Patent Application
20060141029
This invention relates to kits, methods and compositions containing catecholic butanes for the delivery of such to subjects for the treatment of diseases, including but not limited to psoriasis, neurogenerative diseases, for lowering serum triglyceride, serum glucose, serum non-esterified fatty acids, improving insulin sensitivity, and alleviating hypertension. This invention also relates to methods of making the foregoing compositions. In such methods of treatment, one or more catecholic butanes are administered to subjects in need thereof. This invention further relates to compositions comprising one or more catecholic butanes that are formulated appropriately for such modes of delivery and treatment.
Neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, are characterized by transmitter specific loss of neurons and progressive decrease in intellectual and cognitive functions, as described in Frolich, L and Riederer, P. (1995). Alzheimer's disease, in particular, is associated with dementia, impaired mental function, memory loss and forgetfulness. Hermann, C. et al. (1991) indicated at the time that the etiology and pathophysiology of Alzheimer's disease were unknown. Now, more than ten years later, the etiology is still unknown.
Pathologically, two types of lesions are typically present upon microscopic examination in the brain of Alzheimer's disease patients: neuritic plaques and neurofibrillary tangles. Although both types of lesions are observed in the brains of older persons without dementia, they are found in greater numbers in the neocortex and hippocampus of patients with Alzheimer's disease, as mentioned in Bennet D. A. and Evans, D. A. (1992). The most promising therapeutic approaches to cognitive impairment of dementia that are closest to being introduced are cholinergic approaches, such as described by Schneider, L. S. (1998). It would be desirable if other effective therapeutic approaches for treatment of such diseases, disorders or conditions can be found.
Ono, K. et al. (2002) reported that nordihydroguaiaretic acid (“NDGA”) not only inhibited β-amyloid fibrils (“fAp”) formation in vitro, it also dose-dependently broke down fAβ(1-40) and fAβ(1-42) within a few hours at pH 7.5 at 37° C. It is not known, however, whether NDGA is effective for the treatment of Alzheimer's disease or other neurodegenerative diseases.
Moreover, even though NDGA has certain beneficial properties, there have been reports that high doses of Larrea tridentate-containing herbs, the plant from which NDGA is derived, can induce hepatotoxicity and nephrotoxicity in humans, as described in Lambert, J. D. et al. (2002). It is, thus, not known whether NDGA can be used systemically or delivered locally to the brain with minimal toxicity to normal tissues.
It would be desirable if additional therapeutic approaches can be found for treating such neurological diseases, disorders or conditions that are effective and safe.
Further, despite the development of new drugs for the treatment of diabetes, many of these drugs have adverse side effects. For example, troglitazone was taken off the market after FDA approval because of liver toxicity and the resulting deaths in some of the treated patients. It would be desirable to produce other anti-diabetic drugs that would address some of the problems with existing anti-diabetic drugs.
Khandwala et al. in U.S. Pat. No. 5,827,898 and Reed, M. J. et al. (1999) described the use of nordihydroguaiaretic acid (“NDGA”) for reducing serum glucose, serum triglyceride, and serum non-esterified fatty acids in rodents. Gowri, M. S. et al. (1999) described the use of NDGA for reducing hypertension. The mechanism of action of NDGA in these diseases or conditions is unknown. If catecholic butanes can be safely administered to humans, these compounds can be used for treating hypertension or related conditions, for reducing serum glucose, serum triglyceride, and/or serum non-esterified fatty acids.
Catecholic butanes, including nordihydroguaiaretic acid (“NDGA”) and its derivatives, have been used for the inhibition of tumor growth in certain experimental animals. For example, Jordan et al. in U.S. Pat. No. 5,008,294 described the use of a single dose of NDGA on a mammary carcinoma MX-1 xenograft in athymic nude NCr mice. In one experiment, NDGA was injected into the tumor one day following subcutaneous implantation of a 14 mg fragment of the human mammary carcinoma in the axillary region of the mice. Jordan et al. further described topical application of NDGA after day 23 of implantation of human breast adenocarcinomas in athymic mice. Some evidence of inhibition of tumor growth was observed in those experiments, but it is unclear whether the antitumor effect was durable.
Huang et al. in U.S. Pat. No. 6,417,234 and U.S. Pat. No. 6,214,874 described intratumor injection of a NDGA derivative, designated tetra-O-methyl NDGA or M4N, and another NDGA derivative, designated G4N, separately or together into mice implanted with HPV-16 transformed immortal mouse epithelial cells (C3). Huang et al. also found some evidence of suppression of tumor growth by these NDGA derivatives. It is unknown whether compounds such as these NDGA derivatives can be safely administered to other animals such as humans.
Certain of the catecholic butanes, such as M4N, which is a NDGA derivative, are hydrophobic compounds found to be soluble in dimethyl sulfoxide (“DMSO”). When the composition of M4N in DMSO was injected into the tumor, the composition appeared to penetrate most but not all of the tumor tissues. A possible explanation may be that the hydrophobic nature of the compound limits its penetration. It would be desirable if a formulation can be found for safe systemic administration of these hydrophobic compounds so as to improve their efficacy, expand their utility and yet maintain their biological activities, such as anti-tumor activities. It would further be desirable if the catecholic butanes, including the NDGA Compounds, can be safely administered by routes of administration other than by direct injection into the affected tissues or by topical application.
It would also be desirable if the catecholic butanes, such as the NDGA Compounds can be formulated in a manner that would facilitate delivery to targeted tissues and maintenance of a certain range of dose level in the targeted tissues.
It is, thus, one of the objects of the present invention to provide methods and compositions for the prevention or treatment of diseases such as to address the problems in the prior art methods and compositions, for example, those described in the Background.
It is another one of the objects of the present invention to discover novel methods and compositions for the treatment of neurodegenerative diseases, disorders or conditions, such as, for example, Alzheimer's disease, Parkinson's disease, dementia, impaired mental function, memory loss and forgetfulness.
It is another one of the objects of the present invention to provide novel methods and compositions for the treatment of diabetes in a subject in need of such treatment.
It is another one of the objects of the present invention to provide novel methods and compositions for treatment of hypertension in a subject in need of such treatment.
It is yet a further one of the objects of the present invention to provide novel methods and compositions for treatment of hyperglycemia, for reducing serum triglyeride, and for reducing blood glucose.
It is yet another one of the objects of the present invention to provide one or more formulations containing the catecholic butanes, including the NDGA Compounds, that can facilitate and/or optimize distribution of the catecholic butanes, including the NDGA Compounds, to the targeted tissues.
It is another one of the objects of the present invention to provide compositions containing one or more catecholic butanes, including the NDGA Compounds, in formulations appropriate for treatment of the targeted tissues.
In accordance to one of the objects of the present invention, there is provided a pharmaceutical composition for treatment of a disease in a subject, such as an animal, for example, a human, where the composition contains at least one catecholic butane and a pharmaceutically acceptable carrier or excipient, and where the composition is formulated for administration by a route other than by direct injection into or topical application onto an affected tissue.
In accordance to another one of the objects, there is provided a composition as above, where the disease, disorder or condition is other than an inflammatory disease, for example, other than an inflammatory disease that is associated with microglial cell activation or stimulation.
In accordance to another one of the objects, there is provided a composition as above, where the disease is a non-cancer proliferative disease. Such a proliferative disease includes, for example, psoriasis.
In accordance to a further one of the objects, there is provided a composition as above, where the disease results from or is associated with a virus infection, such as, for example, HBV infection, EBV infection, HTLV infection, a JC virus infection, a Varicella-zoster virus infection, an adenovirus infection or a parvovirus infection.
In accordance to another one of the objects, there is provided a composition as above, where the disease is a neurodegenerative disease, disorder or condition, such as, for example, Parkinson's disease, Alzheimer's disease, dementia, memory loss, impaired mental function, and forgetfulness.
In accordance to yet another one of the objects, there is provided a composition as above, where the disease is hypertension or a condition resulting from or associated with hypertension.
In accordance to yet another one of the objects, there is provided a composition as above, where the disease is diabetes, such as type II diabetes, metabolic syndrome or insulin resistance.
In accordance to still another one of the objects, there is provided a composition as above, where the composition is formulated for intranasal administration, oral administration, including through slow release or rapid release capsules, for inhalation administration, for subcutaneous administration, for transdermal administration, for intra-arterial administration, with or without occlusion, for intracranial administration, intraventricular administration, intravenous administration, buccal administration, intraperitoneal administration, intraocular administration, central venous administration, intramuscular administration or for implantation administration.
In accordance to another one of the objects, there is provided a composition as above, where the pharmaceutically acceptable carrier or excipient contains dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), saline, an oil such as, for example, castor oil or corn oil, Cremaphor EL, and ethanol or a mixture containing one or more of such.
In accordance to another one of the objects, there is provided a composition as above, where the pharmaceutically acceptable carrier or excipient contains a lipid based formulation, a liposomal formulation, a nanoparticle formulation, a micellar formulation, a water soluble formulation, or any of the foregoing in a biodegradable polymer.
In accordance to yet another one of the objects, there is provided a composition as above, where the catecholic butane has the structural formula I as follows:
where R1 and R2 are independently —H, a lower alkyl, a lower acyl, an alkylene or an unsubstituted or substituted amino acid residue or salt thereof; R3, R4, R5, R6, R10, R11, R12 and R13 are independently —H or a lower alkyl; and R7, R8 and R9 are independently —H, —OH, a lower alkoxy, a lower acyloxy, or any two adjacent groups together may be an alkyene dioxy, or an unsubstituted or substituted amino acid residue or salt thereof.
In accordance to still another one of the objects, there is provided a catecholic butane as above, where R1 and R2 are independently —H, a lower alkyl, a lower acyl, or an unsubstituted or substituted amino acid residue or salt thereof; R3, R4, are independently a lower alkyl; R5, R6, R10, R11, R12 and R13 are independently —H; and R7, R9 and R9 are independently —H, —OH, a lower alkoxy, a lower acyloxy, or an unsubstituted or substituted amino acid residue or salt thereof.
In accordance to yet another one of the objects, there is provided a catecholic butane as above, where R1 and R2 are independently —H, a lower alkyl, a lower acyl, or an unsubstituted or substituted amino acid residue or salt thereof; R3, R4, are independently a lower alkyl; R5, R6, R7, R10, R11, R12 and R13 are independently —H; and R8 and R9 are independently —OH, a lower alkoxy, lower acyloxy, or an unsubstituted or substituted amino acid residue or salt thereof.
In accordance to still another one of the objects, there is provided the catecholic butane as above, where R1 and R2 are independently —CH3 or —(C═O)CH2N(CH3)2 or a salt thereof.
In accordance to still another one of the objects, there is provided the catecholic butane as above, where R8 and R9 are independently —OCH3 or —O(C═O)CH2N(CH3)2 or a salt thereof.
In accordance to still another one of the objects, there is provided the catecholic butane as above, where R1 and R2 are independently —CH3, —(C═O)CH2N(CH3)2 or —(C═O)CH2N+H(CH3)2.Cl− and R8 and R9 are independently —OCH3, —O(C═O)CH2N(CH3)2 or —O(C═O)CH2N+H(CH3)2.Cl−.
In accordance to still another one of the objects, there is provided the catecholic butane as above, where R1 and R2 are independently —H or —CH3 and R8 and R9 are independently —OH or —OCH3, provided that the catecholic butane is not NDGA.
In accordance to still another one of the objects, there is provided the catecholic butane as above, where R1 and R2 are independently —CH3 and R8 and R9 are independently —OCH3.
In accordance to still another one of the objects, there is provided the catecholic butane as above, where the catecholic butane is NDGA.
In accordance to still another one of the objects, there is provided the catecholic butane as above, where the catecholic butane is other than NDGA.
In accordance to yet another one of the objects, there is provided a method of making a pharmaceutical composition containing a catecholic butane, where the method includes the steps of (a) providing a catecholic butane as above; (b) providing a pharmaceutically acceptable carrier or excipient as above, and (c) combining the catecholic butane with the pharmaceutically acceptable carrier or excipient.
In accordance to a further one of the objects of the present invention, there is provided a method of treating a disease in a subject, where the method of treatment includes providing a pharmaceutical composition as above and administering the composition to the subject, such as a subject in need of such treatment.
