" or "copper stripping agents" are agents that can bind and / or inhibit the ability of copper in any or all of its various forms. Copper stripping agents include chelating agents, agents that reduce total copper, agents that reduce copper value, and agents that reduce the amount of available intracellular copper, including those described herein. Copper stripping agents also include copper modifying agents, i.e., agents that are used to reduce copper by modifying the copper content in the body, including the intracellular content, or by modifying the availability of copper. It is understood that copper is an essential intracellular nutrient, and thus the present invention includes a method for reducing intracellular copper content while maintaining safe patient copper levels. Copper stripping agents include copper removing agents, i.e., agents that remove copper from the body and / or from within cells.

[0109] As used herein, the term "treatment" of or "treating" the signs and / or symptoms of a copper-related disease, disorder or condition in a mammal means, as the context permits, (i) preventing the condition or disease, i.e., avoiding one or more clinical symptoms of the disease; (ii) inhibiting the condition or disease, i.e., arresting the onset or progression of one or more clinical symptoms; and / or (iii) relieving the condition or disease, i.e., causing the regression of one or more clinical symptoms. Thus, "treatment" (and its grammatical variants such as "treat" or "treating") typically refers to clinical intervention in an attempt to alter the natural history of the individual, tissue or cell being treated, and may be performed either for prophylaxis or in the course of clinical pathology. The term does not necessarily imply that the subject is treated until complete recovery. Thus, "treatment" includes reducing, alleviating or reversing the symptoms or severity of a copper-related disease, disorder or condition, or preventing or otherwise reducing the risk of developing a copper-related disease, disorder or condition. This may include maintaining or promoting complete or partial remission of a copper-related disease, disorder or condition. The triethylenetetramine disuccinate dosages described herein are used for treatment.

[0110] As used herein, "related to" merely means that both situations exist and should not be construed as meaning that one is necessarily causally related to the other.

[0111] The term "chelatable copper" includes copper in any of its chelatable forms that can be bound by triethylenetetramine disuccinate, such as copper(II). Thus, the term "copper valence" (e.g., elemental, salt, etc.) refers to copper in any suitable form in the body that is available for such chelation by triethylenetetramine disuccinate (e.g., in extracellular tissue as opposed to intracellular tissue and possibly bound to the outside of cells and / or collagen). Certain methods and compositions of the invention can be used to bind chelatable copper, e.g., chelatable copper(II), while maintaining normal or near-normal copper valence (e.g., within about 70-75% of normal, for example, or other copper valence that is not harmful to the subject).

[0112] The described and claimed triethylenetetramine disuccinate dosages are used to selectively bind or denature copper(II) levels, normalize blood and / or tissue copper levels and prevent unwanted copper accumulation, and are administered to subjects with a disease, disorder or condition treatable by a copper chelator.

[0113] Triethylenetetramine disuccinate includes its prodrugs and the dosage is adjusted to account for the molecular weight of the "pro" portion of the triethylenetetramine disuccinate prodrug.

[0114] The dose of triethylenetetramine disuccinate disclosed herein may be administered alone or in combination with one or more additional ingredients, and may be formulated into a pharmaceutical composition that includes one or more pharma- ceutically acceptable excipients, diluents and / or carriers.In certain embodiments, the present invention provides a combination product that includes (a) a dose of triethylenetetramine disuccinate and (b) one or more anti-inflammatory and / or anti-vascular leakage agents, where components (a) and (b) are suitable for administration simultaneously or sequentially.In certain embodiments of the present invention, the combination product according to the present invention is used in such a manner that at least one of the components is administered while the other components still have an effect on the subject being treated.The doses of triethylenetetramine disuccinate and / or anti-inflammatory and / or anti-vascular leakage agents may be contained in the same or one or more different containers and administered separately, or may be mixed together in any combination and administered simultaneously. Preferably, triethylenetetramine disuccinate and / or the anti-inflammatory agent and / or the anti-vascular leakage agent, or both or all three, are combined in a capsule for oral administration.

[0115] In another embodiment, a fixed dose of triethylenetetramine disuccinate is used in combination with an inhibitor of the metabolism of triethylenetetramine, which is intended to include (but is in no way limited by) inhibitors of the enzymes N-acetylaminotransferase and / or spermine / spermidine N-acetylaminotransferase (SSAT1 and / or SSAT2).

[0116] It is understood that triethylenetetramine acts as a strong chelator of copper (II) ions. Thus, the treatment of Wilson's disease, for example, is believed to work at least in part by enhancing the disappearance of copper (II) ions from the body. However, by reducing the absorption of copper from the gastrointestinal tract, triethylenetetramine can also exert a dual positive effect in treatment. For example, inhibiting the action of copper-dependent enzymes can be optimized by achieving an effective concentration of triethylenetetramine either extracellularly or intracellularly to competitively inhibit the action of these enzymes. Under these conditions, enhancing the disappearance of copper will provide additional therapeutic benefits.

[0117] Syprine® is known to be characterized by very poor intestinal absorption (substantially less than 10% oral bioavailability). This is partially explained by its multiple ionizable (basic) amine groups. Enhanced absorption of triethylenetetramine would likely allow for a substantial reduction in dose, with an improved side effect profile and possibly even better efficacy.

[0118] The rapid metabolism of triethylenetetramine in the liver by N-acetylaminotransferase or spermine / spermidine N-acetylaminotransferase (SSAT1 and / or SSAT2) enzymes--or wherever such enzymes are expressed in sufficient concentrations--is a limiting factor that contributes to the high doses required for Syprine® and other salt forms. Therefore, co-administration of trientine or its various salt forms, including triethylenetetramine disuccinate, with inhibitors of these enzymes can be beneficial for enhancing bioavailability and lowering doses. Thus, triethylenetetramine disuccinate (or triethylenetetramine dihydrochloride or triethylenetetramine tetrahydrochloride) may be formulated or co-administered with inhibitors of N-acetylaminotransferase and / or spermine / spermidine N-acetylaminotransferase (SSAT1 and / or SSAT2). Such agents include molecules such as acetaminophen, diallyl sulfide and caspofungin. In another embodiment, a fixed dose of triethylenetetramine disuccinate is used in combination with an inhibitor of spermidine-spermine-N(1)-acetyltransferase (SSAT1 and / or SSAT2).

[0119] Such combination products can be prepared according to the methods and principles provided herein and as known in the art. Also provided are combination products for use in the methods as described herein.

[0120] In separate or common administration, fixed dose triethylenetetramine disuccinate formulations can be prepared to provide rapid or slow release; immediate, delayed, timed or sustained release; or a combination thereof. The formulations can be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, electuaries, drops (including but not limited to eye drops), tablets, granules, powders, candies, lozenges, capsules, gels, ointments, creams, lotions, oils, foams, sprays, mists or aerosols. For example, gastroretentive or mucoadhesive formulations of triethylenetetramine disuccinate can enhance or extend the absorption of this therapeutic article in the GI tract. Delayed release forms of triethylenetetramine disuccinate will release the drug after it has passed through the stomach, helping to avoid metabolism, sustain and increase absorption, and increase bioavailability. A variety of different means are available to complete these modified release formulations. Such technologies are well known to those of skill in the art, and the particular techniques and excipients are selected to address the issues or challenges posed by the ADME profile of the article in question.

[0121] Mucoadhesive formulations contain specific polymers that adhere to the epithelial lining of the site where they are hydrated. Thus, for example, drugs released in the duodenum after transit through the stomach will adhere to the walls of the GI tract, causing extended and preferential drug release and absorption from this site. The oral cavity, cornea, respiratory system and vaginal tissues are also covered with mucosal tissue and are therefore targets for such formulations. The mucoadhesive properties of most polymers increase with molecular weight, and thus, it is found that MW in the range of, for example, 200,000 to 700,000 correlates with enhanced mucoadhesion of polyoxyethylene polymers and copolymers. Viscosity, pore size and degree of crosslinking are other factors considered when selecting a mucoadhesive polymer. Hydrogen bonding, flexibility, degree of hydration and swelling are also important factors in drug delivery from mucoadhesive polymers. In addition to polyoxyethylene / polyvinyl alcohol, materials composed of polymeric acrylic and methacrylic acid esters, as well as hydroxylated methacrylic acid polymers, are useful for this purpose. Chitosan, cyanoacrylate, hyaluronic acid, hydroxypropylcellulose, gellan, polycarbopole and sodium carboxymethylcellulose are other related polymers that have been used in mucoadhesive formulations. Nasal mucoadhesive formulations are developed with attention to the specific properties of such tissues. Nasal delivery systems include copolymers of methyl vinyl ether, (hydroxypropyl)methylcellulose (HPMC), sodium carboxymethylcellulose, carbopol-934P and Eudragit RL-10. Mucin, gelatin, polycarbophil and poloxamer are examples of polymers used in vaginal or rectal mucoadhesive formulations. Oral delivery systems for GI mucoadhesive systems are represented by chitosan, polyacrylic acid, alginate, polymethacrylic acid and sodium carboxymethylcellulose. Mucoadhesive fixed dose triethylenetetramine disuccinate formulations can be prepared using such compounds.

[0122] Gastroretentive formulations are generally designed for drugs with optimal absorption windows in the stomach and proximal intestine. Hydrodynamically balanced systems, floating microspheres, gas-generating tablets, formulations that swell to prevent passage from the stomach, and formulations that adhere to the stomach wall are examples of such formulations. A "plug" system that expands to a size that cannot easily pass through the pyloric sphincter is an example of a gastroretentive formulation. Low density (floating) or gas-generating (carbon dioxide) formulations are retained for extended periods of time, and such techniques can be used in combination to optimize such performance. Mucoadhesive polymers are often also used to design such effects into formulations. Sodium alginate in combination with sodium carbonate or sodium bicarbonate can create a "rafting" effect, such that the formulation is retained in the stomach based on its buoyancy in gastric juices. Gastroretentive fixed dose triethylenetetramine disuccinate formulations can be prepared using these methods and compounds.

[0123] Preferred copper chelators for use in the methods of the present invention are the fixed doses described herein and the total dosages of triethylenetetramine disuccinate using those fixed doses.

[0124] In one embodiment, the fixed dose of triethylenetetramine disuccinate is substantially pure, including at least about 90% pure, at least about 95% pure, and 100% pure. In one embodiment, the triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydrous. In one embodiment, the triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate or triethylenetetramine disuccinate anhydrous.

[0125] In another embodiment of the present invention, the pharma- ceutically acceptable salt is triethylenetetramine disuccinate polymorph.Triethylenetetramine disuccinate polymorph is described, for example, in US Patent No. 8,067,641.In one embodiment, the fixed dose comprises triethylenetetramine disuccinate polymorph, and the polymorph is a crystal having a structure defined by the coordinates of Table 3B found in US Patent No. 8,067,641.In another embodiment of the present invention, the fixed dose comprises triethylenetetramine disuccinate polymorph, and the polymorph is a crystal having a structure defined by the coordinates of Table 3C found in US Patent No. 8,067,641. In other aspects of the invention, the fixed triethylenetetramine disuccinate dose consists essentially of a triethylenetetramine disuccinate polymorph having a crystal structure defined by the coordinates of Table 3B in U.S. Pat. No. 8,067,641, or consists essentially of a crystalline triethylenetetramine disuccinate polymorph having a structure defined by the coordinates of Table 3C in U.S. Pat. No. 8,067,641.

[0126] Fixed dose amounts and daily or other regular dosing The effective fixed triethylenetetramine disuccinate dose amount is about 400 mg, about 500 mg, about 600 mg, or about 700 mg. A 350 mg fixed dose is also provided. The fixed dose amount is used, for example, to administer a triethylenetetramine disuccinate dose of about 2400 mg per day to about 3000 mg per day, or within other time periods. In one embodiment, the effective amount of triethylenetetramine disuccinate is at least about 95% pure, at least about 99% pure, or 100% pure. In another embodiment, the effective amount of triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate. In yet another embodiment of the method, the effective amount of triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydrate. In yet another embodiment of the method, the effective amount of triethylenetetramine disuccinate is in the form of a fixed dose tablet or capsule. The total dosage may be given in single or divided dosage units (e.g., BID, TID) and preferably will maintain normal urinary and / or plasma copper levels in the subject, or levels not below about 70%-75% of normal. In one preferred embodiment, the fixed dose is administered BID.