In accordance to another one of the objects, there is provided a method of treatment as above, where the disease is other than an inflammatory disease, for example, other than an inflammatory disease that is associated with microglial cell activation or stimulation.
In accordance to another one of the objects, there is provided a method of treatment as above, where the disease is a non-cancer proliferative disease such as psoriasis. For treating psoriasis, it is preferred to administer G4N.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the disease is a neurodegenerative disease, disorder or condition, such as Parkinson's disease, Alzheimer's disease, dementia, memory loss, impaired mental function, and forgetfulness, or stroke or traumatic brain injury.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the disease is hypertension or a condition resulting from or associated with hypertension. For treating hypertension, it is preferred to administer a catecholic butane other than NDGA and 3-O-methyl NDGA.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the disease is diabetes, such as diabetes type I or type II, metabolic syndrome or insulin resistance.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the treatment is to reduce serum glucose in a subject. For treating diabetes and reducing serum glucose, it is preferred to use a “NDGA derivative” as defined herein, wherein for the R groups of formula II in that definition, both of R1 and R2 are —OCH3 and both of R3 and R4 are —OCH3; or R1 and R2 are independently —OCH3 or an unsubstituted or substituted amino acid residue, and R3 and R4 are independently —OCH3 or an unsubstituted or substituted amino acid residue, with the proviso that at least one of R1, R2, R3 and R4 is an unsubstituted or substituted amino acid residue; and wherein R5, and R6 independently are —H or an alkyl; or a salt thereof.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the treatment is to reduce low density lipoprotein cholesterol (LDL-C) in a subject.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the treatment is to reduce serum triglyceride in a subject.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the treatment is to reduce serum non-esterified fatty acid in a subject.
In accordance to a further one of the objects, there is provided a method of treatment as above, where the disease results from or is associated with a virus infection, such as, for example, HBV infection, EBV infection, HTLV infection, a JC virus infection, a Varicella-zoster virus infection, an adenovirus infection, or a parvovirus infection.
In accordance to still another one of the objects, there is provided a method of treatment as above, where the composition is formulated for intranasal administration, oral administration, including through slow release or rapid release capsules, for inhalation, for subcutaneous administration, for transdermal administration, for intra-arterial administration, with or without occlusion, for intracranial administration, intraventricular administration, intravenous administration, buccal administration, intraperitoneal administration, intraocular administration, central venous administration, intramuscular administration or for implantation.
In accordance to another one of the objects, there is provided a method of treatment as above, where the pharmaceutically acceptable carrier or excipient contains dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), saline, an oil, ethanol and any combination of such.
In accordance to another one of the objects, there is provided a method of treatment as above, where the pharmaceutically acceptable carrier or excipient contains a lipid based formulation, a liposomal formulation, a nanoparticle formulation, a micellar formulation, a water soluble formulation, or any of the foregoing in a biodegradable polymer.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the catecholic butane has a formula given above.
In accordance to yet another one of the objects, there is provided a method of treatment as above, where the catecholic butane is tetra-O-methyl NDGA.
In accordance to still another one of the objects, there is provided a method of treatment as above, where the catecholic butane is tetra-dimethylglycinyl NDGA.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is tri-O-methyl NDGA.
In accordance to still another one of the objects, there is provided a method of treatment as above, where the catecholic butane is NDGA.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is other than NDGA.
In accordance to another one of the objects, there is provided a method of treatment as above, where the method includes administering at least two catecholic butanes.
In accordance to another one of the objects, there is provided a method of treatment as above, where the two catecholic butanes are administered substantially contemporaneously.
In accordance to another one of the objects, there is provided a method of treatment as above, where the two catecholic butanes are administered at different times.
In accordance to another one of the objects, there is provided a method of treatment as above, where the two catecholic butanes are selected from the group consisting of tetra-O-methyl NDGA, tri-O-methyl NDGA and tetra-dimethylglycinyl NDGA.
In accordance to another one of the objects, there is provided a method of treatment as above, where the nanoparticle formulation contains at least one selected from the group consisting of poly(DL-lactide-co-glycolide), poly vinyl alcohol, d-α-tocopheryl polyethylene glycol 1000 succinate, and poly(lactide-co-glycolide)-monomethoxy-poly(polyethylene glycol).
In accordance to another one of the objects, there is provided a method of treatment as above, where the liposomal formulation comprises at least one selected from the group consisting of phosphatidylcholine/cholesterol/PEG-DPPE, distearoylphosphatidylcholine/cholesterol/PEG-DPPE, and 1-2-dioleoyl-sn-glycero-3-phosphocholine/1-2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt/cholesterol/triolein/tricaprylin.
In accordance to another one of the objects, there is provided a method of treatment as above, where the method comprises administering the composition more than once.
In accordance to another one of the objects, there is provided a method of treatment as above, where the pharmaceutically acceptable carrier or excipient is an aqueous preparation.
In accordance to another one of the objects, there is provided a method of treatment as above, where the pharmaceutically acceptable carrier or excipient comprises a hydrophobic preparation.
In accordance to another one of the objects, there is provided a method of treatment as above, where the hydrophobic preparation comprises a lipid based vehicle.
In accordance to another one of the objects, there is provided a method of treatment as above, where the pharmaceutically acceptable carrier or excipient comprises at least one selected from the group consisting of castor oil, peanut oil, dimethyl sulfoxide (DMSO), and other dietary fats or oils.
In accordance to another one of the objects, there is provided a method of treatment as above, where the composition is formulated in the form of one selected from the group consisting of a tablet, a powder, a gel capsule, a liquid, and an oral rinse.
In accordance to another one of the objects, there is provided a method of treatment as above, where the pharmaceutically acceptable carrier or excipient comprises a polymer formulation.
In accordance to another one of the objects, there is provided a method of treatment as above, where the polymer formulation is a biodegradable polymer formulation.
In accordance to another one of the objects, there is provided a method of treatment as above, where the pharmaceutically acceptable carrier or excipient allows for high local drug concentration and sustained release over a period of time.
In accordance to another one of the objects, there is provided a method of treatment as above, where the polymer formulation comprises at least one selected from the group consisting of 1,3-bis(p-carboxyphenoxy) propane, sebacic acid, poly(ethylene-co-vinyl acetate), and poly(lactide-co-glycolide).
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is dissolved in saline, DMSO or ethanol prior to administration.
In accordance to another one of the objects, there is provided a method of treatment as above, where the composition is at least one selected from the group consisting of: a powder, an aerosol, an aqueous formulation, a liposomal formulation, a nanoparticle formulation, and a hydrophobic formulation.
In accordance to another one of the objects, there is provided a method of treatment as above, where the composition is administered to a subject daily for a defined period of time.
In accordance to another one of the objects, there is provided a method of treatment as above, where the composition is administered intermittently.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is infused into the subject.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is a water soluble compound.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is a hydrophobic compound.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is formulated as a liquid, an aerosol, an oral rinse, a suspension, a tablet, a powder, or a gel capsule.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is administered in a range of greater than about 10 mg/kg and less than about 375 mg/kg per dose to humans.
In accordance to another one of the objects, there is provided a method of treatment as above, where the range is greater than about 10 mg/kg and less than about 250 mg/kg per dose.
In accordance to another one of the objects, there is provided a method of treatment as above, where the range is greater than about 10 mg/kg and less than about 200 mg/kg per dose.
In accordance to another one of the objects, there is provided a method of treatment as above, where the range is greater than about 10 mg/kg and less than about 150 mg/kg per dose.
In accordance to another one of the objects, there is provided a method of treatment as above, where the range is greater than about 10 mg/kg and less than about 100 mg/kg per dose.
In accordance to another one of the objects, there is provided a method of treatment as above, where the range is greater than about 10 mg/kg and less than about 75 mg/kg per dose.
In accordance to another one of the objects, there is provided a method of treatment as above, where the range is greater than about 10 mg/kg and less than about 50 mg/kg per dose.
In accordance to another one of the objects, there is provided a method of treatment as above, where the composition is administered systemically, such as intravenously, for example.
In accordance to another one of the objects, there is provided a method of treatment as above, where the catecholic butane is tri-O-NDGA or tetra-O-methyl NDGA.
In accordance to still one of the objects, there is provided a kit for treatment of a disease comprising the pharmaceutical composition as above and instructions for administration of the composition.
Further objects, features and advantages of the present invention will be apparent to one of ordinary skill in the art upon reading the present description. Such other objects, features, and advantages are also deemed embodied by the present invention.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.
In the drawings:
The inventors herein have discovered that catecholic butanes of the formula below (formula I):
where R1 and R2 are independently —H, a lower alkyl, a lower acyl, an alkylene or an unsubstituted or substituted amino acid residue or salt thereof; R3, R4, R5, R6, R10, R11, R12 and R13 are independently —H or a lower alkyl; and R7, R8 and R9 are independently —H, —OH, a lower alkoxy, a lower acyloxy, or any two adjacent groups together may be an alkyene dioxy, or an unsubstituted or substituted amino acid residue or salt thereof are useful for the treatment of diseases other than tumor, HIV, HSV, and HPV, such as, for example, hypertension, diabetes, neurodegenerative diseases, disorders or conditions, and other viral infections. Such catecholic butanes can be combined with pharmaceutically acceptable carrier or excipient to produce pharmaceutical compositions that can be formulated for different routes of delivery.
Optionally, in one embodiment of the invention, the disease to be treated is other than obesity. Further optionally, in another embodiment, the disease to be treated is other than an inflammatory disease associated with microglial cell activation or stimulation.
The present inventor has surprisingly discovered that catecholic butanes, such as NDGA derivatives have pleiotropic effects of not only inhibiting tumor growth, but also for the treatment of other metabolic diseases, disorders or conditions. Thus, the present invention provides methods and compositions for reducing blood glucose, serum triglyceride, and serum non-esterified fatty acids and in increasing insulin sensitivity, where the compositions each contain a substantially pure preparation of at least one catecholic butane (formula I) or NDGA derivative (formula II) (any one or more of the NDGA derivatives or NDGA, the “NDGA Compounds”), when administered to an animal, particularly, humans, who are in need of such treatment, where, in formula II, R1, R2, R3 and R4 independently represent —OH, a lower alkoxy, for example, —OCH3, a lower acyloxy, for example, —O(C═O)CH3, or an unsubstituted or substituted amino acid residue or salt thereof but are not each —OH simultaneously; and R5, R6 independently represent —H or an alkyl. In one embodiment of the invention, R5 and R6 can both be —H or an alkyl such as a lower alkyl, for example, —CH3 or —CH2CH3. The NDGA Compounds are useful for treatment of diabetes, such as type II diabetes mellitus.
The present inventors have further found that the catecholic butanes, including the NDGA Compounds are also effective in reducing blood pressure and are useful for the treatment of hypertension, such as vascular systolic and diastolic systemic and pulmonary hypertension. The present inventor has additionally found that the NDGA Compounds are effective in the suppression of food intake and can be administered to a subject for treatment of obesity.
The present inventor has further discovered that the catecholic butanes herein, including the NDGA Compounds, such as NDGA derivatives are also effective for the treatment of neurodegenerative diseases, disorders or conditions. This invention, thus, relates to compositions containing NDGA derivatives and methods using such for the treatment of neurodegenerative diseases, disorders, or conditions, such as, for example, Alzheimer's disease, Parkinson's disease, dementia, impairment of mental function, memory loss, forgetfulness and attention deficit.
For various indicated diseases or conditions as noted in the Summary, it is preferred to administer the specific NDGA derivatives also as noted in the Summary.
In one embodiment of the invention, the catecholic butane has the formula I above where R1 and R2 are independently —H, a lower alkyl, a lower acyl, or an unsubstituted or substituted amino acid residue or salt thereof; R3, R4, are independently a lower alkyl; R5, R6, R10, R11, R12 and R13 are independently —H; and R7, R8 and R9 are independently —H, —OH, a lower alkoxy, a lower acyloxy, or an unsubstituted or substituted amino acid residue or salt thereof.
In a further embodiment of the invention, the pharmaceutical composition has the above formula I where R1 and R2 are independently —H, a lower alkyl, a lower acyl, or an unsubstituted or substituted amino acid residue or salt thereof; R3, R4, are independently a lower alkyl; R5, R6, R7, R10, R11, R12 and R13 are independently —H; and R8 and R9 are independently —OH, a lower alkoxy, lower acyloxy, or an amino acid residue or substituent or salt thereof.
In a further embodiment of the invention, the pharmaceutical composition has the formula above where R1 and R2 are independently —CH3 or —(C═O)CH2N(CH3)2 or a salt thereof.
In another embodiment of the invention, the pharmaceutical composition has the formula above where R8 and R9 are independently —OCH3 or —O(C═O)CH2N(CH3)2 or a salt thereof.
In a further embodiment of the invention, the pharmaceutical composition has the formula above where R1 and R2 are independently —CH3, —(C═O)CH2N(CH3)2 or —(C═O)CH2N+H(CH3)2.Cl− and R8 and R9 are independently —OCH3, —O(C═O)CH2N(CH3)2 or —O(C═O)CH2N+H(CH3)2.Cl−.
In yet another embodiment of the invention, the pharmaceutical composition has the formula above where R1 and R2 are independently —H or —CH3 and R8 and R9 are independently —OH or —OCH3, provided that the catecholic butane is not NDGA.
In a different embodiment of the invention, the pharmaceutical composition has the formula as above where R1 and R2 are independently —CH3 and R8 and R9 are independently —OCH3.
In yet another embodiment of the invention, the catecholic butane is NDGA. In an alternative embodiment, the catecholic butane is other than NDGA, namely a NDGA derivative with the following formula II:
The present inventors have surprisingly discovered that a composition containing a substantially pure preparation of at least one NDGA derivative is effective for the treatment of diseases other than tumors, HIV infection, HSV infection, or HPV infection. The diseases applicable to the present invention include psoriasis, neurodegenerative diseases such as, for example, Parkinson's disease, Alzheimer's disease, dementia, impaired mental function, memory loss or forgetfulness. The present invention is also applicable to diseases such as, for example, hypertension, diabetes, other virus infections such as HBV, EBV, JC virus infection, HTLV, Varicella-zoster virus infection, adenovirus infection or parvovirus infection. The NDGA derivatives preferably have a formula as set forth above (formula II), where R1, R2, R3 and R4 independently represent —OH, a lower alkoxy, for example, —OCH3, a lower acyloxy, for example, —O(C═O)CH3, or an amino acid residue, or a substituent or salt thereof but are not each —OH simultaneously; and R5, R6 independently represent —H or an alkyl such as a lower alkyl, for example, —CH3 or —CH2CH3. In one embodiment, R5 and R6 can both be —H, —CH3 or —CH2CH3.
The inventors have discovered that the present catecholic butane, including the NDGA Compounds, in a suitable formulation can be safely administered to one or more subjects in need of such treatment by intranasal delivery. Optionally, such catecholic butanes or NDGA Compounds can be administered by inhalation. Further optionally, such catecholic butanes or NDGA Compounds can be administered orally, or buccally, or intraocularly. Additionally, the catecholic butanes or NDGA Compounds can be administered as an oral rinse, for example, in a rinse-and-spit treatment one or more times a day.
Moreover, the inventors have discovered that the catecholic butanes or NDGA Compounds in liposomal formulations, nanoparticle formulations, or micellar formulations can additionally be safely administered systemically, such as intravenously, such as by injection into the central vein for example, or intraperitoneally, interstitially, subcutaneously, transdermally, intramuscularly, intra-arterially, intra-cranially, or intra-ventricularly.
Furthermore, the catecholic butanes or NDGA Compounds can be formulated in liposomal formulations, nanoparticles formulations, or micellar formulations, or any formulation embedded in a biodegradable polymer, for administration into a subject, such as one in need of such treatment. Implantation into the brain, for example, can be used for treatment of brain or neurodegenerative diseases, disorders or conditions.
In one embodiment of the invention, the route of administration for purposes herein is other than by parenteral administration, where parenteral administration herein means intravenous, intra-arterial, intramuscular, subcutaneous, transdermal and intraperitoneal administration.
The present invention further features a pharmaceutical composition containing catecholic butanes or NDGA Compounds for treatment of diseases as described above including but not limited to non-cancer proliferative diseases such as psoriasis, neurodegenerative diseases, hypertension, diabetes, and viral diseases where the composition is formulated for delivery or administration as described above such as, for example, in the form of a tablet, a liquid that is either hydrophilic or hydrophobic, a powder such as one resulting from lyophilization, an aerosol, or in the form of an aqueous water soluble composition, a hydrophobic composition, a liposomal composition, a micellar composition, such as that based on Tween 80 or diblock copolymers, a nanoparticle composition, a polymer composition, a cyclodextrin complex composition, emulsions, lipid based nanoparticles termed “lipocores.”
The present invention further features a method of producing the pharmaceutical composition of the present invention, involving making or providing the catecholic butanes or NDGA Compounds in a substantially purified form, combining the composition with a pharmaceutically acceptable carrier or excipient, and formulating the composition in a manner that is compatible with the mode of desired administration.
The present invention still additionally provides for kits comprising compositions or formulations as above for the treatment of diseases where the compositions are formulated for delivery as above, including but not limited to intranasal administration, inhalation, oral administration, intravenous administration, intraperitoneal administration and other parenteral administration, or as an oral rinse, or the like, and instructions for such administration.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention may be better understood in light of the particular meanings as follows.
The term “active agent,” “compound,” and “drug” herein refers to one or more catecholic butanes, including NDGA and NDGA derivatives.
The term “alkylene dioxy” as used herein refers to methylene (or substituted methylene) dioxy or ethylene (or substituted ethylene) dioxy.
The term “unsubstituted or substituted amino acid residue or salt thereof” in reference to one of the R groups in the formula for the catecholic butane herein is an amino acid residue or a substituted amino acid residue or salt of an amino acid residue or salt of a substituted amino acid residue including but not limited to: alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 5-hydroxylysine, 4-hydroxyproline, thyroxine, 3-methylhistidine, ε-N-methyllysine, ε-N,N,N-trimethyllysine, aminoadipic acid, γ-caroxyglutamic acid, phosphoserine, phosphothreonine, phosphotyrosine, N-methylarginine, N-acetyllysine, and an N,N-dimethyl-substituted amino acid residue or a salt thereof, such as a chloride salt.
The term “lower alkyl” means C1-C6 alkyl.
The term “lower acyl” means C1-C6 acyl.
The term “NDGA Compound” refers to NDGA and/or its derivatives, singly or collectively.
The term “NDGA derivative” refers to a derivative of NDGA each having the formula II:
wherein R1, R2, R3 and R4 are independently —OH, lower alkoxy, lower acyloxy, or an unsubstituted or substituted amino acid residue or salt thereof but are not each OH simultaneously; and R5, R6 are independently —H or an alkyl such as a lower alkyl The term includes, for example, a compound in which R1, R2, R3 and R4 are each —OCH3, or are each —O(C═O)CH3; and R5, R6 are each —H or each a lower alkyl. In one embodiment of the invention, R5, R6 are each —CH3 or —CH2CH3.
A “substantially purified” compound in reference to the catecholic butanes or NDGA Compounds herein is one that is substantially free of compounds that are not the catecholic butane or NDGA Compounds of the present invention (hereafter, “non-NDGA materials”). By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of non-NDGA materials.
The “buffer” suitable for use herein includes any buffer conventional in the art, such as, for example, Tris, phosphate, imidazole, and bicarbonate.
As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a condition or disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a condition or disease and/or adverse affect attributable to the condition or disease. “Treatment,” thus, for example, covers any treatment of a condition or disease in a mammal, particularly in a human, and includes: (a) preventing the condition or disease from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease, such as, for example, causing regression of the condition or disease.
A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A “pharmaceutically acceptable carrier” is non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation. For example, the carrier for a formulation containing the present catecholic butane or NDGA Compounds preferably does not include oxidizing agents and other compounds that are known to be deleterious to such. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, buffer, dimethyl sulfoxide, Cremaphor EL, and combinations thereof. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
Pharmaceutically acceptable salts herein include the acid addition salts (formed with the free amino groups of the polypeptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, mandelic, oxalic, and tartaric. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, and histidine.
The term “pharmaceutically acceptable excipient,” includes vehicles, adjuvants, or diluents or other auxiliary substances, such as those conventional in the art, which are readily available to the public. For example, pharmaceutically acceptable auxiliary substances include pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like.
The terms “subject,” “host,” and “patient,” are used interchangeably herein to refer to an animal being treated with the present compositions, including, but not limited to, simians, humans, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
All publications mentioned herein, including patents, patent applications, and journal articles are incorporated herein by reference in their entireties including the references cited therein, which are also incorporated herein by reference.
It must be noted that as used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of such compounds and reference to “the NDGA Compound” includes reference to one or more NDGA Compounds and equivalents thereof known to those skilled in the art.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
The examples described below are given for the purpose of illustration only and are not to be interpreted in any way as limiting the invention.
Preparation of Catecholic Butanes
The catecholic butanes of the present invention can be prepared by any conventional methodologies. For example, such compounds can be made as described in U.S. Pat. No. 5,008,294.
Preparation of the NDGA Compounds
The NDGA Compounds and formulations thereof can be made by any process conventional in the art. For example, the NDGA Compounds can be made as described in, U.S. Pat. No. 5,008,294 (Jordan et al., issued Apr. 16, 1991); U.S. Pat. No. 6,291,524 (Huang et al., issued Sep. 18, 2001); Hwu, J. R. et al. (1998); or McDonald, R. W. et al. (2001).
In one embodiment of the present invention, an NDGA Compound, tetra-O-methyl NDGA, also known as meso-1,4-bis(3,4-dimethoxyphenyl)-2,3-dimethylbutane, or M4N is made as follows: a solution is made containing NDGA and potassium hydroxide in methanol in a reaction flask. Dimethyl sulfate is then added to the reaction flask and the reaction is allowed to proceed. The reaction is finally quenched with water, causing the product to precipitate. The precipitate is isolated by filtration and dried in a vacuum oven. The compound is then dissolved in a solution of methylene chloride and toluene and subsequently purified through an alumina column. The solvents are removed by rotary evaporation and the solid is resuspended in isopropanol and isolated by filtration. The filter cake is dried in a vacuum oven. The resulting tetra-O-methyl NDGA (M4N) is crystallized by refluxing the filter cake in isopropanol and re-isolating the crystals by filtration.
In some embodiments of the present invention, certain NDGA Compounds of the present invention, such as G4N, also known as meso-1,4-bis[3,4-(dimethylaminoacetoxy)phenyl]-(2R,3S)-dimethylbutane or tetra-dimethylglycinyl NDGA, or a hydrochloride salt thereof and similar compounds having amino acid substituents, can also be prepared according to conventional methods, as described in, for example, U.S. Pat. No. 6,417,234.
Compositions
The present invention further provides compositions, including pharmaceutical compositions, comprising the catecholic butanes including the NDGA Compounds and pharmaceutically acceptable carriers or excipients. These compositions may include a buffer, which is selected according to the desired use of the catecholic butanes or NDGA Compounds, and may also include other substances appropriate for the intended use. Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for an intended use. In some instances, the composition can comprise a pharmaceutically acceptable excipient, a variety of which are known in the art. Pharmaceutically acceptable excipients suitable for use herein are described in a variety of publications, including, for example, A. Gennaro (1995); Ansel, H. C. et al. (1999); and Kibbe, A. H. (2000).
The compositions herein are formulated in accordance to the mode of potential administration. Thus, if the composition is intended to be administered intranasally or by inhalation, for example, the composition may be a converted to a powder or aerosol form, as conventional in the art, for such purposes. Other formulations, such as for oral or parenteral delivery, are also used as conventional in the art.
Compositions for administration herein may form solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
Therapeutic Methods
The catecholic butanes, including the NDGA Compound compositions of the subject invention find use as therapeutic agents in situations where one wishes to provide a treatment to a subject who has a non-cancer proliferative disease such as psoriasis, a neurodegenerative disease, condition or disorder, hypertension, diabetes, and where one wishes to provide treatment to viral diseases other than HIV, HPV or HSV, such as, for example, HBV, EBV, HTLV, Varicella-zoster virus infection, JC virus infection, adenovirus infection or parvovirus infection.
A variety of animal hosts are treatable according to the subject methods, including human and non-human animals. Generally such hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., guinea pigs, mice and rats), and other mammals, including cattle, goats, horses, sheep, rabbits, pigs, and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans. Animal models are of interest for experimental investigations, such as providing a model for treatment of human disease. Further, the present invention is applicable to veterinary care as well.