[0127] Other Indications In some embodiments, the present invention provides a cosmetic composition comprising a cosmetically effective amount of a copper sequestering agent or copper stripping agent, e.g., a copper chelating agent, such as a copper(II) chelating agent, e.g., triethylenetetramine disuccinate. In some embodiments, the present invention provides a method for preventing or slowing hair loss or promoting hair growth by administering a composition comprising or consisting essentially of a copper sequestering agent or copper stripping agent. In some embodiments, the present invention provides a method for preventing, reducing or eliminating melasma or promoting normal skin development by administering a composition comprising or consisting essentially of a copper sequestering agent or copper stripping agent. In some embodiments, the present invention provides a method for preventing, reducing or eliminating cellulite or promoting normal skin development by administering a composition comprising or consisting essentially of a copper sequestering agent or copper stripping agent. In some embodiments, the copper sequestering agent or copper stripping agent is a copper chelating agent. In some embodiments, the copper chelating agent is a copper(II) chelating agent, e.g., triethylenetetramine. In some embodiments, triethylenetetramine is triethylenetetramine disuccinate.In some embodiments, the copper sequestering agent or copper stripping agent for preventing or slowing down hair loss or promoting hair growth, preventing, reducing or eliminating melasma or cellulite or promoting normal skin development is administered as a topical pharmaceutical preparation.In some embodiments, the topical preparation is provided in the form of a paste, ointment, oil, cream, lotion, foam, gel, tincture, powder, spray or patch.In some embodiments, the copper sequestering agent or copper stripping agent for preventing or slowing down hair loss or promoting hair growth, preventing, reducing or eliminating melasma or cellulite or promoting normal skin development is administered by microneedle, or by adhesive patch, non-adhesive patch, occlusive patch or micro-electrical patch.In some embodiments, the copper sequestrant or copper stripping agent for preventing or slowing hair loss or promoting hair growth, preventing, reducing or eliminating melasma or cellulite, or promoting normal skin development is administered enterally (e.g., orally, sublingually, bucally, rectally, etc.), parenterally (e.g., intravenously, intramuscularly, subcutaneously, etc.), intranasally, transdermally, ophthalmically, by inhalation, etc. In some embodiments, the copper sequestrant or copper stripping agent for preventing or slowing hair loss or promoting hair growth, preventing, reducing or eliminating melasma or cellulite, or promoting normal skin development is administered using a delayed or sustained release system. The amount of compound contained in the sustained release system will depend, for example, on where the composition is administered, the kinetics and duration of release of the compound of the present invention, and the nature of the condition, disorder and / or disease being treated and / or cared for. Those skilled in the art are aware of the different means by which cosmetic or pharmaceutical compositions containing the compounds of the present invention may be administered.

[0128] In some embodiments, the area being treated to prevent or slow hair loss or promote hair growth, prevent, reduce or eliminate melasma or cellulite, or otherwise promote normal skin development, is pretreated to facilitate transport of the copper sequestrant or copper stripping agent to the desired area of ​​the skin and / or skin-related (e.g., epidermis, dermis, basal layer and / or hair follicle) area. For example, the skin area to be treated may be treated by skin ablation techniques (application of sugar crystals, cellulose-based plant material, frozen CO2, polymeric beads and / or silica granules, etc.), by manual application, etc. Similarly, the skin area to be treated with the copper sequestrant or copper stripping agent may be prepared by application of chemical agents that reduce the thickness or increase the permeability of the stratum corneum, such as surfactants and / or chemical reducing agents. In some embodiments, the skin and / or skin-related area may be prepared by application of pulsed laser light and / or acoustic energy (e.g., via ultrasound) prior to or simultaneously with application of the copper sequestrant or copper stripping agent. Similarly, the skin may be prepared with stripping techniques (such as application and subsequent removal of adhesive film, tape or wax) prior to application of the copper sequestrant or copper stripping agent. The skin may be treated with a copper sequestrant or copper stripping agent-containing composition before, during and / or after such skin preparation treatment. For example, a skin preparation treatment as described above may be applied prior to application of a defensin-containing composition. Alternatively, a skin preparation treatment as described above may be applied after application of a defensin-containing composition to the skin. The copper sequestrant and copper stripping agent may be applied in a continuous or discontinuous manner.

[0129] manufacturing Triethylenetetramine disuccinate suitable for use in the present invention can be obtained from known manufacturers or can be synthesized using methods known in the art.Preparation methods are described, for example, in U.S. Patent No. 9,556,123, which describes the synthesis of triethylenetetramine and useful intermediates in their production.U.S. Patent No. 8,067,641 describes the method for the synthesis of substantially pure triethylenetetramine disuccinate, substantially pure triethylenetetramine disuccinate anhydride, and triethylenetetramine disuccinate polymorphs.

[0130] Other copper sequestering and copper stripping agents, including other copper chelating agents, and compositions and preparations can be obtained from various manufacturers or sources or made according to methods in the art.

[0131] Pharmaceutical Preparations Also provided is a pharmaceutical preparation comprising a fixed dose of triethylenetetramine disuccinate in a pharmaceutically acceptable vehicle.The term "pharmaceutically acceptable" has the meaning specified above, and includes those vehicles approved by federal or state government regulatory agencies for use in mammals, such as humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compound of the present invention is formulated for administration to mammals.

[0132] In one aspect, the disclosure provides a pharmaceutical preparation in which a fixed dose triethylenetetramine disuccinate (alone or together with another active ingredient) is prepared by combining it (or them) with one or more pharma- ceutically acceptable diluents, carriers, adjuvants, etc. in a manner known to those skilled in the art of pharmaceutical formulation. Fixed dosage forms can be prepared by combining it with one or more pharma- ceutically acceptable diluents, carriers, adjuvants, etc. in a manner known to those skilled in the art of pharmaceutical formulation.

[0133] The choice of excipient will be determined in part by the active ingredients, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

[0134] Dosage forms useful herein include any appropriate dosage form known in the art to be suitable for pharmaceutical formulations of compounds suitable for administration to mammals, particularly humans, and particularly (but not exclusively) those suitable for stabilization in solutions, tablets or capsules containing therapeutic compounds for administration to humans.

[0135] The composition may take the form of any standard known dosage form, including those mentioned above, including tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, injectable liquids, transdermal delivery devices (e.g., transdermal patches), or any other suitable compositions. Those skilled in the art to which the present invention pertains will know the most suitable dosage form, taking into account the nature of the condition to be treated and the active agent to be used without undue experimentation. Fixed triethylenetetramine disuccinate doses and dosage ranges are described herein. It should be understood that one or more of the other active agents (e.g., anti-inflammatory agents) may be formulated into a single composition with a fixed dose of triethylenetetramine disuccinate. In certain embodiments, preferred dosage forms include injectable solutions, topical formulations in transdermal patches, and oral formulations. The dosage forms of the present invention include any suitable dosage form now known or later discovered to be suitable for pharmaceutical formulations of compounds suitable for administration to humans.

[0136] An example is, in the case of oral dosage forms, oral delivery forms such as tablets, capsules, lozenges, or any liquid form that can protect the compound from degradation before inducing an effect, for example, in the gastrointestinal tract.

[0137] Particular formulations of the invention are solid forms, particularly tablets or capsules for oral administration.

[0138] Slow or controlled release preparations of triethylenetetramine disuccinate in tablets or capsules are preferred.

[0139] In addition to standard diluents, carriers and / or excipients, the compositions according to the present invention can be formulated with one or more additional components, or in such a manner as to, for example, enhance activity or bioavailability, protect integrity or increase its half-life or shelf life, allow slow release when administered to a subject, or provide other desirable benefits.For example, slow release vehicles include macromers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone) or hydrogels, so as to allow sustained release of product from the matrix over time.As further examples, the compositions can also include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, coating agents, buffers, etc.Those skilled in the art to which the present invention pertains will easily identify additional additives that may be desirable for specific purposes.

[0140] The fixed triethylenetetramine disuccinate dosage of the present invention can be administered by sustained release system.Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules.Sustained release matrices include polylactic acid (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).Sustained release compositions also include liposomally encapsulated (e.g., encapsulated) compounds. Liposomes containing copper chelators (alone or together with antiviral and / or anti-inflammatory agents) can be prepared by known methods, including those described in, for example, DE 3,218,121; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. Typically, liposomes may be used to encapsulate triethylenetetramine disuccinate, and are small (up to about 200-800 angstroms), unilamellar, with lipid content greater than about 30 mole percent cholesterol, with the selected percentage adjusted for the most effective therapy. For example, slow release delivery using PGLA nano- or microparticles or in situ ion-activated gelation systems may be used.

[0141] Another object of the present invention is to provide formulations of triethylenetetramine disuccinate whose pharmacokinetic profile is superior to previous formulations. By superior, it is understood that increasing the absorption, distribution, metabolism or slowing down the elimination of triethylenetetramine may result in enhanced therapeutic efficacy or equivalent efficacy at lower doses. It is further understood that any or all of these same formulations may be practiced with different salt forms or free amine bases of triethylenetetramine so as to also modify or improve their pharmacokinetic profile and provide an improved or more suitable product for the intended use. Such an object may be variously achieved by modifying the formulations of triethylenetetramine disuccinate or other salts of triethylenetetramine to achieve the desired outcome.

[0142] In one such example, the desired salt form can be formulated using a gastric retention dosage form (GRDF). Such delivery forms are formulated with the intention of sustaining gastric retention time, thus enhancing absorption. Such strategies may use, for example, 1) transit retardants, 2) large single unit dosage forms, 3) bioadhesive drug delivery systems, 4) heavy pellets, and 5) buoyant forms. Polymers such as carbopol, chitosan, sodium alginate, HPMC, polyacrylic acid, polyethylene glycol, and modified forms of these polymers are variously used to achieve gastric retention, as some examples among others.

[0143] In the second example of the modified dosage form for non-immediate release, the product is formulated to delay the release of drug until after the dosage form leaves the stomach.In the delayed release form, the release profile is similar or equal to that of the immediate release form, but the actual release of drug is delayed, for example, by enteric coating, so that the active ingredient is not released from the dosage form granulation until after the transit through the stomach is completed.Enteric coating, as an example of this strategy, is accomplished by using, for example, (meth)acrylic acid polymers that do not dissolve in aqueous medium until the pH is above 5.5, thus achieving a dosage form that transits through the stomach without releasing the active ingredient.

[0144] Extended release dosage forms differ from delayed release in that the release profile of the drug is extended beyond that of immediate release products. Mechanisms for extended release include delayed dissolution, diffusion, osmotic delivery from intact dosage forms, maintaining hydrological or hydrodynamic equilibrium, and ion exchange. The traditional means of obtaining extended release delivery is to formulate in a matrix of non-ionic cellulose ether (such as HPMC; see US8865778B2) in the presence of a selected amount of a non-crosslinked swelling agent, such as carboxymethyl starch or sodium starch glycolate. Other approaches to achieve the same result are known. For example, a drug delivery formulation core containing an osmotic agent and a water-swellable polymer is easily used as a driving force to deliver the drug in a controlled extended manner.

[0145] Therapeutic formulations for use in preparing the methods and compositions of the present invention may be prepared by any method well known in the art of pharmacy. For example, Gilman et al. (eds.) GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS (8th ed.) Pergamon Press (1990); and Remington, THE SCIENCE OF PRACTICE AND PHARMACY, 20th Edition. (2001) Mack Publishing Co., Easton, Pa.; Avis et al. (eds.) (1993) See PHARMACEUTICAL DOSAGE FORMS: PARENTERAL MEDICATIONS Dekker, NY; Lieberman et al. (eds.) (1990) PHARMACEUTICAL DOSAGE FORMS: TABLETS Dekker, NY; and Lieberman et al. (eds.) (1990) PHARMACEUTICAL DOSAGE FORMS: DISPERSE SYSTEMS Dekker, NY. Compositions may be formulated according to standard techniques as found in standard references such as, for example, Gennaro AR: Remington: The Science and Practice of Pharmacy, 20.sup.th ed., Lippincott, Williams & Wilkins, 2000.

[0146] Certain formulations of the invention are in the form of transnasal administration, such as nanoemulsions. Other formulations of the invention are in the form of transdermal patches.

[0147] Other formulations of the invention consist essentially of a fixed dose of triethylenetetramine disuccinate in the amounts described herein. Preferred are formulations that consist essentially of a fixed dose of substantially pure triethylenetetramine disuccinate anhydrate, i.e., at least about 90% pure, at least about 95% pure, or 100% pure.

[0148] Manufacturing Articles / Kits The present invention also includes an article of manufacture, or a "kit" containing materials useful for treating a subject for a copper-related disease, disorder, or condition is provided. The kit includes a container containing or consisting essentially of a fixed dose of triethylenetetramine disuccinate, preferably substantially pure triethylenetetramine disuccinate anhydrous. The kit may further include a label or package insert on or associated with the container (or noted as available online or in the cloud, or in a flash drive, or in another storage mechanism). The term "package insert" is used to refer to instructions for use that are customarily included in commercial packages of therapeutic products (or available online), which contain information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Suitable containers include, for example, bottles, blister packs, and the like. The containers can be formed from a variety of suitable materials, including, for example, plastic. The container may be a package containing the composition in the form of a tablet or capsule, the latter being preferred, and the fixed dose triethylenetetramine disuccinate is provided, for example, in a blister pack. The label or package insert indicates that the composition is used to treat a subject having (or suspected of having) a disease, disorder, or condition associated with excess or unwanted copper or otherwise treatable with a copper chelator. In one embodiment, the instructions state that triethylenetetramine disuccinate should be administered to a patient with Wilson's disease who has previously received triethylenetetramine dihydrochloride or DPA. In another embodiment, the instructions state that triethylenetetramine disuccinate should be administered to a patient with heart failure, diabetic cardiomyopathy, left ventricular hypertrophy, cancer, or another disease, disorder, or condition described herein. The instructions will refer to one or more of the doses or dosing regimens described herein.

[0149] In another embodiment of the present invention, an article of manufacture contains a dose (single or multiple doses) of triethylenetetramine disuccinate useful for treating the disorders described herein. The article of manufacture includes a container, a label, and a package insert. Suitable containers include, for example, bottles, blister packs, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a dose of triethylenetetramine disuccinate composition effective for treating a condition. A label on or associated with the container indicates that the triethylenetetramine disuccinate dose composition is used to treat a selected condition. In certain embodiments, the patient has Wilson's disease. In some embodiments, the patient has previously been treated for Wilson's disease and is intolerant to the patient's previous therapy. In certain embodiments, the patient has heart failure. In certain embodiments, the patient has diabetic cardiomyopathy. In certain embodiments, the patient has left ventricular hypertrophy. In certain embodiments, the patient has cancer. The package insert may optionally contain some or all of the clinical trial results found, for example, on clinicaltrials.gov, or those subsequently published.

[0150] Evaluation of fixed dose triethylenetetramine disuccinate therapy can be accomplished by reference to the copper titer available in mammals, including humans. References herein to "elevated" in relation to the presence of copper titer include humans having at least about 10 mcg free copper / dL of serum as measured. Measurement of free copper, which is equal to total plasma copper minus ceruloplasmin-bound copper, can be made using a variety of procedures. A preferred procedure is disclosed in the Merck & Co datasheet (www.Merck.com) for SYPRINE (trientine hydrochloride) capsules, a compound used to treat Wilson's disease, in which a 24-hour urinary copper analysis is performed to determine the cooper in serum by calculating the difference between quantitatively determined total copper and ceruloplasmin copper. EXAMPLES

[0151] The present invention relates to and describes methods related to the discoveries surrounding the fixed dose triethylenetetramine disuccinate invention described and claimed herein.

[0152] The following examples are presented so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are complete or exclusive.

[0153] Example 1 is described.

[0154] Example 1 describes in vitro studies using standard assays that predicted that triethylenetetramine disuccinate would have good absorption in humans (estimated to be approximately 70%).

[0155] Example 2 is a quantitative in vivo study of tissue distribution of triethylenetetramine disuccinate after oral administration to male albino and male pigmented rats. In both species, significant tissue penetration was observed throughout 42 different body tissues, including brain, heart, lung, and liver. In male pigmented rats, maximum tissue concentrations of radioactivity were evenly distributed between the 1- and 8-hour time points. The highest levels of radioactivity were observed in various tissues, including lung, at 1 hour post-dosing, with lung penetration continuing for the entire 8 hours. At 24 hours post-dosing, elimination was ongoing in male pigmented rats, with approximately half of the tissues measured having levels of radioactivity below the limit of quantification. At 72 hours post-dosing, elimination of radioactivity in male pigmented rats was nearly complete, with approximately 65% ​​of the tissues below the limit of quantification.

[0156] A novel dosing regimen for the copper-depleting compound triethylenetetramine disuccinate is described in Examples 3 and 4, which describe human population pharmacokinetic and pharmacodynamic modeling of triethylenetetramine, its two major metabolites, and copper excretion following oral two-way crossover administration of triethylenetetramine disuccinate and triethylenetetramine dihydrochloride to healthy adult volunteers, and determine the bioavailability of triethylenetetramine disuccinate.

[0157] Example 1 Assessment of Pgp involvement in compound permeability through the use of the Caco-2 in vitro model for oral bioavailability The presence of the p-glycoprotein (Pgp) efflux pump in mammalian intestinal tissue has been demonstrated and plays a central role in the active transport of drugs.

[0158] The aim of this study was to evaluate the involvement of Pgp in the permeability and metabolism of the test compound triethylenetetramine disuccinate (PX811019) using the Caco-2 in vitro model of the human intestinal barrier.

[0159] As a highly soluble and low-toxicity compound in vitro, and based on the in vitro Papp values ​​described below, it is predicted that triethylenetetramine disuccinate will have good absorption in humans (estimated to be approximately 70%).

[0160] The most commonly used models in intestinal transport studies are human intestinal cell lines, specifically HT29 and Caco-2 cell lines derived from colon carcinoma (Wils P., et al. Differentiated intestinal epithelial cell lines as in vitro models for predicting the intestinal absorption of drugs. Cell Biol. Toxicol. 10:393, 1994;Boulenc X. Intestinal Cell Models: Their use in evaluating the metabolism and absorption of xenobiotics. STP.Pharma. Sciences 7:259, 1997), with Caco-2 cells being the most widely used to evaluate intestinal absorption in pharmaceutical research. Meunier V, et al. The human intestinal epithelial cell line Caco-2; Pharmacological and pharmacokinetic applications. Cell Biol. Toxicol. 11:187, 1995. Cultured for 21 days on solid permeable membranes under conditions that enhance their polarization, this colon carcinoma-derived cell line achieves a confluent monolayer of well-differentiated cells with apical microvilli mimicking the intestinal lumen and a fully differentiated basolateral surface, equivalent to the cell surface that is usually in contact with the blood system. Moreover, they display phenotypic and physiological characteristics that closely resemble enterocytes derived from the human small intestinal epithelium.Zweibaum A., et al. Use of cultured cell lines in studies of intestinal cell differentiation and function. In: M. Field and RA Frizzel (eds), Handbook of physiology. The gastrointestinal system. Vol IV: Intestinal absorption and secretion. American Physiological Society, Washington DC. 223, 1991. Artursson P., and Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in humans See intestinal epithelial (Caco-2) cells. Biochem. Biophy. Res. Com. 175:880, 1991.

[0161] The main objective of this project is to 14 C] Radiolabeled test substance, triethylenetetramine disuccinate ([2- 14 The objective of this study was to address Pgp involvement in the metabolism and permeability of [C]PX811019, and to further determine whether the unlabeled triethylenetetramine disuccinate test article (PX811019) represents a Pgp inhibitor or substrate. To this end, polarized cultures of Caco-2 cells, assessed for monolayer integrity and functionality, were used as an in vitro model for the GI barrier.

[0162] Assessment of unlabeled triethylenetetramine disuccinate (PX811019) cytotoxicity on Caco-2 cultures: For this purpose, the WST-1 assay was performed. This standard assay for measuring cell proliferation, cell viability and cytotoxicity in mammalian cells utilizes the measurement of mitochondrial succinate dehydrogenase activity as an indicator of mitochondrial damage and is accepted as one of the most sensitive to detect early cytotoxic events. Caco-2 cells were cultured at 5 × 10 5 cells / cm 2 After 48 hours of culture, unlabeled PX811019 was added to HBSS(×1)-Ca 2+ Mg 2+ -Applied at eight different concentrations (1, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0.0078 mM) in pH=7.4 buffer and incubated for 2 hours at 37° C. Cells were then checked for viability by WST-1 application and measurement of absorbance at 450 nm in an ELISA plate reader. Each concentration was tested in triplicate.

[0163] The Caco-2 barrier model [2- 14 C]Triethylenetetramine disuccinate (PX811019) permeability assessment: Once cytotoxicity on Caco-2 cultures was assessed, [2- 14 [C]PX811019 was incubated on 21-day-old Caco-2 polarized cultures in transwell filters (diameter 6.5 mm; 0.4 μm pores). TEER measurements and permeability to Lucifer Yellow (low permeability marker) and antipyrine (high permeability marker) were first performed to check for barrier integrity and quality. Digoxin was used as a marker to confirm Pgp activity in the cultures. In parallel, unlabeled PX811019 was applied together with Lucifer Yellow in the apical compartment of control transwell filters at the concentrations used in the permeability assay to determine the effect of the test compound on barrier integrity. [2- 14C]PX811019 was placed in the donor compartment in HBSS(×1)-Ca 2+ Mg 2+ -Applied at one non-toxic concentration (1 μCi / ml; 0.02 mM) in pH=7.4 buffer and incubated alone or in the presence of verapamil or sodium azide for 1 h at 37°C. Samples were collected from the receptor compartment after 0, 15, 30, 45, 60 and 120 min and further analyzed by liquid scintillation counting. Samples were also collected from the donor compartment at time 0 and 120 min for mass balance evaluation. Each condition was performed in three replicate transwell filters in the presence of a Caco-2 barrier. Based on the dpm primary data obtained from sample analysis by scintillation counting, the permeability coefficient (Papp, in cm / s) was calculated in both AB (apical-basal) and BA (basolateral-apical) directions and expressed as [2- 14 C]PX811019 permeability was assessed under each experimental condition.

[0164] Evaluation of unlabeled triethylenetetramine disuccinate (PX811019) effect on Pgp activity in the Caco-2 barrier model: Once its permeability to Caco-2 cultures was assessed, 3 [H]-digoxin was incubated alone or together with unlabeled triethylenetetramine disuccinate (PX811019) on 21-day-old Caco-2 polarized cultures in transwell filters (6.5 mm diameter; 0.4 μm pores). TEER measurements and permeability to Lucifer Yellow (low permeability marker) and antipyrine (high permeability marker) were first performed to confirm barrier integrity and quality. In parallel, unlabeled PX811019 was applied together with Lucifer Yellow in the apical compartment of control transwell filters at the concentrations used in the permeability assay to determine the effect of test compounds on barrier integrity. [H]-digoxin was then incubated alone or together with unlabeled triethylenetetramine disuccinate (PX811019) on 21-day-old Caco-2 polarized cultures in transwell filters (6.5 mm diameter; 0.4 μm pores). TEER measurements and permeability to Lucifer Yellow (low permeability marker) and antipyrine (high permeability marker) were first performed to confirm barrier integrity and quality. In parallel, the effect of test compounds on barrier integrity was determined by applying unlabeled PX811019 together with Lucifer Yellow in the apical compartment of control transwell filters at the concentrations used in the permeability assay. To assess the effect of test compounds on Pgp activity, [ 3H]digoxin (4 μCi / ml; 0.2 mM) was added alone or in the presence of a similar and non-toxic concentration of PX811019 test compound (0.2 mM) in HBSS (× 1)-Ca 2+ Mg 2+ - pH = 7.4 buffer into the donor compartment and incubated at 37 °C for 1 hour. Samples were collected from the acceptor compartment after 0, 15, 30, 45, 60 and 120 minutes and further analyzed by liquid scintillation counting. Samples were also collected from the donor compartment at time 0 and 120 minutes for mass balance evaluation. Each condition was performed in triplicate transwell filters in the presence of a Caco-2 barrier. Based on the dpm primary data obtained from sample analysis by scintillation counting, [ 3 The permeability coefficient (Papp, in cm / s) of [H]digoxin was calculated in both the AB (apical-basal) and BA (basolateral-apical) directions, and the effect of PX811019 on Pgp-dependent digoxin permeability was assessed under each experimental condition.

[0165] Key results Unlabeled triethylenetetramine disuccinate (PX811019) did not exhibit any cytotoxicity towards Caco-2 cells at any of the concentrations tested. The Caco-2 polarized monolayers used in this study met the quality criteria for barrier conditions required for predictive in vitro permeability assays: in both experiments, TEER values ​​were 1000 ohm.cm 2 for Lucifer Yellow (a low-transmittance marker) the Papp value was 1 × 10 -6 cm / s, and the Papp value for antipyrine (a high permeability marker) is 1 × 10 -6 The velocity was higher than cm / s. Digoxin has a low Papp value (0.75±×10 -6 cm / s) and a moderate Papp value (6.08×10 -6The asymmetry index, which indicates Pgp activity in the system, was 8.06. In contrast, the [2- 14 [C]Triethylenetetramine disuccinate (PX811019) exhibited moderate to high AB permeability values ​​at the non-toxic concentrations tested, with a mean value of 9.87 × 10 -6 The flow rates were 100-250 nm / s and no permeability was observed in either the BA direction or in the presence of verapamil or sodium azide. Mass balances were between 70 and 120% for all conditions tested. In the presence of the test compound triethylenetetramine disuccinate (PX811019), the Papp values ​​of digoxin in either the AB or BA orientation were similar to those obtained with digoxin alone when the test compound was applied apically (0.66 ± 0.89 × 10, respectively). -6 cm / s and 12.7 ± 6.76 × 10 -6 cm / s), whereas both were slightly reduced when applied to the basolateral side (0.2 ± 0.35 × 10 -6 cm / s and 5.68 ± 3.03 × 10 -6 In both cases, however, the asymmetry index was maintained and even increased, indicating that the digoxin transport pathway and therefore Pgp activity were not affected by application of the unlabeled test substance, triethylenetetramine disuccinate (PX811019), regardless of the compartment to which it was applied.