Formulations, Dosages, and Routes of Administration
As mentioned above, an effective amount of the active agent is administered to the host, where “effective amount” means a dosage sufficient to produce a desired result. In some embodiments, the desired result is at least a reduction or inhibition of progression of neurodegenerative diseases, hypertension, diabetes, and viral infections.
Typically, the compositions of the instant invention will contain from less than about 1% up to about 99% of the active ingredient, that is, the catecholic butanes including the NDGA Compounds herein; optionally, the instant invention will contain about 5% to about 90% of the active ingredient. The appropriate dose to be administered depends on the subject to be treated, such as the general health of the subject, the age of the subject, the state of the disease or condition, the weight of the subject, the size of the lesion, for example. Generally, between about 0.1 mg and about 500 mg or less may be administered to a child and between about 0.1 mg and about 5 grams or less may be administered to an adult. The active agent can be administered in a single or, more typically, multiple doses. Preferred dosages for a given agent are readily determinable by those of skill in the art by a variety of means. Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials establishing dose response curves. The amount of agent will, of course, vary depending upon the particular agent used.
The frequency of administration of the active agent, as with the doses, will be determined by the care giver based on age, weight, disease status, health status and patient responsiveness. Thus, the agents may be administered one or more times daily, weekly, monthly or as appropriate as conventionally determined. The agents may be administered intermittently, such as for a period of days, weeks or months, then not again until some time has passed, such as 3 or 6 months, and then administered again for a period of days, weeks, or months.
The catecholic butanes or active agents of the present invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the catecholic butanes of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, aerosols, liposomes, nanoparticles, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
As such, administration of the active agents can be achieved in various ways, such as oral, buccal, rectal, intranasal, intravenous, intra-arterial, intra-tracheal, intraventricular, intracranial, interstitial, transdermal, etc., or by inhalation or implantation.
In particular, nanoparticle, micelle and liposomal preparation can be administered systemically, including parenterally and intranasally, as well as interstitially, orally, topically, transdermally, via inhalation or implantation, such as for drug targeting, enhancement of drug bioavailability and protection of drug bioactivity and stability. Nanoparticle bound drugs herein are expected to achieve prolonged drug retention in the subject after administration.
In pharmaceutical dosage forms, the active agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the active agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. For oral rinses, the preparations can be made in a manner conventional in the art, such as described in, for example, Epstein, J. B. et al. (2002) and Pitten, F. et al. (2003).
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are conventional in the art. Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents or emulsifying agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.
The active agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, including corn oil, castor oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The active agents can be utilized in aerosol formulation to be administered via inhalation.
The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the active agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
Kits with multiple or unit doses of the active agent, are included in the present invention. In such kits, in addition to the containers containing the multiple or unit doses of the compositions containing the catecholic butanes or NDGA derivatives will be an informational package insert with instructions describing the use and attendant benefits of the drugs in treating pathological condition of interest.
While the description herein refers to methods of formulating NDGA, NDGA Compounds or derivatives, it is to be understood that these are illustrative examples of certain catecholic butanes within the present invention.
Preparation of NanoParticles (“NP”)
The present invention includes formulations of catecholic butanes, including NDGA Compounds, in a NP preparation. A number of different NP formulations suitable for use herein can be made depending on the method of delivery. The NP formulation can differ based on the drug release profile desired, by controlling the molecular weight, the copolymer ratio, the drug loading, the microparticle size and porosity and the fabrication conditions. The NP formulations can also differ on the basis of polymers, stabilizers, and surfactants used in the production process. Different excipients may also have different effects on drug uptake, drug distribution throughout the body and persistence of the drug in plasma. A person having skills conventional in the art will be able to determine the desired properties or characteristics, and accordingly determine the appropriate NP formulation to use.
The polymeric matrix of the NP must meet the criteria of biocompatibility, bioavailability, mechanical strength and ease of processing. The best known polymers for this purpose is the biodegradable poly(lactide-co-glycolide)s (“PLGAs”).
NP herein can be made by any process conventional in the art. In one embodiment, the NP can be made as described in, for example, Lockman, P. R., et al. (2002). The types of manufacturing process include, for example, emulsion polymerization, interfacial polymerization, desolvation evaporation and solvent deposition.
In the emulsion polymerization process of making the NP herein, the polymerization process consists of building a chain of polymers from a single monomer unit, as described in, for example, Kreuter, J. (1994). Polymerization occurs spontaneously at room temperature after initiation by either free radical or ion formation, such as by use of high-energy radiation, UV light, or hydroxyl ions. Once polymerization is complete the solution is filtered and neutralized. The polymers form micelles and droplets consisting of from about 100 to 107 polymer molecules. Surfactants and stabilizers are generally not need in this process. Also, this process can be accomplished in an organic phase rather than an aqueous phase.
The NP herein can also be made by an interfacial polymerization process as described in, for example, Khouri, A. I., et al. (1986). In this process, monomers are used to create the polymer and polymerization occurs when an aqueous and organic phase are brought together by homogenization, emulsification, or micro-fluidization under high-torque mechanical stirring. For example, polyalkylcyanoacrylate nanocapsules containing the catecholic butanes, such as the NDGA Compounds, can be made by combining the lipophilic NDGA Compounds and the monomer in an organic phase, dissolving the combination in oil, and slowly adding the mixture through a small tube to an aqueous phase with constant stirring. The monomer can then spontaneously form 200-300 nm capsules by anionic polymerization. A variation of this process involves adding a solvent mixture of benzyl benzoate, acetone, and phospholipids to the organic phase containing the monomer and the drug, as described in, for example, Fessi, H., et al. (1989). This creates a formulation in which the drug is encapsulated and protected against degradation until it reaches the target tissue.
Macromolecules such as albumin and gelatin can be used in oil denaturation and desolvation processes in the production of NPs. In the oil emulsion denaturation process, large macromolecules are trapped in an organic phase by homogenization. Once trapped, the macromolecule is slowly introduced to an aqueous phase undergoing constant stirring. The nanoparticles formed by the introduction of the two immiscible phases can then be hardened by crosslinking, such as with an aldehyde or by heat denaturation.
Alternatively, macromolecules can form NPs by “desolvation.” In the desolvation process, macromolecules are dissolved in a solvent in which the macromolecules reside in a swollen, coiled configuration. The swollen macromolecule is then induced to coil tightly by changing the environment, such as pH, charge, or by use of a desolvating agent such as ethanol. The macromolecule may then be fixed and hardened by crosslinking to an aldehyde. The NDGA Compounds can be adsorbed or bound to the macromolecule before crosslinking such that the derivatives become entrapped in the newly formed particle.
Solid lipid NP can be created by high-pressure homogenization. Solid lipid NPs have the advantage that they can be sterilized and autoclaved and possess a solid matrix that provides a controlled release.
The present invention further includes NP with different methods of drug loading. The NP can be solid colloidal NP with homogeneous dispersion of the drug therein. The NP can be solid NP with the drug associated on the exterior of the NP, such as by adsorption. The NP can be a nanocapsule with the drug entrapped therein. The NP can further be solid colloidal NP with homogeneous dispersion of the drug therein together with a cell surface ligand for targeting delivery to the appropriate tissue.
The size of the NPs may be relevant to their effectiveness for a given mode of delivery. The NPs typically ranges from about 10 nm to about 1000 nm; optionally, the NPs can range from about 30 to about 800 μm; further typically, from about 60 to about 270 nm; even further typically, from about 80 to about 260 nm; or from about 90 to about 230 nm, or from about 100 to about 195. Several factors influence the size of the NPs, all of which can be adjusted by a person of ordinary skill in the art, such as, for example, pH of the solution used during polymerization, amount of initiation triggers (such as heat or radiation, etc.) and the concentration of the monomer unit. Sizing of the NPs can be performed by photon correlation spectroscopy using light scattering.
The NPs herein, such as polysaccharide NPs or albumin NPs, may optionally be coated with a lipid coating. For example, polysaccharide NPs can be crosslinked with phosphate (anionic) and quarternary ammonium (cationic) ligands, with or without a lipid bilayer, such as one containing dipalmitoyl phosphatidyl choline and cholesterol coating. Other polymer/stabilizer include, but is not limited to: soybean oil; maltodextrin; polybutylcyanoacrylate; butylcayanoacrylate/dextran 70 kDa, Polysorbate-85; polybutylcyanoacrylate/dextran 70 kDa, polysorbate-85; stearic acid; poly-methylmethylacrylate.
The NP preparations containing the catecholic butanes, such as the NDGA Compounds, such as by adsorption to the NPs, can be administered intravenously for treatment of diseaes, for example, in the brain, heart and reticuloendothelial cell (“RES”) containing organs, such as liver, spleen and bone marrow. To avoid undesirable uptake of these NP preparations by the reticuloendothelial cells, the NPs may be coated with a surfactant or manufactured with a magnetically responsive material.
Thus, optionally, a surfactant may be used in conjunction with the NP. For example, polybutylcyanoacrylate NPs can be used with a dextran-70,000 stabilizer and Polysorbate-80 as a surfactant. Other surfactants include, but not limited to: Polysorbate-20, 40, or 60; Poloxamer 188; lipid coating-dipalmitoyl phosphatidylcholine; Epikuron 200; Poloxamer 338; Polaxamine 908; Polaxamer 407. For example, Polyaxamine 908 may be used as a surfactant to decrease uptake of NPs into the RES of the liver, spleen, lungs, and bone marrow.
The magnetically responsive material can be magnetite (Fe3O4) which can be incorporated into the composition for making the NP. These magnetically responsive NPs can be externally guided by a magnet.
In another embodiment, the NPs herein can be made as described in Mu, L. and Feng, S. S. (2003), using a blend of poly(lactide-co-glycolide)s (“PLGAs”) and d-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS). The latter can also act as an emulsifier, in addition to being a matrix material.
Preparation of Micelle Forming Carriers
The present invention includes catecholic butanes, including the NDGA Compounds, formulated in micelle forming carriers, where the micelles are produced by processes conventional in the art. Examples of such are described in, for example, Liggins, R. T. and Burt, H. M. (2002); Zhang, X. et al. (1996); and Churchill, J. R. and Hutchinson, F. G. (1988). In one such method, polyether-polyester block copolymers, which are amphipathic polymers having hydrophilic (polyether) and hydrophobic (polyester) segments, are used as micelle forming carriers.
Another type of micelles is, for example, that formed by the AB-type block copolymers having both hydrophilic and hydrophobic segments, which are known to form micellar structures in aqueous media due to their amphiphilic character, as described in, for example, Tuzar, Z. and Kratochvil, P. (1976); and Wilhelm, M. et al. (1991). These polymeric micelles are able to maintain satisfactory aqueous stability irrespective of the high content of hydrophobic drug incorporated within the micelle inner core. These micelles, in the range of approximately <200 nm in size, are effective in reducing non-selective RES scavenging and shows enhanced permeability and retention at the targeted sites.
Further, for example, poly(D,L-lactide)-b-methoxypolyethylene glycol (MePEG:PDLLA) diblock copolymers can be made using MePEG 1900 and 5000. The reaction can be allowed to proceed for 3 hr at 160° C., using stannous octoate (0.25%) as a catalyst. However, a temperature as low as 130° C. can be used if the reaction is allowed to proceed for about 6 hr, or a temperature as hight as 190° C. can be used if the reaction is carried out for only about 2 hr.
In one embodiment, N-isopropylacrylamide (“IPAAm”) (Kohjin, Tokyo, Japan) and dimethylacrylamide (“DMAAm”) (Wako Pure Chemicals, Tokyo, Japan) can be used to make hydroxyl-terminated poly(IPAAm-co-DMAAm) in a radical polymerization process, using the method of Kohori, F. et al. (1998). The obtained copolymer can be dissolved in cold water and filtered through two ultrafiltration membranes with a 10,000 and 20,000 molecular weight cut-off. The polymer solution is first filtered through a 20,000 molecular weight cut-off membrane. Then the filtrate was filtered again through a 10,000 molecular weight cut-off membrane. Three molecular weight fractions can be obtained as a result, a low molecular weight, a middle molecular weight, and a high molecular weight fraction. A block copolymer can then be synthesized by a ring opening polymerization of D,L-lactide from the terminal hydroxyl group of the poly(IPAAm-co-DMAAm) of the middle molecular weight fraction. The resulting poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) copolymer can be purified as described in Kohori, F., et al. (1999).