[0166] In conclusion, triethylenetetramine disuccinate (PX811019) did not exhibit any cytotoxicity towards Caco-2 cells at any of the concentrations tested (1, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0.0078 mM). The Caco-2 polarized monolayers used in this study met the quality criteria of the barrier profile required for predictive in vitro permeability assays: TEER values ​​of 1000 ohm.cm. 2 Higher than; Papp Lucifer Yellow 1×10 -6cm / s, Papp-antipyrine is 10 × 10 6 Furthermore, the Papp values ​​and asymmetry index obtained for digoxin indicated that the levels of Pgp activity were within the acceptable range for this cell model.

[0167] In addition, triethylenetetramine disuccinate (PX811019) did not affect monolayer integrity at the concentrations used in both assays. Trientine disuccinate (PX811019) applied to these Caco-2 monolayers exhibited moderate to high permeability values ​​at the concentrations tested, with a mean value of 9.87 × 10 in the absorptive (AB) direction. -6 The absorption rate of the compound was 1.2 g / cm / s. Based on the in vitro Papp value, as well as the highly soluble and low toxicity of the compound in vitro, it can be predicted that the compound will have good absorption in humans (estimated to be approximately 70%). No permeability was observed in the secretory (BA) direction.

[0168] Compared with the permeability data from the digoxin Pgp substrate, the Papp data acquired in both AB and BA directions suggested that triethylenetetramine disuccinate (PX811019) uses a Pgp-independent polarized transport pathway to cross the Caco-2 barrier in the absorptive direction. Triethylenetetramine disuccinate (PX811019) AB transport through Caco-2 monolayers was fully inhibited in the presence of sodium azide and verapamil. As sodium azide is an ATP synthesis inhibitor, this suggested that the transport of the test substance (PX811019) was ATP-dependent. Verapamil is normally used in permeability assays as a Pgp inhibitor through inhibition of the ATP binding cassette. However, this is not consistent with the Ca 2+These have been extensively described as channel blockers, more specifically L-type channels. Because the AB polarization and asymmetry indices of triethylenetetramine disuccinate transport indicated that Pgp was not involved, the data in the presence of verapamil would suggest that this agent acts as a specific transporter blocker for triethylenetetramine disuccinate. Based on these data, the possible mechanisms of transport of the test substances across the intestinal barrier were active ATP-dependent and / or Ca 2+ This may be via a dependent transporter pathway.

[0169] Data obtained from permeability assays using digoxin alone and in the presence of test substances showed that triethylenetetramine disuccinate did not affect Pgp activity. Furthermore, it did not compete with digoxin and therefore is unlikely to be a Pgp substrate, further supporting the data showing that its permeability is not Pgp-dependent in the cell model and experimental conditions used in the study.

[0170] Example 2 [2- 14 Quantitative tissue distribution of total radioactivity in rats after a single oral dose of [C]PX811019 Study Objective: The objective of this in vivo study was to provide quantitative information on the tissue distribution of the copper-depriving compound triethylenetetramine disuccinate following oral administration to male albino and male pigmented rats. Whole-body phosphorimaging (WBPI) was performed on whole-body sections taken from three albino male rats sacrificed at 1, 3, 8, and 24 hours post-dosing and from one pigmented rat at 1, 8, 24, 72, 168, and 336 hours post-dosing. Tissue radioactivity concentrations within individual sections were quantified using a phosphorimager system. Annotated images of selected sections at each time point were generated using a supplementary software package designed for this purpose. Terminal blood samples were taken from all animals immediately prior to sacrifice and analyzed for radioactivity. The study was conducted in compliance with Good Laboratory Practice (GLP).

[0171] Test material: Supplied by Selcia as a solid of 99.6% radiochemical purity [2- 14 [C]PX811019 (radiolabeled triethylenetetramine disuccinate). Authenticity and radiochemical purity were determined using high performance liquid chromatography (HPLC) at Aptuit prior to use in this study.

[0172] Analytical reagents: Liquid scintillant, Gold Star™, was obtained from Meridian (Epsom, Surrey, UK), Ultima Gold™ and Permafluor® E+ were obtained from PerkinElmer LAS (UK) Ltd. CO2 absorbing solution Carbo Sorb® E was also obtained from PerkinElmer LAS (UK) Ltd. Unless otherwise stated, all other analytical reagents were of at least standard analytical laboratory reagent grade and were primarily obtained from VWR International Ltd (Poole, Dorset, UK) and Sigma-Aldrich Company Ltd (Poole, Dorset, UK). Deionized water was prepared in-house.

[0173] [Table 9]

[0174] Animals were uniquely identified by marking their tails with indelible ink and animal numbers were allocated arbitrarily. All studies were conducted in accordance with the Animals (Scientific Procedures) Act 1986, UK Home Office guidance on the implementation of the act and all applicable codes of practice for the care and housing of laboratory animals. The facilities used were fully accredited by the Association for the Accreditation of Laboratory Animal Care (AAALAC). Animal health was assessed prior to the study and all animals were deemed healthy and suitable for experimental use.

[0175] Study design: Each rat was administered 10 mg / kg free base at a target dose level of [2- 14Each rat received a single oral dose of [C]PX811019. Quantitative whole-body phosphorimaging (QWBPI) was performed on whole-body sections taken from three albino male rats sacrificed at 1, 3, 8, and 24 hours post-dosing, and from one pigmented rat at 1, 8, 24, 72, 168, and 336 hours post-dosing. Tissue radioactivity concentrations within individual sections were quantified and annotated, and representative images of selected sections were generated at each time point. Terminal blood samples were taken from all animals immediately prior to sacrifice and analyzed for radioactivity.

[0176] result

[0177] Radiochemical purity: Before use, [2- 14 The radiochemical purity of [C]PX811019 was determined to be 97.7% with a single impurity of 0.9%. The mean radioactive concentration of the formulation was determined to be 22.2 μCi / g (0.82 MBq / g) at the time of dosing and was formulated with [2- 14 The mean specific activity of [C]PX811019 was determined to be 22.2 μCi / mg (0.82 MBq / mg).

[0178] Administered doses: Administered doses ranged between 9.96 and 10.2 mg / kg for PX811019. Radiation doses ranged from 8.14 to 8.32 MBq / kg.

[0179] Animal observation and environmental control: During the survival period, 14 No animal observations were made that could be attributed to administration of [C]PX811019. During the survival phase, temperature and relative humidity in the rooms housing the animals ranged between 20°C and 22°C and 67% and 90%, respectively.

[0180] Tissue distribution of radioactivity after oral administration: [2- 14 The mean tissue concentrations of radioactivity in male albino rats following oral administration of [C]PX811019 are presented in Table 1. [Table 1-1] [Table 1-2]

[0181] At the target dose level of 10 mg / kg free base, 14 Tissue concentrations of radioactivity in male pigmented rats following oral administration of [C]PX811019 are presented in Table 2. [Table 2-1] [Table 2-2]

[0182] Tissue:blood ratios in male albino and pigmented rats are presented in Tables 3 and 4, respectively. [Table 3] [Table 4]

[0183] Presumably, high concentrations of radioactivity were found in the gastrointestinal tract after oral dosing (473267 ng equivalents / g in colon contents 8 hours post-dose, 437329 ng equivalents / g in cecal contents 3 hours post-dose, 317629 ng equivalents / g in stomach contents 1 hour post-dose, and 268536 ng equivalents / g in small intestinal contents 1 hour post-dose). Urinary concentrations were also high, with a maximum level of 104341 ng equivalents / g observed 3 hours post-dose. All values ​​reported above are for male albino rats and are generally representative of pigmented animals.

[0184] In male albino rats, maximum tissue concentrations of radioactivity were achieved, as measured by cellular uptake, involving approximately 44% of the tissues measured at 1 hour post-dose and an additional 30% at 3 hours post-dose. At 1 hour post-dose, absorption appeared to be ongoing in approximately half of the tissues. The highest levels of radioactivity were found in the kidney medulla, prostate, liver, pituitary gland and lungs (6760, 5361, 3034, 866 and 801 ng equivalents / g, respectively), compared to a cardiac blood concentration of 694 ng equivalents / g. The liver and kidney medulla had their greatest concentrations at this time point. At 3 hours post-dose, the highest levels of radioactivity were associated with the prostate, kidney cortex, kidney medulla, liver and submandibular salivary gland (6632, 6617, 4444, 2231 and 1018 ng equivalents / g, respectively), compared to a cardiac blood concentration of 290 ng equivalents / g. Levels of radioactivity in the prostate appeared high at this time point, but this was considered to be the result of urinary contamination. At 8 hours post-dose, radioactivity concentrations in tissues were lower than their maximum values, with the exception of the Harderian gland (535 ng equivalents / g), thymus (845 ng equivalents / g), and thyroid (683 ng equivalents / g), which had their maximum tissue concentrations at this time point. The highest levels of radioactivity were associated with the kidney cortex, kidney medulla, liver, prostate, thymus, and thyroid (3783, 3733, 1416, 920, 845, and 683 ng equivalents / g, respectively), compared with a cardiac blood concentration of 215 ng equivalents / g. At 24 hours post-dose, clearance of radioactivity was ongoing, with approximately 80% of the tissues at or below the limit of quantification. The highest levels of radioactivity were observed in the kidney medulla, kidney cortex, and thymus (1054, 1036, and 438 ng equivalents / g, respectively).

[0185] Tissue:blood ratios in male albino rats, where calculable, ranged between 0.28 (testes) and 22.9 (prostate) at 1 and 3 hours post-dosing, respectively. The majority of tissues had tissue:blood ratios greater than 1, with the highest ratios calculated at 3, 3, 8, and 3 hours post-dosing in the prostate (22.9), kidney cortex (22.8), kidney medulla (17.4), and liver (7.69), respectively. As detailed above, it was thought that prostate levels were likely high in relation to urinary contamination. However, the majority of tissue:blood ratios at 1 hour post-dosing were less than 1, and the ratios tended to increase over time. This may indicate slower uptake and release by tissues compared to blood.

[0186] The distribution of radioactivity in male pigmented rats was similar to that observed in male albino rats. In male pigmented rats, maximum tissue concentrations of radioactivity were evenly distributed between the 1- and 8-hour time points. Maximum levels were achieved in approximately 40% of the tissues measured at 1 hour post-dose and an additional 45% at 8 hours post-dose. At 1 hour post-dose, absorption appeared to be ongoing in approximately half of the tissues. The highest levels of radioactivity were seen in the kidney cortex, kidney medulla, and liver (4797, 2648, and 1299 ng equivalents / g, respectively), compared with a cardiac blood concentration of 410 ng equivalents / g. The liver and kidneys had their maximum concentrations at this time point. At 8 hours post-dose, the highest levels of radioactivity were associated with kidney cortex, kidney medulla, spleen, submandibular salivary gland, liver, Harderian gland, and thymus (1293, 1236, 905, 651, 615, 590, and 572 ng equivalents / g, respectively), compared with a cardiac blood concentration of 151 ng equivalents / g. At 24 hours post-dose, clearance was ongoing, with approximately half of the tissues measured having levels of radioactivity below the limit of quantification. The highest levels were associated with kidney medulla and kidney cortex (727 and 565 ng equivalents / g, respectively). At 72 hours post-dose, clearance of radioactivity was nearly complete, with approximately 65% ​​of the tissues below the limit of quantification. The highest levels of radioactivity were observed in kidney cortex, kidney medulla, and pancreas (277, 233, and 200 ng equivalents / g, respectively).

[0187] At 168 hours post-dose, elimination appeared to be complete and all levels of radioactivity in tissues were below the limit of quantification.

[0188] Tissue:blood ratios in male pigmented rats, where calculable, ranged between 0.33 (white fat) and 11.9 (kidney medulla) at 1 and 24 h post-dose, respectively. The majority of tissues had tissue:blood ratios greater than 1, with the highest ratios calculated at 24, 1, 8, and 8 h post-dose in kidney medulla (11.9), kidney cortex (11.7), spleen (5.99), and liver (4.07), respectively. However, the majority of tissue:blood ratios at 1 h post-dose were less than 1, and the ratios tended to increase over time. This may indicate slower uptake and release by tissues compared to blood.