The catecholic butanes, such as the NDGA Compounds, can be loaded into the inner cores of micelles and the micelles prepared simultaneously by a dialysis method. For example, a chloride salt of the NDGA Compounds can be dissolved in N,N-dimethylacetamide (“DMAC”) and added by triethylamine (“TEA”). The poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) block copolymer can be dissolved in DMAC, and distilled water can be added. The solution of NDGA Compounds and the block copolymer solution can be mixed at room temperature, followed by dialysis against distilled water using a dialysis membrane with 12,000-14,000 molecular weight cut-off (SpectralPor®2, spectrum Medical Indus., CA. U.S.A.) at 25° C. Poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) micelles incorporating NDGA Compounds can be purified by filtration with a 20 nm pore sized microfiltration membrane (ANODISC™, Whatman International), as described in Kohori, F., et al. (1999).
Preparation of Multivesicular Liposomes Containing NDGA Compounds
Multivesicular liposomes (“MVL”) can be produced by any method conventional in the art, such as, for example, the double emulsification process as described in Mantripragada, S. (2002). Briefly, in the double emulsification process, a “water-in-oil” emulsion is first made by dissolving amphipathic lipids, such as a phospholipid containing at least one neutral lipid, such as a triglyceride, in one or more volatile organic solvents, and adding to this lipid component an immiscible first aqueous component and a hydrophobic catecholic butane, such as a hydrophobic NDGA Compound. The mixture is then emulsified to form a water-in-oil emulsion, and then mixed with a second immiscible aqueous component followed by mechanical mixing to form solvent spherules suspended in the second aqueous component, forming a water-in-oil-in-water emulsion. The solvent spherules will contain multiple aqueous droplets with the catecholic butane, such as the NDGA Compound dissolved in them. The organic solvent is then removed from the spherules, generally by evaporation, by reduced pressure or by passing a stream of gas over or through the suspension. When the solvent is completely removed, the spherules become MVL, such as DepoFoam particles. When the neutral lipid is omitted in this process, the conventional multilamellar vesicles or unilamellar vesicles will be formed instead of the MVL.
Formulation of Catecholic Butanes, such as NDGA Compounds for Oral Delivery
Some catecholic butanes, such as NDGA Compounds are water-soluble, hydrophilic compounds, such as G4N. This invention includes formulation of hydrophilic compounds in a pharmaceutically acceptable carrier or excipient and delivery of such as oral formulations, such as in the form of an aqueous liquid solution of the compound, or the compounds can be lyophilized and delivered as a powder, or made into a tablet, or the compounds can be encapsulated.
The tablets herein can be enteric coated tablets. The formulations herein can be sustained release, either slow release or rapid release formulations.
The amount of the catecholic butanes, such as NDGA Compounds, to be included in the oral formulations can be adjusted depending on the desired dose to be administered to a subject. Such an adjustment is within the skill of persons conventional in the art.
Some catecholic butanes, including some NDGA Compounds, are hydrophobic or lipophilic compounds, such as M4N. The absorption of lipophilic compounds in the gut can be improved by using pharmaceutically acceptable carriers that can enhance the rate or extent of solubilization of the compound into the aqueous intestinal fluid. Lipidic carriers are known in the art, such as, for example, as described in Stuchlik, M. and Zak, S. (2001) The formulations herein can be delivered as oral liquids or can be encapsulated into various types of capsules.
The present invention includes, in one embodiment, a formulation containing the lipophilic NDGA Compounds that are formulated for oral delivery by dissolution of such compounds in triacylglycerols, and the formulation is then encapsulated for oral delivery. Triacyglycerols are molecules with long chain and/or medium chain fatty acids linked to a glycerol molecule. The long chain fatty acids range from about C14 to C24, and can be found in common fat. The medium chain fatty acids range from about C6 to C12, and can be found in coconut oil or palm kernel oil. Triacylglycerols suitable for use herein include structured lipids that contain mixtures of either short-chain or medium chain fatty acids or both, esterified on the same glycerol molecule.
In another embodiment of the present invention, one or more surfactants can be added to a mixture of catecholic butanes, including NDGA Compounds, and lipidic carrier such that the drug is present in fine droplets of oil/surfactant mix. The surfactants can act to disperse the oily formulation on dilution in the gastrointestinal fluid.
The present invention also includes a formulation for oral delivery of the catecholic butanes, including NDGA Compounds, in the form of a micro-emulsion consisting of hydrophilic surfactant and oil. The micro-emulsion particles can be surfactant micelles containing solubilized oil and drug.
Also suitable for oral administration are formulations of the catecholic butanes, including NDGA Compounds, in a solid lipid nanoparticle preparation. Solid lipid nanoparticles can be prepared in any manner conventional in the art, such as, for example, as described in Stuchlik, M. and Zak, S. (2001).
In one embodiment, the solid lipid nanoparticle can be prepared in a hot homogenization process by homogenization of melted lipids at elevated temperature. In this process, the solid lipid is melted and the catecholic butane, such as the NDGA Compound, is dissolved in the melted lipid. A pre-heated dispersion medium is then mixed with the drug-loaded lipid melt, and the combination is mixed with a homogenisator to form a coarse pre-emulsion. High pressure homogenization is then performed at a temperature above the lipids melting point to produce a oil/water-nanoemulsion. The nanoemulsion is cooled down to room temperature to form solid lipid nanoparticles.
In another embodiment of the present invention, the solid lipid nanoparticles can be prepared in a cold homogenization process. In this process, the lipid is melted and the catecholic butane, such as the NDGA Compound, is dissolved in the melted lipid. The drug-loaded lipid is then solidified in liquid nitrogen or dry ice. The solid drug-lipid is ground in a powder mill to form 50-100 μm particles. The lipid particles are then dispersed in cold aqueous dispersion medium and homogenized at room temperature or below to form solid lipid nanoparticles.
The present invention also includes formulation of the lipophilic catecholic butanes, such as NDGA Compounds, in liposomes or micelles for oral delivery. These formulations can be made in any manner conventional in the art. Micelles are typically lipid monolayer vesicles in which the hydrophobic drug associates with the hydrophobic regions on the monolayer. Liposomes are typically phospholipids bilayer vesicles. The lipophilic catecholic butane, such as the lipophilic NDGA Compounds, will typically reside in the center of these vesicles.
Intra-Arterial Administration
The present invention includes formulation of the catecholic butanes, as exemplified by the NDGA Compounds, for intra-arterial administration as is conventional in the art, as described in, for example, Doolittle, N. D. et al. (2000); and Cloughesy, T. F. et al. (1997), with or without accompanying blood brain barrier disruption (“BBBD”), and with or without occlusion, such as in hepatic artery chemoemobolization, as described in Drougas, J. G. et al. (1998); and Desai, D. C. et al. (2001). Briefly, where NDGA Compounds are administered intra-arterially with occlusion, primary arteries leading to the target site are catheterized and the NDGA Compounds are applied through a catheter. Embolization of the arteries, in order to retain the NDGA Compounds at the target site for a longer period, is performed using polyvinyl alcohol particles alone or in combination with coils. Intra-arterial delivery of the NDGA Compounds is limited to water soluble compositions. Water soluble NDGA Compounds, such as G4N, for example, liposomal formulations of hydrophobic NDGA Compounds, such as M4N, for example, or nanoparticle formulations of hydrophobic NDGA Compounds are particularly suited for this type of delivery. The drugs or agents herein can be dissolved in saline prior to intra-arterial injection and such injection may be preceded by heparin treatment and sedation.
Osmotic disruption of the blood brain barrier (“BBB”) as conventional in the art may accompany intra-arterial delivery of the agents herein as described in, for example, Doolittle, N. D. et al. (2000); Sato, S. et al., Acta Neurochir (Wien) 140: 1135-1141; disc 1141-1132 (1998); and Bhattacharjee, A. K. et al. Brain Res Protocol 8: 126-131 (2001). Such a procedure can be used to increase the transfer of drugs into the central nervous system (“CNS”) preferably just prior to intra-arterial delivery. For such disruption, a catheter is placed into an artery, usually the superficial temporal artery, leading to the brain and the BBB is disrupted with a solution of mannitol. This invasive procedure is typically performed while the patient is under general anesthesia. Such treatment may require prior hydration and administration of anticonvulsants and/or atropine.
Formulation of NDGA Compounds for Intranasal Delivery
The present invention includes formulations of catecholic butanes, as exemplified by the NDGA Compounds, for intranasal delivery and intranasal delivery thereof. Intransal delivery may advantageously build up a higher concentration of the active agents in the brain than can be achieved by intravenous administration. Also, this mode of delivery avoids the problem of first pass metabolism in the liver and gut of the subject receiving the drug.
The amount of the active agents that can be absorbed partly depends on the solubility of the drug in the mucus, a composition that consists of about 95% water solution of serum proteins, glycoproteins, lipids and electrolytes. Generally, as lipophilicity of the active agents herein increases, the drug concentration in the CSF also increases. See, for example, Minn, A. et al. (2002).
The hydrophilic NDGA Compounds can be dissolved in a pharmaceutically acceptable carrier such as saline, phosphate buffer, or phosphate buffered saline. In one embodiment, a 0.05 M phosphate buffer at pH 7.4 can be used as the carrier, as described in, for example, Kao, H. D., et al. (2000).
Intranasal delivery of the present agents may be optimized by adjusting the position of the subject when administering the agents. For example, the head of the patient may be variously positioned upright-90°, supine-90°, supine-45°, or supine-70°, to obtain maximal effect.
The carrier of the composition of NDGA Compounds may be any material that is pharmaceutically acceptable and compatible with the active agents of the composition. Where the carrier is a liquid, it can be hypotonic or isotonic with nasal fluids and within the pH of about 4.5 to about 7.5. Where the carrier is in powdered form it is also within an acceptable pH range.
The carrier composition for intranasal delivery may optionally contain lipophilic substances that may enhance absorption of the active agents across the nasal membrane and into the brain via the olfactory neural pathway. Examples of such lipophilic substances include, but are not limited to, gangliosides and phosphatidylserine. One or several lipophilic adjuvants may be included in the composition, such as, in the form of micelles.
The pharmaceutical composition of active agents for intranasal delivery to a subject for treatment of the diseases, disorders, or conditions herein can be formulated in the manner conventional in the art as described in, for example, U.S. Pat. No. 6,180,603. For example, the composition herein can be formulated as a powder, granules, solution, aerosol, drops, nanoparticles, or liposomes. In addition to the active agents, the composition may contain appropriate adjuvants, buffers, preservatives, salts. Solutions such as nose drops may contain anti-oxidants, buffers, and the like.
Delivery by Implantation
The catecholic butanes herein, as exemplified by the NDGA Compounds, may be delivered to a subject for treatment by surgical implantation into a desired site, such as by implantation of a biodegradable polymer containing the NDGA Compounds. In one embodiment, this method of treatment can be performed, for example, as described in, Fleming, A. B. and Saltzman, W. M., Pharmacokinetics of the Carmustine Implant, Clin. Pharmacokinet, 41: 403-419 (2002).
Thus, the biodegradable polymer herein can be any polymer or copolymer that would dissolve in the interstitial fluid, without any toxicity or adverse effect on host tissues. Preferably, the polymer or monomers from which the polymer is synthesized is approved by the Food and Drug Administration for administration into humans. A copolymer having monomers of different dissolution properties is preferred so as to control the dynamics of degradation, such as increasing the proportion of one monomer over the other to control rate of dissolution.
In one embodiment, the polymer is a copolymer of 1,3-bis-(p-carboxyphenoxy)propane and sebacic acid [p(CPP:SA)], as described in Fleming A. B. and Saltzman, W. M., Pharmacokinetics of the Carmustine Implant, Clin. Pharmacokinet, 41: 403-419 (2002); and Brem, H. and Gabikian, P. (2001). In another embodiment, the polymer is a copolymer of polyethylene glycol (“PEG”) and sebacic acid, as described in Fu, J. et al., (2002).
Polymer delivery systems are applicable to delivery of both hydrophobic and hydrophilic NDGA Compounds herein. The NDGA Compounds are combined with the biodegradable polymers and surgically implanted at the desired or affected site. Some polymer compositions are also usable for intravenous or inhalation therapy herein.