[0189] The levels of radioactivity in blood measured by QWBPI were compared with values ​​obtained by sample combustion of blood samples taken immediately before sacrifice. Similar trends and magnitudes were evident between the values ​​obtained by QWBPI measurements and by sample combustion. Blood levels at 1 hour after administration were 694 and 527 ng equivalents / g by QWBPI quantification and by sample combustion, respectively, for male albino rats, and 410 and 332 ng equivalents / g by QWBPI quantification and by sample combustion, respectively, for male pigmented animals.

[0190] To summarize some aspects of this study, it is noted that high concentrations of radioactivity were observed in the gastrointestinal tract contents after oral administration. In male albino rats, the absorption of radioactivity was rapid, with measurable levels of radioactivity present in the majority of tissues at 1 hour after dosing. Maximum levels of radioactivity were reached in approximately 44% of tissues at 1 hour after dosing, and further reached maximum levels in 30% of tissues at 3 hours after dosing. Maximum tissue concentrations of radioactivity were reached in kidney medulla, prostate, kidney cortex, and liver (6760, 6632, 6617, and 3034 ng equivalents / g, respectively) at 1, 3, 3, and 1 hour after dosing, respectively. Although the level of radioactivity in the prostate appeared to be high, this was considered to be the result of urinary contamination.

[0191] The concentration and distribution of radioactivity in male pigmented rats was similar to that seen in male albino rats. Maximal levels of radioactivity were reached in approximately 40% of the tissues at 1 hour post-dose, and further reached maximum levels in 45% of the tissues at 8 hours post-dose. The highest levels were associated with the kidney cortex, kidney medulla, and liver (4797, 2648, and 1299 ng equivalents / g, respectively), all at 1 hour post-dose.

[0192] Conclusions: The distribution and concentration of total radioactivity in male albino and male pigmented rats were similar. No binding to melanin in pigmented tissues was evident.

[0193] PX811019 was rapidly absorbed and distributed, with nearly half of the tissues having maximum concentrations of radioactivity one hour after dosing, although absorption was ongoing in approximately half of the tissues. The tissue:blood ratio in the majority of tissues reached values ​​greater than 1 after the one hour time point, which may indicate slower uptake and release by tissue compared to blood.

[0194] Tissues associated with biotransformation and elimination (eg, liver and kidney) and secretory glands (eg, pancreas, submandibular salivary gland, thymus, and thyroid) tended to have higher concentrations of radioactivity.

[0195] Clearance of radioactivity from tissues was generally rapid, with a decline in tissue levels observed 24 hours after dosing and appearing to be complete in most tissues by 72 hours.

[0196] Example 3 A single-center, randomized, double-blind, single-dose, two-way crossover, dose-escalation study of the pharmacokinetics and pharmacodynamics of triethylenetetramine disuccinate (PX811019) compared with triethylenetetramine dihydrochloride in normal healthy volunteers This human clinical study provides population pharmacokinetic and pharmacodynamic modeling of triethylenetetramine, its two major metabolites, and copper excretion following oral two-way crossover administration of triethylenetetramine disuccinate and triethylenetetramine dihydrochloride to healthy adult volunteers.

[0197] The population PK analysis included samples from the study (TETA doses, 166, 499, 832 mg free base in each of the three cohorts) in which each subject received triethylenetetramine disuccinate (PX811019) and triethylenetetramine dihydrochloride (Syprine®) in a two-way crossover design. Triethylenetetramine dihydrochloride (Syprine®) is a potent copper chelator and was approved by the FDA in 1985 for the second-line treatment of Wilson's disease. Triethylenetetramine disuccinate (PX811019) is an alternative and superior salt form of triethylenetetramine, but its target dosing is unknown and cannot be known from the prior art.

[0198] A population pharmacokinetic / pharmacodynamic (PK / PD) model was used to describe the concentrations of triethylenetetramine (TETA), its two major metabolites (monoacetylated (MAT) and diacetylated (DAT) forms), and copper excretion in urine. A model with first-order absorption and two-compartment kinetics for TETA, catenary curve formation of MAT and DAT, and copper excretion directly controlled by TETA in plasma was further used to estimate absorption-related parameters separately for PX811019 and Syprine® to identify differences between the studied TETA formulations. The effects of subject-specific covariates and dose on PK / PD parameters were examined based on standard chi-square statistics. Population PK / PD modeling was performed using NONMEM software.

[0199] Objectives: The objectives of this study were to compare the pharmacokinetic (PK) profile and determine the dose relationship of triethylenetetramine (TETA) disuccinate (PX811019) compared to TETA dihydrochloride (Syprine®), and to characterize the pharmacodynamic (PD) profile of urinary copper excretion in response to the study drug.

[0200] Simplified PK / PD, Half-life, Absorption Kinetics and Bioavailability Summary: In conducting this study, it was discovered that the relative bioavailability of PX811019 compared to Syprine® was equal to 74.5%. The lag times (0.083 and 0.239 hours) and absorption rate constants (1.74 and 1.19 hours) were also significantly improved. -1) differences between Syprine and PX811019 were observed. Analysis of covariance did not identify major PK / PD differences related to dose, sex, weight, or renal function. The compounds exhibited highly stable and consistent PK / PD profiles. Some dose-dependence of TETA volume of distribution was found to produce somewhat longer half-lives at higher doses. Differences in absorption kinetics between the forms were modest, with a lower bioavailability of PX811019 (74.5%) compared to Syprine®. It was discovered that administration of approximately 134% of the dose of PX811019 would produce plasma concentrations of TETA, MAT, and DAT, as well as copper excretion rates essentially identical to Syprine®. See also Example 3.

[0201] Methodology: This study was a phase 1, prospective, randomized, double-blind, dose escalation, two-way crossover design. Up to four cohorts of six subjects per cohort were planned to be enrolled. PX811019 or Syprine® doses were to be administered to subjects within each cohort at approximately molar equivalent doses of TETA free base (approximately 166-167 mg free base per capsule). Cohort dosing is shown in Table 5. [Table 5]

[0202] After completion of each cohort, the sponsors and investigators reviewed the plasma concentration-time profiles of TETA and its acetyl metabolites [monoacetyl TETA (MAT) and diacetyl TETA (DAT)] and safety and tolerability data before approving escalation to the next cohort. Based on analysis of interim PK data, the decision was made to stop enrollment after completion of three cohorts.

[0203] There were three clinic visits: a screening visit occurring within 28 days prior to the first dose, and two treatment visits. Subjects were screened and enrolled based on medical history, clinical laboratory results, physical examination findings, vital signs assessment, and resting 12-lead electrocardiogram assessment. Eligible subjects were admitted to the study site by 20:00 the evening prior to dosing at each treatment visit. After an overnight fast, subjects were randomized to receive a single oral dose of PX811019 or a single oral dose of Syprine® on day 1 and the alternative treatment on day 8. Subjects remained confined to the study site for 48 hours after each dose (until the morning of day 3 or day 10).

[0204] Safety evaluations included adverse event (AE) assessments, physical examinations, clinical laboratory tests, and vital signs (blood pressure and pulse rate) assessments.Blood samples for determination of plasma TETA, MAT, and DAT levels were collected at time 0 (within 30 minutes before dosing), 5, 15, 30, 60, 90, 120 minutes after dosing, and 3, 4, 5, 6, 8, 10, and 12 hours thereafter on days 1 and 8, then at 16, 20, 24, 30, 36, 42, and 48 hours after dosing on days 2-3 and 9-10. Urinary copper excretion in urine collected on days 1 and 8 was measured at the following intervals: pre-dose up to -2 to 0 hours and up to 0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 12, and 12 to 16 hours post-dose, then at 16 to 20, 20 to 24, 24 to 30, 30 to 36, 36 to 42, and 42 to 48 hours post-dose on days 2 to 3 and 9 to 10.

[0205] Protocol Amendments: There was one protocol amendment during the study. Protocol amendment 1 extended the screening window from 14 to 28 days to dosing on day 1 from the screening visit to allow sufficient time for review of safety and PK data before escalation to the next cohort.

[0206] Number of Subjects: A total of 18 subjects (9 males, 9 females) were enrolled and randomized to receive a single oral dose of PX811019 and Syprine®.

[0207] Inclusion Criteria: To be eligible, subjects must have completed an IRB-approved informed consent that was administered appropriately prior to the performance of any study-related procedures and must have a body weight between 18 and 30 kg / m2, inclusive. 2 Participants had to be healthy adult men or women between 18 and 60 years of age, including the borderline age group, with a body mass index (BMI) between 0.01 and 0.05, and had to have normal renal function calculated by a creatinine clearance greater than 90 mL / min. Women of childbearing potential had to have a negative pregnancy test at the screening visit and on each admission to the study site, be willing to use an effective means of contraception for 4 weeks prior to study drug administration, and be non-lactating. Men had to be willing to use an effective barrier method of contraception for 4 weeks after study drug administration. Subjects were excluded if they were smokers, had a history of drug or alcohol abuse, had participated in a clinical research study within 30 days prior to the first dose of study drug, or had donated a pint or more of blood within 56 days prior to the first dose of study drug or plasma within 14 days; had used iron, copper or other dietary supplements within 2 weeks prior to the first dose of study drug or during the study; had required prescription or over-the-counter medications or herbal or dietary supplements within 1 week prior to the first dose of study drug or during the study; had a history of systemic lupus erythematosus, sideroblastic anemia, dystonia, muscle spasms or myasthenia gravis, or a history of therapeutic anticoagulation; had a known allergy to TETA or formulation excipients; had pulmonary abnormalities evident on clinical examination; or had clinical laboratory results at the Screening Visit indicating any of the following: iron deficiency based on plasma iron, iron binding capacity and ferritin levels, copper deficiency based on low levels of plasma copper or ceruloplasmin, abnormal liver function test results, or a ≥100×10 6 Patients were excluded if they had a clinical diagnosis of a platelet count less than / L.

[0208] Test products, doses, and mode of administration: TETA disuccinate (PX811019) was supplied in 435 mg capsules, and TETA dihydrochloride (Syprine®) was supplied in 250 mg capsules, each equivalent to an approximately equimolar dose of TETA free base. Capsules of the two formulations were similar in size and shape, but not identical in appearance. To maintain the integrity of the blinding, subjects were blinded to study medication administered by a designated pharmacist or subinvestigator who was not otherwise involved in the conduct of the study, and subjects were not allowed to directly handle the capsules. Capsules were administered with 240 mL of water at approximately 08:00 on days 1 and 8 after an overnight fast.

[0209] Duration of Treatment: The study included a screening visit within 28 days prior to the first dose of study medication, and two treatment visits separated by 7 days, each of which required 3 consecutive nights.

[0210] Evaluation criteria: PK profiles of PX811019 and Syprine® were evaluated by analysis of plasma concentrations of TETA and its metabolites, MAT and DAT, after a single oral dose of both formulations. Pharmacodynamic parameters were evaluated by determination of urinary copper excretion after a single oral dose of both formulations. Safety was evaluated by assessing the frequency of treatment-emergent adverse events (AEs), discontinuations due to AEs, physical examination findings, changes in vital signs, and clinical laboratory test results.

[0211] Statistical Methods: PK parameters (C max , T max , AUC 0-24 , AUC 0-t , AUC 0-inf The dose-response relationships of PX811019 and Syprine® were analyzed by non-compartmental methods. The plasma concentration-time curves and C for TETA, MAT, and DAT for both formulations were analyzed. maxand the AUC for plasma TETA based on equimolar doses of TETA free base. 0-t Summary statistics for the pharmacokinetic parameters and mean urinary Cu excretion were calculated for each formulation. AUC 0-24 , AUC 0-t , AUC 0-inf and C max Summary statistics (mean, median, standard error, minimum and maximum) for plasma concentrations and urinary Cu excretion were calculated for each formulation at the appropriate sample collection times.

[0212] Safety data including adverse events, vital sign assessments, clinical laboratory evaluations and physical examinations were summarized by formulation and dose cohort. Adverse events were coded using the MedDRA dictionary. Subject-specific adverse event data listings are provided, including verbatim terms, preferred terms and system organ class, as well as severity, relationship to treatment and actions taken. Concomitant medications are listed by subject and coded using the WHO drug dictionary. Descriptive statistics (arithmetic mean, standard error, median, minimum and maximum) were calculated using SAS.

[0213] Results: A total of 18 eligible subjects (9 males and 9 females) between the ages of 20-48 years were enrolled and randomized to receive study medication. Seventeen (94.4%) subjects completed the study, with one (5.6%) subject in cohort 3 withdrawing due to an adverse event following administration of PX811019 during the first treatment visit.

[0214] Demographics: Enrolled subjects were representative of the healthy adult population, ranging from 20 to 48 years. The overall mean (SD) age of enrolled subjects was 34.3 (8.14) years, and the racial distribution was 4 (22.2%) White, 6 (33.3%) Black, 7 (38.9%) Latino / Hispanic, and 1 (5.6%) American Indian / Alaska Native. Mean (SD) height and weight were 169.5 (8.46) cm and 73.9 (11.41) kg, respectively, and the mean (SD) BMI was 25.7 (3.27) kg / m.

[0215] Safety results: Treatment-emergent AEs: Five subjects reported treatment-emergent adverse events, 3 of 17 (17.6%) subjects reported AEs after receiving Syprine®, and 2 of 18 (11.1%) subjects reported AEs after receiving PX811019. AEs reported after administration of Syprine® included headache, diarrhea, and nausea. AEs reported after administration of PX811019 included headache, diarrhea, and elevated liver enzymes. All AEs were mild or moderate in intensity and resolved before withdrawal from the study, and no serious AEs were reported. One subject in cohort 3 discontinued the study due to mild, reversible elevated liver enzymes after administration of PX811019 (2175 mg) during the first treatment visit.

[0216] Other Safety Assessments: Based on sitting blood pressure and pulse rate, no clinically significant hemodynamic effects attributable to study drug were observed.

[0217] No clinically significant changes in laboratory test parameters were observed, except for one subject receiving PX811019 (2175 mg) who reported having mild reversible elevations in liver enzymes that were deemed possibly related to study drug.

[0218] Pharmacokinetic Results: The mean pharmacokinetic parameters for TETA, MAT and DAT following a single equimolar oral dose in Cohorts 1, 2 and 3 are listed in Table 6 below and further described in Example 3. [Table 6]

[0219] TETA's penalty kick: C of TETA relative to Syprine® following administration of PX811019 to subjects in cohorts 1, 2, and 3 max The ratios were 0.58, 0.59 and 0.55, respectively, and the AUC 0-t and AUC 0-inf The ratios were 0.66-0.68, 0.64-0.65, and 0.55, respectively. AUC of TETA vs. Syprine® after PX811019 for subjects in all three dose cohorts 0-24 The ratio was also low.

[0220] The mean elimination t1 / 2 of TETA following administration of PX811019 and Syprine® to subjects in cohort 1 was 8.4 and 18.8 hours, respectively, and ranged from 21.8 to 26.9 hours following administration of PX811019 and Syprine® to subjects in cohorts 2 and 3. Effective t1 / 2 values ​​were approximately one-third to one-quarter of the elimination t1 / 2 values ​​in the three dose cohorts and were independent of formulation, with the exception of PX811019 in cohort 1, which was approximately half (8.4 hours vs. 4.5 hours). Median T max Values ​​ranged between 1.25 and 2.0 hours for all three dose cohorts.

[0221] MAT PK: C of MAT following administration of PX811019 to subjects in cohorts 1, 2, and 3 relative to Syprine® maxThe ratios were 0.87, 0.75, and 0.91, respectively. AUC vs. Syprine® after administration of PX811019 0-t and AUC 0-inf The ratios were 0.74-0.76, 0.74-0.75, and 0.84, respectively. The mean t1 / 2 values ​​for MAT following administration of PX811019 and Syprine® to subjects in cohort 1 were 16 and 22 hours, respectively, and 17-18 hours following administration of PX811019 and Syprine® to subjects in cohorts 2 and 3. Exposure to MAT as measured by AUC was approximately 2-3 times higher compared to TETA at all three dose levels. The C of MAT max C of TETA following administration of PX811019 and Syprine® to subjects in cohort 1 max The median MAT T max The T for both formulations was 5.0-5.5 hours for subjects in all three dose cohorts, and was longer than the T for the parent compound. max It happened later than that.

[0222] DAT's PK: DAT C max The AUCs of DAT were generally 2-3 times lower than those of TETA and 3-4 times lower than those of MAT. The AUCs of DAT were also lower than those of MAT for both parent drugs and both formulations. The C of DAT for PX811019 formulations versus Syprine® max The ratios ranged from 0.71 (cohort 1) to 0.88 (cohort 3), while the AUC ratios ranged from 0.72 (cohort 1) to 0.84 (cohort 3). max The value is T for MAT max (5.0-6.0 hours).

[0223] Pharmacodynamic results The majority of cupuria occurred during the first 6 hours after dosing for all dose groups. The level of cupuria increased with increasing dose of Syprine® from 250 to 1250 mg, was similar to that of PX811019 at 435 and 1305 mg, and was increased at the highest dose of 2175 mg.

[0224] conclusion Single oral doses of Syprine® (250, 750, 1250 mg) and PX811019 (435, 1305, 2175 mg) were both safe and well tolerated in these healthy adult male and female subjects.

[0225] Adverse events were reported in three (17.6%) subjects after administration of Syprine® and in two (11.1%) subjects after administration of PX811019 and included headache, nausea, diarrhea, and elevated liver enzymes. Adverse events were either mild or moderate in intensity; no serious adverse events were reported.

[0226] One subject discontinued participation in the study due to mild, reversible increases in liver enzymes following treatment with 2175 mg of PX811019.

[0227] Based on sitting blood pressure and pulse rate, no clinically significant hemodynamic effects attributable to study drug were observed.

[0228] No clinically significant changes in laboratory test parameters were observed, except for one subject receiving 2175 mg of PX811019 who reported having mild reversible elevations in liver enzymes that were considered possibly related to study drug.

[0229] The majority of cupriuria occurred during the first 6 hours after dosing for all dose groups, and cupriuria increased with increasing dose level. No clear differences in urinary copper excretion between formulations were evident.

[0230] TETA's Cmax was 41-45% lower following single oral doses of PX811019 formulations at all three dose levels tested, compared with administration of an equimolar dose of Syprine®.

[0231] AUC 0-t and AUC 0-inf was 34-45% lower following single oral doses of PX811019 formulations at all three dose levels compared with administration of an equimolar dose of Syprine®.

[0232] C for metabolites MAT and DAT max and AUC values ​​were lower following single oral doses of PX811019 formulations at all three dose levels compared to administration of an equimolar dose of Syprine®.

[0233] Overall conclusion Single oral doses of PX811019 (435, 1305, 2175 mg) and Syprine® (250, 750, 1250 mg) were safe and well tolerated in these healthy adult male and female subjects. Adverse events were either mild or moderate in intensity, and no serious adverse events were reported. One subject discontinued participation in the study due to a mild, reversible increase in liver enzymes following treatment with 2175 mg of PX811019. The C of TETA was significantly increased following single oral doses of the PX811019 formulations at the three dose levels tested, compared to administration of an equimolar dose of Syprine®. max was 41-45% lower, and the AUC of TETA 0-t and AUC 0-inf The urinary excretion of copper was 34-45% lower in the two preparations.

[0234] Triethylenetetramine disuccinate 1200 mg / day given as 600 mg twice daily would be expected to produce a significant cupuric effect with minimal side effects and negligible adverse effects on serum copper levels or other laboratory test parameters throughout the dosing interval.

[0235] Example 4 Population pharmacokinetic and pharmacodynamic modelling of triethylenetetramine The data analyzed in this report were obtained in Study Example 3, a double-blind, dose-escalation, two-way crossover design study comparing TETA disuccinate (PX811019) with TETA dihydrochloride (Syprine®). Study Example 3 demonstrated that administration of TETA as the disuccinate salt resulted in lower exposure indices (C max It has been demonstrated that population-based modeling provides a comparative absorption kinetics and a more comprehensive assessment of the relative bioavailability of the two salt forms of TETA in the context of the study design established in Example 3.

[0236] In the analysis of Example 3, model-based population analysis is applied to the data to obtain an overall assessment of the pharmacokinetics of TETA, MAT and DAT, to further assess the pharmacodynamics of urinary copper excretion, to consider potential covariates with PK / PD parameters such as gender, age and dose, and in comparing the PK / PD of Syprine® and PX811019 from Example 2, particularly with respect to bioavailability.

[0237] Study Analysis: A population PK / PD model for TETA and its metabolites was developed based on data obtained from the study in Example 3 (a single-center, randomized, double-blind, single-dose, two-way crossover, dose-escalation study of the pharmacokinetics and pharmacodynamics of triethylenetetramine disuccinate (PX811019) compared to triethylenetetramine dihydrochloride in normal healthy volunteers), in which subjects were randomized to receive either a single oral dose of PX811019 or a single oral dose of Syprine® on day 1 and the alternative treatment on day 8. Data from three cohorts of six subjects per cohort were available. Only day 1 PD data was available for one subject in cohort 3. PX811019 or Syprine doses were administered to subjects within each cohort at approximately molar equivalent doses of TETA free base (approximately 166-167 mg free base per capsule) at doses specified in Table 5 in Example 2.

[0238] Blood samples for determination of plasma TETA, MAT and DAT concentrations were collected at time 0 (within 30 min prior to dosing), 5, 15, 30, 60, 90, 120 min post-dose and 3, 4, 5, 6, 8, 10, 12, 16 and 20 h thereafter on days 1 and 8, then at 24, 30, 36, 42 and 48 h post-dose on days 2-3 and 9-10. Urinary copper excretion was measured via urine collection on days 1 and 8 at the following intervals: -2 to 0 h (pre-dose) and 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-16, 16-20 and 20-24 h post-dose, then at 24-30, 30-36, 36-42 and 42-48 h post-dose on days 2-3 and 9-10.

[0239] Population pharmacokinetic / pharmacodynamic analysis Data handling for PK / PD analysis: PK samples were analyzed using a validated bioanalytical LC / MS / MS method for the simultaneous determination of triethylenetetramine and its two major metabolites in human serum. Triethylenetetramine (TETA) and the two major TETA-derived metabolites: N1-acetyltriethylenetetramine (MAT) and N1,N10-diacetyltriethylenetetramine (DAT) were measured. Assay LLOQs were 0.005 mg / L for TETA, MAT, and DAT. Urine samples were collected for copper analysis, which served as the pharmacodynamic endpoint. Concentrations below the limit of quantification (BLQ) were handled using the Beal M3 method with the F-flag option. Beal, SL, Ways to fit a PK model with some data below the quantification limit. J Pharmacokinet Pharmacodyn. 2001, 28:481-504.

[0240] Population PK / PD Methods: Population nonlinear mixed-effects modeling was performed using NONMEM (version 7.3.0, Icon Development Solutions, Ellicott City, MD, USA) with the GFortran compiler 9.0. NONMEM operations were performed using Wings for NONMEM (WFN730, http: / / wfn.sourceforge.net). The Laplacian estimation method was used. The differential equations of the model were solved using the ADVAN6 PREDPP subroutine. NONMEM data processing and plotting were performed with Matlab® software version 7.0 (The MathWorks, Inc., Natick, MA, USA).

[0241] The minimum of the NONMEM objective function, typical goodness-of-fit diagnostic plots, and assessment of precision and variability estimates of pharmacokinetic parameters were used to distinguish between different models during the model building process.

[0242] Population PK / PD Model: A PK / PD model (Figure 1) was used to describe TETA, MAT and DAT concentrations and the amount of copper excreted in urine. This is a first-order absorption, two-compartment pharmacokinetic model for TETA and a catenary curve one-compartment pharmacokinetic model for MAT and DAT. A series of transition compartments was used to describe the delay between TETA and MAT concentrations. The following equation was used for PK:

number

number

[0243] The initial conditions for equations 1-8 are: A T (0)=D;C P,T (0) = 0; C T,T (0) = 0; C P,M (0)=0;A 1,M (0)=0;A 2,M (0)=0;A 3,M (0)=0; and C P,D (0)=0. F represents the estimated bioavailability of TETA (Syprine); F P / S shows the relative bioavailability of PX811019 to Syprine; P,T、 C P,M、 C P,D are the concentrations of TETA, MAT and DAT in plasma; C T,T is the concentration of TETA in the peripheral compartment; CL T , C.L. M , C.L. D is the total body clearance of TETA, MAT and DAT; Q T is the distribution clearance of TETA; V P,T , V P,M , V P,D are the volumes of distribution for TETA, MAT and DAT; V T,Tis the peripheral distribution volume for TETA; MTT is the mean transit time that describes the delay between TETA and MAT concentrations. The model was tested with 0 to 3 transition steps. M and fr T is the fraction of TETA metabolized to MAT and MAT metabolized to DAT. The molecular masses for TETA, MAT, and DAT (146, 188, and 233 g / mol) were used to convert the mass changes (i.e., TETA equivalents) between the parent compound and metabolites. CONV was equal to 0.5721 (250 / 435) for PX811019 and 1 for Syprine and was used to convert the mass of PX811019 to Syprine equivalents.