Delivery Through Inhalation
The catecholic butanes herein, as exemplified by the NDGA Compounds, may be delivered systemically and/or locally by administration to the lungs through inhalation. Inhalation delivery of drugs has been well accepted as a method of achieving high drug concentration in the pulmonary tissues without triggering substantial systemic toxicity, as well as a method of accomplishing systemic circulation of the drug. The techniques for producing such formulations are conventional in the art. Efficacy against pulmonary diseases may be seen with either hydrophobic or hydrophilic NDGA Compounds delivered in this manner.
For pulmonary delivery via inhalation, the NDGA Compounds herein may be formulated into dry powders, aqueous solutions, liposomes, nanoparticles, or polymers and administered, for example, as aerosols. Hydrophilic formulations may also be taken up through the alveolar surfaces and into the bloodstream for systemic applications.
In one embodiment, the polymers containing the active agents herein are made and used as described in Fu, J. et al. (2002). For example, the polymers herein can be polymers of sebacic acid and polyethylene glycol (“PEG”), or can be poly(lactic-co-glycolic) acid (“PLGA”), or polymers of polyethyleneimine (“PEI”) and poly-L-lysine (“PLL”).
In another embodiment, the NDGA Compounds for inhalation delivery may be dissolved in saline or ethanol before nebulization and administered, as described in Choi, W. S. et al. (2001).
In a further embodiment, the agents herein are also effective when delivered as a dry powder, prepared in the manner conventional in the art, as described in, for example, Patton, J. S. et al., Inhaled Insulin, Adv. Drug Deliv. Rev., 35: 235-247 (1999).
The present invention includes delivery of the NDGA Compounds with the aid of microprocessors embedded into drug delivery devices, such as, for example, SmartMist™ and AERx™, as described in, for example, Gonda, I., et al. (1998).
After reading the present disclosure, those skilled in the art will recognize other disease states and/or symptoms which might be treated and/or mitigated by the administration of formulations of the present invention.
The following examples are put forth so as to provide those 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 experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Examples in the present tense are prophetic examples.
Tetra-O-Methyl-NDGA, referenced herein as M4N, was synthesized by the reaction of NDGA with excess dimethyl sulfate in the presence of base, such as potassium hydroxide. The product was isolated by the addition of water causing precipitation of the product. The reaction product was passed through a plug of basic alumina to remove traces of phenolic impurities, primarily various species of di-O-methyl and tri-O-methyl-substituted NDGA. After the solution of the reaction mixture had passed through the alumina plug, the solvent was removed on a rotary evaporator giving a solid product. This was triturated with 2-propanol, filtered and dried in a vacuum oven to give crude tetra-O-methyl-NDGA. Crystallization from 2-propanol gave tetra-O-methyl-NDGA with a purity of greater than or equal to 99.66%.
Step 1: Synthesis of Crude Preparation of Tetra-O-Methyl-NDGA
A 22 L flask fitted with a mechanical stirrer, condenser and inlet for inert atmosphere was set up in a tub for use as a cooling bath. The flask was placed under an argon atmosphere, and was charged with 484.3 grams of NDGA (Western Engineering & Research Co, El Paso, Tex.), and 4850 mL of methanol and stirred. To the stirred slurry was added a solution of 387.5 grams of potassium hydroxide in 1210 mL of deionized water. The flask containing this reaction mixture was cooled using an ice bath, and dimethyl sulfate (1210 mL) was slowly added (dropwise). The addition was controlled to avoid an exotherm. At the end of the addition, the temperature was about 13° C. The pH of the reaction was monitored, and a 50% KOH solution was added in portions during the day to maintain a basic pH; a total of 1400 mL of 50% KOH solution was added. The reaction mixture with excess base gave a pH of about 12, as detected using pH indicating strips. The solution was dark at basic pH, but became light colored at neutral or acidic pH.
At the end of the day, an additional 600 mL of dimethyl sulfate was added, and the reaction mixture was allowed to stir overnight. The next morning, the reaction was still basic, and the reaction had progressed to about 90%.
The reaction mixture was quenched by the addition of 4850 mL of deionized water, causing the product to precipitate. The product was isolated by filtration, the filter cake washed thoroughly with water, and the product dried in a vacuum oven at 50° C. for approximately 65 hr to give 539.5 g of the crude product. This product was dissolved in 750 mL of methylene chloride, and to this solution was added 375 mL of toluene. This solution was passed through a short column of 2215 g of basic alumina. The alumina was eluted with 12,000 mL of a methylene chloride/toluene solution (2:1). Removal of the solvent in vacuo on a rotary evaporator gave a solid residue. This was triturated with 1 L of 2-propanol. The resulting slurry was filtered to isolate the solid product. This was dried in a vacuum oven at 50° C. under high vacuum for approximately 21 hr to give 426.7 g (74% yield) of crude tetra-O-methyl-NDGA.
Step 2—Crystallization of Tetra-O-Methyl-NDGA
A 3 L flask with mechanical stirrer, condenser, and inlet was placed in a heating mantle, and was charged with 415.4 g of the product. The flask was charged with 1245 mL of 2-propanol, and the stirred mixture was heated to give a mild reflux; a solution was obtained. The heat was turned off, and the mixture was allowed to cool overnight. The crystalline product was isolated by filtration, and the filter cake washed with 200 mL of cold 2-propanol. The product was dried in a vacuum oven at 50° C. under high vacuum to constant weight giving 404.7 g (70.5% yield overall from NDGA).
The NDGA Compounds can be formulated as a nanoparticle preparation in any manner conventional in the art. For example, the nanoparticles can be prepared as described in Lamprecht, A. et al. (2001a); and Lamprecht, A. et al. (2001b) and as follows.
The biodegradable polymer poly[DL-lactide-co-glycolide] 50/50 (PLGA) (mol. wt. 5,000 or 20,000) can be purchased from Wako (Osaka, Japan). About 40 mg of a NDGA Compound can be dissolved in 4 ml of methylene chloride containing 250 mg of the polymer poly [DL-lactide-coglycolide] 50/50 (mol. wt. 5,000 or 20,000). This solution can thereafter be poured into 8 ml of aqueous polyvinyl alcohol solution (1%) and homogenized with an ultrasonifier (Ultrasonic Disruptor model UR-200P; Tomy Seiko Co., Ltd., Tokyo, Japan) in an ice bath for 3 min. The methylene chloride can be evaporated under reduced pressure, and the polymer precipitated. The nanoparticles can be separated from the non-encapsulated drug and free surfactant by centrifugation (14,000 g for 5 min). Nanoparticles can be redispersed and centrifuged three times in distilled water before lyophilization. Before oral administration, the nanoparticles can be re-dispersed in phosphate buffer at pH 6.8.
The nanoparticles can be analyzed for their size distribution and their surface potential using a Photal laser particle analyzer LPA 3100 (Otsuka Electronics, Osaka, Japan) and a Zetasizer II (Malvern Instruments, Worcestershire, U.K.) respectively. The external morphology of the nanoparticles can be analyzed with a JEOL JSM-T330A scanning microscope (Tokyo, Japan).
NPs containing PLGA and another matrix material, d-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS), can be made as described in Mu, L. and Feng, S. S. (2003), a modified oil-in-water single emulsion solvent evaporation/extraction method. In this method, known amounts of mass of polymer and NDGA Compounds are added into methylene chloride (dichloromethane). The polymer, for example, poly(DL-lactide-co-glycolide (PLGA; L/G=50/50, MW 40,000-75,000; L/G=75/25, MW 90,000-120,000; and L/G=85/15, MW 90,000-120,000), can be purchased from Sigma (USA). Vitamin E TPGS can be obtained from Eastman Chemical, USA. The mixture is stirred to ensure that all the materials are dissolved. The solution of organic phase is then slowly poured in the stirred aqueous solution with or without emulsifier and sonicated simultaneously at 50 W in pulse mode (Misonix, USA). The formed o/w emulsion can be gently stirred at room temperature (22° C.) by a magnetic stirrer overnight to evaporate the organic solvent. The resulting sample can be collected by centrifugation, such as at 10,000 rpm, 10 min. 16° C. (Eppendorf model 5810R, Eppendorf, Hamburg, Germany) and washed once or twice with deionized water for some samples. The produced suspension can be freeze dried (Alpha-2, Martin Christ Freeze Dryers, Germany) to obtain a fine powder of nanoparticles, which can be placed and kept in a vacuum dessicator.
The NDGA Compounds, such as the lipophilic drugs, can be encapsulated in long acting liposomes by processes conventional in the art. One such method is described in, for example, Sharma, U. S. et al. (1997).
Long-acting liposomes have extended blood circulation time. They are typically composed of high phase-transition Tm lipids, high cholesterol content, and a component such as phosphatidyl inositol, monosialoganglioside (GM1), or synthetic phospholipids bearing a polyethylene glycol (PEG) headgroup, which provides a steric barrier against plasma protein access to the liposome surface.
In an example, liposomes composed of phosphatidylcholine (“PC”): cholesterol (“Chol”): polyethylene glycol conjugated to dipalmitoylphosphatidylethanolamine (“PEG-DPPE”) in a molar ratio of 9:5:1 can be prepared. The lipids are initially mixed in chloroform, and a thin film of lipid can be produced by evaporation of the solvent. The lipids are then hydrated in a buffer consisting of NaCl (145 mM), Tris[Hydroxymethyl]-2-aminoethane-sulfonic acid (TES: 10 mM), and ethylenediamine tetraacetate (EDTA: 0.1 mM) buffer, pH 7.2. The liposomes can then be extruded several times through 0.08 μm polycarbonate filters.
In another example, liposomes composed of distearoylphosphatidylcholine (“DSPC): Chol: PEG-DSPE in at a molar ratio of 9:5:1 can be prepared using a “remote loading” method as described in Madden, T. D., et al. (1990). This remote loading method allows for encapsulation of high concentration of NDGA Compounds within the liposome aqueous core. Briefly, a thin film of lipids can be hydrated in ammonium sulfate (250 mM, pH 5.5). The lipid suspension can be extruded through 0.08 μm polycarbonate filters at 60° C. and dialyzed overnight against isotonic sucrose to remove free ammonium sulfate. Hydrophilic NDGA Compounds can be hydrated in 10% (w/v) sucrose and incubated with the preformed liposomes for 1 hr at 65° C. The preparation can be dialyzed against isotonic sucrose to remove the minor residual fraction of unencapsulated drug. This method may yield encapsulation efficiencies of greater than or equal to 90% of the initial NDGA compounds.
Poly(lactide-co-glycolide)-monomethoxy-poly(polyethylene glycol) (PLGA-mPEG) copolymers of different molar ratios can be prepared by a melt polymerization process under vacuum using stannous octoate as catalyst, as described in Beletsi, A et al. (1999); and Avgoustakis, K. et al. (2002).
The NDGA Compounds can be formulated as a dry powder or an aerosol for intranasal delivery by any methods conventional in the art, such as, for example, as described in Marttin, E. et al. (1997).
In one embodiment, the NDGA Compound is formulated as a solution with randomly methylated β-cyclodextrin (“RAMEB”) (degree of substitution 1.8)(Wacker, Burghausen, Germany), mannitol or glucose in MQ water, water that is filtered by a Mili-Q UF plus ultrapure water system from Millipore (Etten-Leur, The Netherlands). This formulation may be administered as a spray or as drops. The dose of NDGA Compound in the liquid formulation may be from about 1 mg/ml to about 1500 mg/ml, or optionally from about 10 mg/ml to about 1200 mg/ml, or further optionally from about 100 mg/ml to about 1000 mg/ml, or still optionally, from about 200 mg/ml to about 800 mg/ml, or any value that falls between these ranges. These liquid formulations can be sprayed into the nostril or applied as drops.
In another embodiment, the present invention includes lyophilized powder formulations of NDGA Compounds, prepared by dissolving the NDGA Compounds and various amounts of RAMEB, lactose, or mannitol in MQ water, and lyophilizing the mixture, such as, for example, overnight.