[0244] The actual parameters of the generated model are: CL for TETA T / F, Q T / F, V P,T / F, V T,T / F;MAT is CL M / F / f rM and V P,M / F / f rM ; and CL for DAT D / F / f rM / fr D and V P,D / F / f rM / f rD which reflects the administration of an oral dose with uncertain bioavailability (F) and the conversion of TETA to MAT and MAT to TETA (f r ) was due to the inability to identify additional lag times (t lag ) was used to account for the delay in the ascending phase of the TETA concentration-time profile observed after oral dosing. The half-life value (t 0.5 ) was calculated from these parameters.

[0245] Pharmacodynamics were modeled assuming a linear relationship between TETA plasma concentrations and urinary copper excretion (Cho HY, Blum RA, Sunderland T, Cooper GJS, and Jusko WJ, Pharmacokinetic and pharmacodynamics modeling of a copper-selective chelator (TETA) in healthy adults, J Clin Pharmacol 2009, 49:916-928):

number

[0246] From equation (9), the mass of copper excreted (Cu(t)) was integrated over the copper excretion rate for each urine collection interval. In graphical representation, ER(t) was approximated as the amount of copper excreted (experimental or model predicted) over the urine collection interval:

number

[0247] Inter-individual (IIV) and inter-ocular (IOV) variability for PK parameters was modeled assuming a log-normal distribution:

number

[0248] In the analysis, k th i in time th For individuals, TETA, MAT, and DAT concentrations and urinary copper content were randomly determined. th Observation, time t j C measured at ijk teeth:

number

number

number

number

number

number

number

number

[0249] Visual Prediction Check: Model performance was assessed using visual predictive check (VPC). VPC was calculated based on 1000 datasets simulated with the final parameter estimates [7-9]. VPC allows the comparison of predicted data against observed data over time. In this study, the 10th, 50th and 90th percentiles were used to summarise the data and VPC predictions. VPC allows the comparison of confidence intervals obtained from the predictions with observed data over time. If the corresponding percentiles from the observed data fall outside the 90% confidence interval derived from the predictions, this indicates a misconfiguration of the model.

[0250] Analysis of covariance: One objective of this work was to characterize possible PK / PD differences for TETA given as PX811019 versus Syprine®. Thus, all absorption-related parameters (lag time and k a ) were estimated separately for each formulation. Other parameters were assumed to be identical between drug formulations unless some evidence to the contrary was found during the model building process.

[0251] Other possible relationships were sought using standard analysis of covariance, where individual (post-hoc) estimates of PK / PD parameters (eta (η) or kappa (κ)) were plotted against available covariates (weight, age, eGFR, sex, dose, sequence, duration, formulation) to identify their potential effects. If relationships were found, all recorded values ​​were fit to the following regression model: Pik =θ P1 (1+θ P2 (COV ik -COV median ))exp(η P,i +κ P,k ) (Equation 17) where θ P1 and θ P2 are the regression coefficients. Continuous variables are centered around their median, COVmedian, and therefore, θ P1 allows us to express parameter estimates for a typical patient with median covariates. Models based on indicator variables included categorical covariates (e.g., sex):

number

[0252] Results and Discussion Data analyzed from 18 subjects contained 714 plasma concentration measurements for each of TETA, MAT, and DAT, and 455 copper measurements in urine. There were 124 (17.4%) TETA, 113 (15.8%) MAT, and 187 (26.2%) DAT measurements below the limit of quantification (BQL).

[0253] Table 7 presents an overview of subject characteristics and available covariates. [Table 7]

[0254] The group of nine men and nine women had a median age of 34 years, with a range of 20 to 48 years. Body weight ranged from 57.3 to 93.6 kg. All subjects had normal renal function, with estimated glomerular filtration rates (eGFR) ranging from 91.3 to 158.8 ml / min.

[0255] Table 6 in Example 2 provides an overview of the main exposure indices for TETA, MAT, and DAT using traditional non-compartmental (NCA) analysis. max From the and AUC values, it is clear that these equimolar doses of TETA produce lower concentrations of these three compounds when administered as PX811019. However, since the NCA does not adequately account for the BLQ values ​​at later times, any parameter that depends on such (e.g., t 0.5 ) may be skewed. Because this study included a range of doses and joint measurements of the parent drug, two metabolites, and copper excretion, this population-based analysis was designed to compare the two salt forms in this overall, more generalized fashion.

[0256] First, a PK / PD model was used to describe data from a multiple-dose study in healthy volunteers. This is a two-compartment pharmacokinetic model with first-order absorption for the PK of TETA. The metabolism of TETA was modeled assuming a hanging curve metabolism (TETA→MAT→DAT). In addition, three transition steps were used to model the delay between TETA and MAT concentrations. A one-compartment pharmacokinetic model was assumed for both MAT and DAT plasma concentrations. Urinary copper was modeled as a direct linear connection with TETA plasma concentrations as previously found. Cho, HY, et al., Pharmacokinetic and pharmacodynamics modeling of a copper-selective chelator (TETA) in healthy adults, J Clin Pharmacol 2009, 49:916-928.

[0257] The following modifications were applied to the Cho, HY, et al. model: (i) no additional parts of the residual error model were required; (ii) an estimate of inter-individual variability for ER0 (baseline copper excretion rate) was included due to the larger variability in this study; (iii) reparameterization of equation (9) to ER0 and SL ER0 as independent parameters; (iv) inclusion of correlations between apparent volume of distribution and clearance for MAT and DAT; (v) modeling the IOV for absorption rate constants as part of the central objective of this study; and (vi) dose-dependence of TETA peripheral volume of distribution was seen. Each of those steps significantly improved the model fit as judged by the NONMEM objective function and visual prediction checks.

[0258] The experimental data and model fits of plasma concentrations of TETA, MAT and DAT and copper excretion throughout the study were graphed for each of the subjects (subject graphs).

[0259] Modeling time-interval variability in estimated systemic bioavailability (F) and absorption rate constants was used as a surrogate to account for overall within-subject variability in TETA pharmacokinetics. Such inclusion of time-interval variability avoids bias in population parameter estimates. Bergstrand, M, et al., Prediction-corrected visual predictive checks for diagnosing nonlinear mixed-effects models AAPS J. 2001, 13: 143-151.

[0260] Model fitting on object graphs demonstrated that the final PK model accurately described the measured concentrations and PD responses. Representative goodness-of-fit diagnostic plots were prepared for the final model. Individual and population predictions to observed concentrations were relatively symmetrically distributed around the line of identity, and individual weighted residuals to individual predicted concentrations and over time did not show any trends and were relatively evenly distributed around zero, indicating good model performance in quantifying the PK data.

[0261] VPC plots for TETA, MAT, and DAT concentrations and the amount of copper excreted in urine were used to assess the quality of the model and fitted parameters. The VPC plots indicated that not only were both the central tendency of the data and the variability at specific sample collection times recaptured, but most of the data points were within the 90% confidence interval. There were no major misspecifications in the model fits for measurements and percentages of concentrations below the LLOQ.

[0262] The model fitted population PK / PD parameters for TETA and metabolites are listed below in Tables 8A-8D. [Table 8-1] [Table 8-2]

[0263] PK / PD parameters, between-subject, time-period and residual error variance were all well estimated with CV values ​​less than 57.6%. The apparent mean central volume of distribution was 326 L for TETA, 426 L for MAT and 197 L for DAT. The corresponding apparent clearances were 141, 76.2 and 306 L / h. For TETA, the apparent peripheral volume was 1210 L at the 750 mg dose and the apparent distribution clearance was 39.2 L / h. The relative bioavailability of PX811019 to Syprine® was 74.5%. The absorption rate constant was 1.19 h for PX811019. -1 and 1.74 hours for Syprine® -1 and the time lags were 0.239 and 0.083 hours. The mean transit time (MTT) associated with the conversion of TETA to MAT was 0.381 hours, reflecting a short delay in the appearance of MAT. The baseline copper excretion rate was 0.581 μg / hour. Copper excretion was 41.8 (mg / L). -1 The concentration of TETA increased linearly with a slope (SL) of 0.01.

[0264] Interindividual variability (IIV) was generally modest to moderate and could be identified for all central distribution volumes (16.3%, 55.1% and 86.8% for TETA, MAT and DAT), for all apparent total body clearances (10.3%, 41.5% and 55.9% for TETA, MAT and DAT), for the volumes of the peripheral compartment for TETA (13.5%), for ER0 (112%) and for SL ER0 (58.1%). For other parameters, IIV was fixed at zero since it either tended to be zero or a large (>50%) reduction was estimated during the model building process.

[0265] Repeated administration of a drug leads to the occurrence of IOV. When this process was included in the model building process, it substantially improved the model fit. The IOV for the estimated F and absorption rate constants were moderate and equal to 44% and 70%.

[0266] V T,T A significant relationship was found between / F and TETA dose expressed in Syprine equivalents (p=9.0224e-05, ΔOFV=15.331). Model predicted V T,T / F increased by 6.37% for every 100 mg difference from the 750 mg dose of TETA. This factor may account for the modest increase in half-life with dose (Table 6 in Example 13). In the final model, the individual values ​​of parameters with IIV were closely estimated, as indicated by the very low reduction of less than 20% (V, whose reduction was equal to 31%). T,T / F). Savic, RM and Karlsson, MO. The importance of shrinkage in empirical Bayes estimates for diagnostics: problems and solutions. AAPS J. 2009; 11: 558-69.This suggests that the data are informative about individual predicted parameters that allow the search for other covariate relationships. Relationships between weight, age, eGFR, sex and sequence factors were determined using individual estimates for ETA of TETA PK / PD parameters related to subject sex based on ETA plots (deviation of individual estimates from the population mean). Similarly, relationships between formulation and time factors were determined based on KAPPA plots (deviation of estimates at specific times (visits) from the individual mean PK parameters) of TETA PK / PD parameters related to formulation and time. The lack of any trends in these data indicates that these individual covariates do not explain the remaining unexplained inter-subject variability in PK / PD parameters.

[0267] Individual TETA, MAT, and DAT concentrations and copper excretion rates versus time were also graphed jointly for six representative subjects. Drug and metabolite concentrations and copper excretion rates over the entire study period appear to be similar and consistent for all subjects.

[0268] Model fitted half-life values ​​were added to Table 6 in Example 2 for comparison with NCA values. Beta for TETA 0.5 is the V with dose T The increased V increased with dose from 18 to 28 to 37 hours. The LLOQ of the assay was increased, allowing for more extended and reliable measurements during the washout period in this study. T The increase in t is usually explained by either nonlinear plasma protein binding or an increase in tissue binding with drug concentration. 0.5 The values ​​reflect the theoretical disposition rates that would be expected for these compounds if administered directly. Their actual terminal slopes are dominated by the "formation rate limited disposition" from TETA and as such are determined in a process of global congruent fitting of the data.

[0269] The PK / PD model applied to copper excretion showed a highly consistent relationship between TETA concentration and copper excretion that was superimposable for Syprine® and PX811019 in each subject across all doses. One subject had an abnormally high baseline and copper excretion rate that was affected by TETA.

[0270] Overall, administration of TETA as the succinate salt produces a generally linear profile and PK / PD profile indistinguishable from TETA given as the dihydrochloride salt, except for the lower systemic exposure reflected as a relative bioavailability of 74.5%. The absorption kinetics of the two forms differ, but only slightly. The lower C observed in a preliminary analysis of these data with PX811019 (Example 2, Table 6) maxand AUC values ​​can be compensated by administering 134% (1 / 0.745) of the succinate formulation of the present invention. The concentration of TETA over time that can be expected after such a triethylenetetramine disuccinate dose adjustment should be the same as the triethylenetetramine dihydrochloride profile. ***

[0271] The invention described and claimed herein has many attributes and embodiments, including but not limited to those set forth or described or referenced in this detailed disclosure, which is not intended to be comprehensive, and the invention described and claimed herein is not limited by or to the features or embodiments identified in this detailed disclosure, which are included for purposes of illustration only and not limitation.

[0272] All patents, publications, scientific papers, websites, and other documents and materials referred to or mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each such reference document and material is hereby incorporated by reference into this specification to the same extent as if it were individually incorporated by reference in its entirety or set forth in its entirety herein. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific papers, websites, electronically available information, and other reference materials or documents. The reference to any application, patent, and publication in this specification is not, and should not be taken as, an agreement or any form of suggestion that they constitute pertinent prior art or form part of the general common knowledge in any country in the world.

[0273] The specific methods and compositions described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art in view of this specification, and are encompassed within the spirit of the invention as defined by the claims. Thus, for example, in each example herein, and in embodiments or examples of the invention, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms herein. The methods and processes illustratively described herein may suitably be practiced with different sequences of steps, and are not necessarily limited to the sequence of steps indicated herein or in the claims. Also, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Under no circumstances may the patent be construed as being limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be construed as being limited by any statement made by any examiner or any other officer or employee of the Office unless such statement is specifically and expressly adopted without qualification or reservation in the applicants' reply.