The NDGA Compounds herein can be incorporated into a biodegradable polymer for implantation into a desired or affected site. Such biodegradable polymer can be made by any method conventional in the art, such as described in Fleming, A. B. and Saltzman, W. M. (2002). One or more wafers of this biodegradable polymer can be implanted at one time depending on the dose of the compounds desired. The biodegradable matrix of the polymer can be made up of polifeprosan 20, a copolymer of 1,3-bis-(p-carboxyphenoxy)propane and sebacic acid [p(CCP:SA)] in a 20:80 molar ratio. To form the polymer for implant, p(CPP:SA) and a compound herein can be co-dissolved in dichloromethane and spray dried to form spherical particles with a size range of about 1 to about 20 μm. The resulting “microspheres” are compression moulded to form wafers of any desired size, such as, for example, about 14 mm in diameter and about 1 mm in thickness. The wafers have a homogeneous structure consisting of densely packed microspheres surrounded by small gaps. Concentration of the NDGA Compounds can be in any amount appropriate for the subject to be treated, such as, for example, 3.8% active compound.
PLGA-mPEG nanoparticles containing the NDGA Compounds can be prepared using the double emulsion method described by Song C. X. et al (1997), with minor modifications. Here, an aqueous solution of the NDGA Compounds can be emulsified in dichloromethane in which the copolymer is dissolved, using probe sonication (Bioblock Scientific, model 75038). This water/oil emulsion can be transferred to an aqueous solution of sodium cholate and the mixture can be probe sonicated. The resulting water/oil/water emulsion formed can be gently stirred at room temperature until evaporation of the organic phase is complete. The nanoparticles made in this way can be purified by centrifugation and reconstituted with deionized and distilled water. The nanoparticles can then be filtered such as through a 1.2-μm filter (Millex AP, Millipore).
Pluronic is a triblock PEO-PPO-PEO copolymer, with PEO representing poly(ethylene oxide), and PPO representing poly(propylene oxide). The hydrophobic central PPO blocks form micelle cores, while the flanking PEO blocks form the shell or corona, which protects the micelles from recognition by the reticuloendothelial system (“RES”). Pluronic copolymers are commercially available from BASF Corp, and ICI. The NDGA Compounds can be introduced into the Pluronic micelles by any method conventional in the art, as described in, for example, Rapoport, N. Y., et al. (1999).
Briefly, the NDGA Compounds can be dissolved in PBS or RPMI medium, followed by a short, such as 15 sec, sonication in a sonication bath operating at 67 kHz. The solution can be kept for about 2 hr at 37° C., upon which the non-solubilized drug can be removed by dialysis through a 1000 D cutoff membrane at 37° C. for about 12 hr against PBS or RPMI medium (dialysis to be done only for 10 and 20 wt % Pluronic solutions).
Implantation of the NDGA Compounds herein can be done in any manner conventional in the art. In one embodiment, implantation is performed as described in Brem, H., and Gabikian, P. (2001). Further, the dura should be closed in a water-tight fashion to eliminate cerebrospinal fluid leakage and to decrease risk of infection. It is also desirable to use preoperative anti-convulsants and high dose steroids as necessary for neurologic compromise. It is further desirable to continue steroid therapy for at least 2 weeks post-operatively.
The NDGA Compounds herein can be delivered via inhalation using any formulation conventional in the art, including as dry powders or as aqueous solutions. The former has the advantage of stability, low susceptibility to microbial growth and high mass per puff. Aqueous solutions offer better reproducibility and avoid the issue of clumping.
In one embodiment, certain of the NDGA Compounds are delivered according to the method as described in Choi, W. S. et al. (2001). Depending on the particular compound and the solubility thereof, the compounds can be formulated to an appropriate concentration in ethanol, such as, for example in a range of from about 1 mg/ml to about 1000 mg/ml, or any intervening values in-between, such as, for example, between about 2 mg/ml and about 800 mg/ml, or between about 4 mg/ml and about 100 mg/ml, or between about 5 mg/ml and about 50 mg/ml. Aerosol particles of 1-3 μm size can be generated for maximal deep lung delivery. For better solubility of the compounds in ethanol, the compounds herein can be first lyophilized, then acidified if necessary or desirable, such as with H3PO4. The pH of the resulting composition can be adjusted with NaOH, if desired, such as to pH 7.4. The resulting composition can then be lyophilized, suspended in ethanol, sonicated and stirred to produce appropriate submicron size particles. The aerosolized compounds can then be administered using a standard commercial nebulizer, such as a compressor (air jet) or an ultrasonic type, or a metered dose inhaler. An example is a PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, Calif.) in conjunction with a PARI PRONEB compressor. A volume of about 9 ml can be charged in the reservoir of the nebulizer and nebulized for up to about 10 min.
In another embodiment, the formulation for inhalation can be prepared as described in Wang, D. L., et al. (2000). For example, powdered NDGA Compounds can be dissolved in 10:90 (v/v) polyethylene glycol 300:100% ethanol containing 0.5% (w/v) ascorbic acid and 0.5% (w/v) phosphatidylcholine. The drug formulation can then be aerosolized using a Pari LC-plus nebulizer (Pari, Richmond, Va.) and a subject to be treated can be exposed to the aerosol generated for varying lengths of time, depending on the dose of the formulation and the desired concentration to be achieved. Such periods of time can be about 5 minutes, 10 minutes, 15 minutes or longer.
The NDGA Compounds can also be formulated in a number of other pharmaceutically acceptable carriers for inhalation purposes. In this example, certain of the compounds herein can be delivered according to the method of Enk, A. H. et al. (2000). Such compounds can be dissolved in a solution containing about 5% glucose and 2% human albumin. Inhalation can then be performed using a specially designed inhalator. (Jetair, Fa. Hoyer, Germany).
The delivery of NDGA derivatives to the oral cavity involves the use of an oral rinse using excipients that are conventional in the art, such as, for example, that described in Armstrong W. B., et al. (2000). The NDGA derivatives are dispensed as a powder that is reconstituted in an appropriate delivery fluid, such as Roxane Saliva Substitute (Roxane Laboratories, Columbus, Ohio), immediately before use. Patients then hold the NDGA derivative suspension in the mouth for about 1 minute before expectorating or swallowing the drug mixture. This procedure is carried out at least once daily for local delivery of NDGA derivatives to the oral cavity.
Alternatively, the delivery of NDGA derivatives to the oral cavity can involve an oral rinse formulation such as described in Epstein, J. B., et al. (2001). Briefly, the NDGA derivatives are prepared in an oral rinse containing about 0.1% alcohol and sorbitol. Patients are provided with a suitable volume, such as about 5 ml of the rinse, to be rinsed in the mouth for about 1 minute and expectorated. This procedure is carried out at least once daily for local delivery of NDGA derivatives to the oral cavity.
In this example, the inventors demonstrated clear safety and efficacy of NDGA derivative delivery in humans as a therapy for head and neck cancer. This example describes the results of two separate clinical studies that spanned range of patient ages, stages of disease development, and two different treatment methods. This example demonstrates the following: (1) M4N can be delivered by escalating doses up to about 495 mg weekly for three weeks or at dosages of 20 mg per day for up to five days without drug-related toxicity. This daily delivery of M4N can be with or without concomitant therapy with G4N at a dose of 20 mg per day for up to five days and is followed by surgical resection of the lesion; (2) Both of these treatment methods delivered over 80% efficacy in terms of induction of necrosis in patients that completed the treatments; (3) In long-term follow ups of the ex-US study 64% of patients remained disease free and also free from long term effects of NDGA-derivative exposure.
US Phase I Intratumoral Head and Neck Cancer Study:
A Phase I clinical study has been completed under a US IND. Mean subject age was 66 years (range 53 to 82 years). Eight male subjects and one female subject participated. Mean weight was 139 lbs. (range 102 to 219 lbs.). All patients were diagnosed with refractory head and neck carcinomas.
Nine (9) subjects were dosed with an intratumoral dose of M4N given weekly for three weeks, at doses of 5 mg/cm3 tumor volume (2 subjects), 10 mg/cm3 (2 subjects) 15 mg/cm3 (3 subjects) and 20 mg/cm3 (2 subjects). Doses up to 495 mg weekly for three weeks were administered.
Three subjects completed the study per protocol. Two subjects died on study from causes considered unlikely to be related to study medication. One subject withdrew consent after receiving three doses of M4N as he could not travel to meet protocol requirements. One withdrew consent after experienced severe radiating pain on injection associated with an accidental perineural dose. One was withdrawn after a single dose as his tumor was considered to be too close to the carotid artery to allow a safe second dose. One was withdrawn as a result of tumor progression. Dosing related adverse events were otherwise minor and included mild or moderate pain on injection (4 subjects). No other adverse events were attributed to M4N administration. Sporadic, and non-reproducible mild elevations in LFTs were seen in 2 subjects, which resolved while still on therapy. No changes attributable to drug were seen in hematology parameters.
Six (6) Serious Adverse Events (SAE) were reported in four (4) subjects. Serious adverse events included supraventricular tachycardia (two episodes on separate occasions in one subject), pneumonia, dehydration and death from tumor progression (one subject), and death 19 days after study (cause unknown). In all cases, the SAE's were considered unlikely or not related to study medication.
In 5 of 6 subjects receiving three doses, drug related tumor necrosis occurred after injection. No damage occurred to healthy tissue surrounding the tumor. Fistula formation developed where tumors were full thickness. Tumors also were noted to have softened, or “pancaked”, but residual tumor at the margins continued to grow, suggesting that systemic administration may be more appropriate. Tumor volume reduction was radiologically confirmed in three of the six patients completing three doses. Dosing was generally well tolerated.
In this example the Maximal Tolerable Dose (MTD) of Dimethyl Sulfoxide (DMSO)] to male and female Beagle Dogs was determined. This example shows that M4N was safely administered by intravenous infusion into dogs over four hours at doses up to 100 mg/kg with a DMSO vehicle. Blood levels of up to 14,000 ng/ml M4N were achieved with these formulations with minimal toxicity.
Vascular Access Port (VAP) Implantation Surgery for M4N-CET Group
VAPs were implanted into beagle dogs such that the tip of the infusion catheter was situated at the level of the superior vena cava. Dogs were treated prophylactically with an analgesic and antibiotic on the day of surgery and with antibiotics and/or analgesics following surgery (according to Gene Logic Inc. SOP Nos. 324.0.2, 325.0.1, and 326.0.2, as appropriate.) Other treatments were provided as recommended by the staff veterinarian. The catheter lines were flushed with saline during the postoperative recovery period with a frequency deemed appropriate by the Study Director.
Although VAPs were implanted into dogs that were assigned to receive infusion of M4N-DMSO, however, DMSO was found to be not compatible with the infusion catheter attached to the VAP inside the animals. Thus the M4N-DMSO group animals were administered with M4N-DMSO with eight intravenous injections via the non-VAP jugular vein every 30 minutes over a 4-hour period. This frequency of delivery mimicked the delivery of the test article using the infusion pump.
a8 intravenous injections every 30 minutes over 4 hours
bAdditional animals to determine potential toxicity, M received 3 injections, F received 2 injections
The dogs in the DMSO group were observed throughout the jugular vein injection period and for at least one hour following the last (eighth) injection.
Blood Sample Collection for Toxicokinetic (TK) Analysis
Blood samples from the DMSO group animals were collected via the cephalic vein on SD 1, SD 3, SD 6, and SD 8 at the following timepoints: predose, 0.25, 0.5, 1, 2, 4, 8, and 16 hours following the final injection dose of M4N-DMSO. Blood samples collected from the animals were processed for plasma and serum for TK analysis.
TK Analysis
The plasma and serum samples were sent to MedTox Laboratories, the Sponsor's designated laboratory for TK analysis. TK analysis of M4N plasma and serum concentration-time data was performed using a validated method (M200406) by MedTox Laboratories and analyzed by noncompartmental methods to obtain estimates of toxicokinetic parameters (where data allow), but not necessarily limited to, Cmax, Tmax and AUC.
Study Day 1 (SD1):
M4N-DMSO Group
Male dog: He reacted to DMSO with slight erythema, slight itchiness, otherwise normal. His behavior was normal soon following end of last injection. Female dog: She reacted similarly to the male dog. Her behavior was normal soon following end of last injection.
All 4 dogs survived the infusion of their respective vehicle treatment. They all appeared fine and behaved normally following treatment.
Study Day 3 (SD3):
M4N-DMSO Group
Male dog: there was no adverse clinical reaction exhibited by this dog. There was, as expected, some irritation at the injection sites along the jugular vein.
Female dog: there was no adverse clinical reaction exhibited by this dog. There was, as expected, some irritation at the injection sites along the jugular vein.