[0274] The terms and expressions which have been employed are used as terms of description and not of limitation, and in the use of such terms and expressions there is no intention to exclude any equivalents or portions thereof of the features shown and described, but it is recognized that various modifications are possible within the scope of the invention as claimed.Thus, while the present invention has been specifically disclosed by preferred embodiments and features as required, it will be understood that modifications and variations of the concepts disclosed herein may be exercised by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the present specification as defined by the appended claims.

[0275] Other embodiments are within the following claims. Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual members or subgroups of members of the Markush group.

Claims

1. An article of manufacture comprising a single dose capsule or tablet containing a single fixed dose of triethylenetetramine disuccinate, said fixed dose being selected from the group consisting of about 350 mg, about 584 mg and about 701 mg of triethylenetetramine disuccinate.

2. 10. The article of manufacture of claim 1, further comprising a package insert instructing a user to administer said fixed dose to a patient having a disease treatable with a copper chelator.

3. 10. The article of manufacture of claim 1, wherein the disease treatable with a copper chelator is characterized by an excess of copper.

4. 3. The article of manufacture of claim 2, wherein the disease is selected from the group consisting of Wilson's disease, heart failure, diabetic cardiomyopathy, diabetes mellitus, Alzheimer's disease, Parkinson's disease, idiopathic pulmonary fibrosis, cancer and copper toxicity.

5. 1. An article of manufacture comprising a number of fixed dose capsules equivalent to one or more daily doses of triethylenetetramine disuccinate, said daily dose being selected from the group consisting of about 1050 mg per day to about 2300 mg per day, about 1400 mg per day to about 3500 mg per day, about 2300 mg per day to about 2800 mg per day, and about 2800 mg per day to about 5600 mg per day of triethylenetetramine disuccinate, optionally wherein said fixed dose is selected from the group consisting of about 350 mg, about 400 mg, about 500 mg, about 584 mg, about 600 mg, and about 701 mg of triethylenetetramine disuccinate.

6. 2. The article of manufacture of claim 1, wherein the triethylenetetramine disuccinate has a purity of at least about 95%.

7. 2. The article of manufacture of claim 1, wherein the triethylenetetramine disuccinate has a purity of at least about 99%.

8. 2. The article of manufacture of claim 1, wherein the triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate.

9. 2. The article of manufacture of claim 1, wherein the triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydrate.

10. 2. The article of manufacture of claim 1, wherein said fixed dose of triethylenetetramine disuccinate is about 350 mg.

11. 2. The article of manufacture of claim 1, wherein said fixed dose of triethylenetetramine disuccinate is about 584 mg.

12. 2. The article of manufacture of claim 1, wherein said fixed dose of triethylenetetramine disuccinate is about 600 mg.

13. 2. The article of manufacture of claim 1, wherein said fixed dose of triethylenetetramine disuccinate is about 700 mg.

14. 10. The article of manufacture of claim 1, wherein the fixed dose triethylenetetramine disuccinate is in the form of a capsule.

15. 2. The article of manufacture of claim 1, wherein the fixed dose triethylenetetramine disuccinate is in the form of a tablet.

16. 10. The article of manufacture of claim 1, wherein the capsule or tablet is packaged in a blister pack.

17. The article of manufacture of claim 1 , wherein the capsule or tablet is packaged in a bottle.

18. 10. The article of manufacture of claim 1, wherein the capsule or tablet is formulated to provide delayed release.

19. 10. The article of manufacture of claim 1, wherein the capsule or tablet is formulated to provide sustained release.

20. 10. The article of manufacture of claim 1, wherein the capsule or tablet is formulated in combination with a pharmacokinetic enhancer (PKE) that provides improved absorption of the triethylenetetramine disuccinate.

21. 1. An article of manufacture for managing a subject having a disease treatable with a copper chelator, said article of manufacture comprising a fixed dose of triethylenetetramine disuccinate, said fixed dose ranging from about 350 to about 700 milligrams.

22. 22. The article of manufacture of claim 21, wherein the fixed dose of triethylenetetramine disuccinate is about 350 mg, 400 mg, about 500 mg, about 600 mg, or about 700 mg.

23. 22. The article of manufacture of claim 21, wherein a fixed dose of triethylenetetramine disuccinate is administered to the subject in an amount ranging from about 1050 mg per day to about 2300 mg per day, about 1400 mg per day to about 3500 mg per day, about 2300 mg per day to about 2800 mg per day, about 2400 mg per day to about 3000 mg per day, and about 2800 mg per day to about 5600 mg per day.

24. 22. The article of manufacture of claim 21, wherein the triethylenetetramine disuccinate is at least about 95% pure.

25. 22. The article of manufacture of claim 21, wherein the triethylenetetramine disuccinate is at least about 99% pure.

26. 22. The article of manufacture of claim 21, wherein the triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate.

27. 22. The article of manufacture of claim 21, wherein the triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydrate.

28. 27. The article of manufacture of claim 26, wherein the triethylenetetramine disuccinate is a triethylenetetramine disuccinate polymorph.

29. 22. The article of manufacture of claim 21, wherein the fixed dose of triethylenetetramine disuccinate is in the form of a capsule for oral administration.

30. 22. The article of manufacture of claim 21, wherein the fixed dose of triethylenetetramine disuccinate is in the form of a tablet for oral administration.

31. 22. The article of manufacture of claim 21 , wherein the subject is a human.

32. 22. The article of manufacture of claim 21, wherein one or more symptoms or diagnostic markers of the disease are reduced.

33. 22. The article of manufacture of claim 21, wherein said fixed dose of triethylenetetramine disuccinate lowers copper titer content and / or reduces intracellular copper in said subject.

34. 22. The article of manufacture of claim 21, wherein the fixed dose of triethylenetetramine disuccinate reduces total copper.

35. 22. The article of manufacture of claim 21, wherein the fixed dose of triethylenetetramine disuccinate reduces intracellular copper.

36. 22. The article of manufacture of claim 21, wherein the disease treatable with a copper chelator comprises diabetes and the fixed dose of triethylenetetramine disuccinate produces a cupruria effect.

37. 37. The article of manufacture of claim 36, wherein the diabetes is type 2 diabetes and the fixed dose of triethylenetetramine disuccinate attenuates left ventricular hypertrophy.

38. 22. The article of manufacture of claim 21, wherein the disease treatable with a copper chelator comprises diabetic cardiomyopathy.

39. 22. The article of manufacture of claim 21, wherein the disease treatable with a copper chelator comprises heart disease and the subject has type 2 diabetes.

40. 40. The article of manufacture of claim 39, wherein the cardiac condition comprises heart failure.

41. 22. The method of claim 21, wherein the disease treatable with a copper chelator comprises Wilson's disease.

42. 22. The article of manufacture of claim 21, wherein the fixed dose of triethylenetetramine disuccinate is administered in combination with one or more drugs that reduce or prevent inflammation or vascular leakage.

43. 43. The article of manufacture of claim 42, wherein the blood vessel is a capillary.

44. 22. The article of manufacture of claim 21, wherein the fixed dose of triethylenetetramine disuccinate is administered twice per day.

45. 22. The article of manufacture of claim 21, wherein the fixed dose is administered 1 to 4 times per day.

46. An article of manufacture comprising triethylenetetramine disuccinate and an inhibitor of N-acetylaminotransferase.

47. An article of manufacture comprising triethylenetetramine disuccinate and an inhibitor of spermine / spermidine N-acetyltransferase (SSAT1).

48. An article of manufacture comprising triethylenetetramine disuccinate and an inhibitor of spermine / spermidine N-acetyltransferase (SSAT2).

49. An article of manufacture comprising triethylenetetramine disuccinate and a polyamine membrane transport promoter, including bergamottin, maringenin, quercetin, other psoralens, piperine or tetrahydro-piperine, which acts as a membrane permeability enhancer for increased absorption.

50. 6. The article of manufacture of claim 1 or 5, wherein the fixed-dose triethylenetetramine disuccinate capsule or tablet has a shelf life of at least about 12 months at room temperature.

51. 51. The article of manufacture of claim 50, wherein the minimum purity of said triethylenetetramine disuccinate over said shelf life is at least about 98.5%, has no more than about 0.5% decomposition products, and has no more than about 0.1% new unidentified impurities.

52. 51. The article of manufacture of claim 50, wherein the shelf life is about 12 months.

53. A pharmaceutical formulation comprising a fixed dose of about 350 mg, about 584 mg, or about 701 mg of triethylenetetramine disuccinate in a mucosal adhesive formulation.

54. The pharmaceutical formulation of claim 53, wherein the mucoadhesive formulation comprises a compound selected from the group consisting of polymeric acrylates, methacrylates, hydroxylated methacrylate polymers, polyacrylic acid, alginates, polymethacrylic acid, chitosan, cyanoacrylates, hyaluronic acid, hydroxypropyl cellulose, gellan, polycarbophil, sodium carboxymethylcellulose, mucin, gelatin, polycarbophil, and poloxamer.

55. The pharmaceutical formulation of claim 53, wherein the mucoadhesive formulation is a nasal mucoadhesive formulation.

56. The pharmaceutical formulation of claim 55, wherein the nasal mucoadhesive formulation comprises a compound selected from the group consisting of copolymers of methyl vinyl ether, (hydroxypropyl)methylcellulose, sodium carboxymethylcellulose, Carbopol-934P and Eudragit RL-10.

57. A pharmaceutical formulation comprising a fixed dose of about 350 mg, about 584 mg, or about 701 mg of triethylenetetramine disuccinate in a gastric retentive formulation.

58. The pharmaceutical formulation of claim 57, wherein the triethylenetetramine disuccinate is substantially pure.

59. The pharmaceutical formulation of claim 57, wherein the triethylenetetramine disuccinate is at least about 90% pure, at least about 95% pure, or about 100% pure.

60. The pharmaceutical formulation of claim 57, wherein the triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydrate.

61. The pharmaceutical formulation of claim 57, wherein the triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate or anhydrous triethylenetetramine disuccinate.

62. The pharmaceutical formulation of claim 59, wherein the triethylenetetramine disuccinate or triethylenetetramine disuccinate anhydrous is at least about 90% pure.

63. A pharmaceutical formulation for use in treating a subject for a disease treatable with a copper chelator, comprising about 350 mg, about 584 mg, or about 701 mg of triethylenetetramine disuccinate and a pharma- ceutically acceptable carrier.

64. The pharmaceutical formulation of claim 63, wherein the disease is selected from the group consisting of Wilson's disease, heart failure, diabetic cardiomyopathy, diabetes mellitus, Alzheimer's disease, Parkinson's disease, idiopathic pulmonary fibrosis, cancer and copper toxicity.

65. The pharmaceutical formulation of claim 63 or 64, wherein the triethylenetetramine disuccinate is selected from the group consisting of anhydrous triethylenetetramine disuccinate, a crystalline form of triethylenetetramine disuccinate, and a polymorph of triethylenetetramine disuccinate.

66. The pharmaceutical formulation of claim 63, wherein the disease is type 2 diabetes and the triethylenetetramine disuccinate is anhydrous triethylenetetramine disuccinate in a form suitable for oral administration.

67. The pharmaceutical formulation of claim 66, wherein the triethylenetetramine disuccinic acid salt anhydrous is in a capsule or tablet suitable for oral administration.

68. The pharmaceutical preparation of any one of claims 63 to 66 or 67, wherein the subject is a human.

69. The use of triethylenetetramine disuccinate in the manufacture of an article of manufacture according to any one of claims 1 to 51 or 52.

70. Use of triethylenetetramine disuccinate in the manufacture of a pharmaceutical formulation according to any one of claims 53 to 61 or 62.

71. Use of triethylenetetramine disuccinate in the manufacture of a pharmaceutical formulation according to any one of claims 63 to 66 or 68.

72. Use of triethylenetetramine disuccinate in the manufacture of a pharmaceutical formulation for use in treating a subject having type 2 diabetes, the pharmaceutical formulation comprising about 350 mg, about 584 mg or about 701 mg of triethylenetetramine disuccinate and a pharma- ceutically acceptable carrier, wherein the triethylenetetramine disuccinate is in a form suitable for oral administration.

73. The use of claim 72, wherein the triethylenetetramine disuccinate is selected from the group consisting of anhydrous triethylenetetramine disuccinate, a crystalline form of triethylenetetramine disuccinate, and a polymorph of triethylenetetramine disuccinate.

74. The use described in claim 72 or 73, wherein triethylenetetramine disuccinate is in the form of a capsule or tablet suitable for oral administration.

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