All 4 dogs survived following administration of their respective test article treatment. They all appeared fine and behaved normally following treatment.
Study Day 6 (SD6):
M4N-DMSO Group
Male dog: This dog was successfully injected intravenously with M4N-DMSO via the non-VAP jugular vein for the first 3 dosing intervals (½ hr between doses). As with the previous dosing days, this dog did not show any adverse clinical signs or symptoms following each injection. Prior to the fourth injection, the technicians noticed a swelling “the size of an egg” around the injection site. Subcutaneous misdose could be ruled out because it would have been easily detected during the 3rd injection. It was most likely a hematoma as a result of slow extravasation of blood through the injection site. This animal did not receive any more dosing following the third injection, however, blood samples were collected, the exact time points of the blood collection post-third injection dose was clearly documented. The hematoma resolved within two hours and gentle massaging of the injection site area did not irritate the animal.
Female dog: This dog was successfully injected with M4N-DMSO for the entire 8 repeated injections over 4 hours. Similar to the previous dosing days, this dog did not show any adverse clinical signs or symptoms.
Study Day 8 (SD8):
M4N—DMSO Group
Both the male and female dogs received full dose. Their reactions to the high dose were similar to those on previous dosing days. There appeared to be more G.I. irritation as both dogs showed some retching reaction without vomiting, they were more lethargic than usual. However, both dogs survived the full high-dose administration regimen and appeared to have recovered following the end of dosing.
Additional dose (200 mg/kg) for M4N-DMSO Group
Since animals dosed with M4N-DMSO at 100 mg/Kg did not show adverse clinical signs or symptoms, two spare dogs (1 male, 1 female) were dosed with M4N-DMSO at 200 mg/Kg. At 200 mg/Kg, the female dog experienced difficulty breathing (with nasal frothing) after only the first of eight doses, she soon collapsed but was able to recover for the second dose. After the second dose, her reaction was similar but even more severe. Thus the staff veterinarian suggested euthanizing the female dog. The male dog was slightly more tolerant but exhibited similar difficulty breathing signs and collapsing symptoms. He received a total of three doses and the staff veterinarian suggested further dosing be stopped. There were no post-dose TK analysis for this additional dosing, all pre-dose blood samples collected today were discarded.
TK Analysis
In general, repeated intravenous injection of M4N-DMSO at different dose levels for 4 hours resulted in extremely high serum concentrations. The serum concentration data reported in Table 2 are the results following systematic dilution of the serum to accommodate detection range. The results showed that in general, the serum concentration of M4N-DMSO was high in the early time points and peaked at 30 minutes following the last injection. The serum concentrations of the test article reduced over the next 15 hours. Based on the serum concentrations from this group, the half-life of M4N-DMSO, when administered by repeated intravenous injection, was approximately 1.5 to 2 hours. It is noteworthy that from the pre-dose serum concentrations of M4N-DMSO over the course of this MTD phase, there was a slight build-up of the test article in the blood, but this retention was generally less than 0.3% of the highest serum concentration.
The purpose of the MTD phase of this study was to determine the maximum tolerable dose of Dimethyl Sulfoxide (DMSO) to male and female Beagle Dogs. Animals that received repeated injections of M4N-DMSO showed some irritation at the injection site and minor retching at 100 mg/kg. However, both animals collapsed following 2 or 3 injections of M4N-DMSO at 200 mg/kg. TK analysis from this group suggested a half life for M4N-DMSO in the range of 1.5-2 hours. There was minor build up of the test article over the course of the MTD phase, however, this retention only amounted to less than 0.3% of the maximum serum concentration. In conclusion, the MTD phase of this study was a success, as the dose level that resulted in significant adverse clinical signs and symptoms was identified, thus the objective of this phase was achieved.
The ability of NDGA derivatives to inhibit the formation of protein deposits known as amyloids, which are characteristic of Alzheimer's disease, can be measured using an in vivo murine model of amyloid formation, such as that described in U.S. Pat. No. 5,164,295. Briefly, mice of the CD strain are injected subcutaneously with AgNO3 and intravenously with amyloid enhancing factor. The treated animals are then given daily administration of one or more NDGA derivatives in suitable formulations and doses, depending on the particular NDGA derivative, for a period of six days. At the end of six days, the experiment is terminated, and the animals are sacrificed by cervical dislocation. The spleens, livers, and kidneys are fixed and embedded in paraffin. Sections of the paraffin are stained with Congo Red and viewed under polarized light. Image analysis is conducted to determine the percent of spleen occupied by amyloid. Those animals given NDGA derivatives have reduced formation of amyloid as demonstrated by a lower percentage of spleen occupied by amyloid as compared to the untreated controls.
The ability of NDGA derivatives to protect neurons from neurotoxic Alzheimer's Aβ amyloid fibrils can be measured using an in vitro model, such as that described in DeFelice, F. G. et al. (2001). Amyloid fibrils are formed in vitro by dissolving full-length Aβ peptide in aqueous buffer. This fibril solution is then added to primary cultures of E18 rat hippocampal neurons. Finally, appropriate amounts of NDGA derivatives are added to the culture media and the cells are incubated under standard conditions for five days. The results of the experiment are viewed by light microscopy. The cultures given NDGA derivatives protect against amyloid fibril neurotoxicity and contain large cell bodies with long, branched neurites, whereas unprotected cultures exhibit significant neuronal degeneration and cell death.
The ability of NDGA derivatives to protect cells from cytotoxic Alzheimer's Aβ amyloid fibrils can be measured using an in vitro model, such as that described in Klunk, W. E. et al. (1998). Amyloid fibrils are formed in vitro by dissolving Aβ peptide in distilled deionized water and incubating at 37° C. for seven days. This fibril solution is then combined with rat pheochromocytoma PC12 cells, and appropriate amounts of NDGA derivatives are added to the culture media. After 24 hours, the viability of the cells is assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cultures given NDGA derivatives that protect against amyloid fibril cytotoxicity absorb more light at 560 nm than unprotected samples.
The ability of NDGA derivatives to inhibit the formation of β-amyloid fibrils from α-amyloid peptide can be measured using an in vitro model of amyloid fibril formation, such as that described in Howlett, D. R., et al. (1999). Briefly, β-amyloid peptide and NDGA derivatives are diluted in PBS and incubated at 37° C. in microtiter plates. Aliquots of peptide/NDGA derivative incubates are stained with uranyl acetate and subsequently examined by transmission electron microscopy. Samples where β-amyloid fibril formation is prevented by treatment with NDGA derivatives show much shorter fibrils than untreated controls.
The ability of NDGA derivatives to inhibit inflammation and the formation of Alzheimer's disease related plaques can be measured using an in vivo murine model of Alzheimer's disease progression, such as that described in Lim, G. P. et al. (2001). Ten-month-old male and female transgenic APPSw mice (Tg2576) are randomly split between treatment groups and are fed either mouse chow or mouse chow supplemented with the NDGA derivatives. After six months, the mice are perfused with saline and HEPES buffer containing the protease inhibitors leupeptin and aprotinin. Brain regions from the animals are dissected and snap frozen in liquid nitrogen.
Hemibrain cryostat sections cut from the hippocampus are incubated with antibodies directed against amyloid peptide fibrils or antibodies against phosphotyrosine (PT) which serves as a rodent microglia marker. Following the primary antibody, secondary biotinylated goat anti-rabbit antibody is used to boost the responding signal. Chemiluminescent substrate is added to the samples and the signal is visualized using photographic emulsion. Final images are viewed by light microscopy and analyzed with NIH Image software. Brain tissue protected by NDGA derivatives is clearly discernable from vulnerable tissue by the absence of areas of inflammation and concentrated immunostaining (plaques).
The ability of the NDGA derivatives to reduce serum glucose, triglyceride, and non-esterified fatty acids is measured in a rat model of type II diabetes by conventional methods, such as one described by Reed, M. J. et al. (1999). In this method, six week old Male Sprague-Dawley rats are fed a high fat diet consisting about 20% fat, 46% carbohydrate, and 20% protein (w/v) (Harlan Teklad, Madison, Wis., USA). After 2 weeks on this diet, animals are anesthetized, such as with ketamine (about 65 mg/kg) and xylazine (about 7 mg/kg), and injected with streptozotocin (about 0.19 mmol/kg) into the tail vein. For three days after streptozotocin injection, diabetic animals are given free access to food and water. Starting the next day and for the next 3 days, that is, experimental days 1-4, the diabetic animals are treated as follows: food is removed in the morning (such as at 0700 hr), the animals are dosed twice with at least one NDGA derivative dissolved in a suitable vehicle via oral gavage (for example at 1100 hr and 1600 hr), then food is returned to the animals (for example at 1700 hr).
Blood from the animals can be sampled daily by tail milking about three hours post drug dose, such as at 1400 hr. The removed serum can be separated by centrifugation in serum separator tubes (Becton Dickinson, Franklin Lakes, N.J., USA) and various parameters are analyzed using commercially available enzymatic colorimetric assays, such as, glucose (Trinder method, Sigma Diagnostics, Sigma Chemical Co., St. Louis, Mo., USA), insulin (RIA, Linco Research, St. Charles, Mo., USA), glycerol and triglyceride (GPO-Trinder method, Sigma), and non-esterified fatty acids. The extent of NDGA derivative-mediated glucose, triglyceride, and non-esterified fatty acid reduction is visualized by spectroscopic measurements of resultant color intensity.
Insulin-mediated glucose disposition can then be determined on the fifth day of dosing. Food is removed in the morning, such as at 0700 hr, and animals are dosed with at least one NDGA derivative at 0930 hr. At 1000 hr, animals are anaesthetized, such as with sodium pentobarbital (65 mg/kg i.p.) and a cannula (0.064 cm internal diameter, Braintree Scientific, Braintree, Mass., USA) is placed in the jugular vein. The animals are infused with epinephrine (0.08 mg.kg.min−) and propranolol (1.7 mg.kg.min−1) for 1 hr and then are additionally infused with glucose (0.22 mmol.kg.min−1) and insulin (30 pmol.kg.min−1) for 4 hr. Sodium pentobarbital is reinjected i.p. as necessary to maintain anesthesia. Blood samples are taken periodically, such as every 30 min during the final hour of the infusion. Under these conditions, glucose and insulin concentrations are in a steady state during the last hr of infusion. The steady state plasma glucose concentration achieved by each animal is a measure of its whole body insulin-mediated glucose disposal.
Basal and insulin-stimulated glucose clearance by isolated adipocytes can also be determined, such as by conventional methods. For example, epididymal fat pads from normal rats are removed, minced with scissors, and placed in plastic flasks in Krebs bicarbonate buffer with 2% bovine serum albumin (“BSA”), 3 mmol/l glucose, and 1 mg collagenase/ml. Collagenase digestion is carried out at 37° C. in a gyratory shaker for 1 hr. Cells are then washed three times in fresh Krebs buffer with 2% BSA and allowed to separate from the infranatant by floatation. Isolated adipocytes (2% lipocrit) are incubated for 90 min in 200 μl Krebs buffer with 2% BSA, NDGA derivative (about 30 μmol/l), and tracer (300 nmol/l) amounts of D-[U-14C]-glucose in the absence and presence of insulin (8 nmol/l). The cell suspension is incubated in a water bath at 37° C. for 1 hr with continuous shaking at 40 rpm, followed by addition of 200 μl of cell suspension to a microcentrifuge tube containing 200 μl of silicone oil. The assay is terminated by centrifugation of the microcentrifuge tube. The supernatant layer of the tube, containing the fat cells and incorporated glucose, was placed in a scintillation vial, and the amount of radioactivity associated with the adipocytes (and the total radioactivity in the incubation medium) is determined by liquid scintillation.
Isolated adipocytes are fixed with 2% osmium tetroxide, washed and diluted in saline. Cells in a 100 μl aliquot are counted using a Coulter electronic cell counter (model ZB; Coulter Electronics, Inc., Hialeah, Fla., USA) with a 400 μm aperture equipped with logarithmic range-expanded channelyser.
Serum glucose of rats treated with NDGA derivatives is found to be lower than that in control rats. Serum triglyceride concentration in rats receiving NDGA derivative is also found to be lower than that in control rats. Further, serum non-esterified fatty acid concentration in rats receiving NDGA derivative is also lower than that in control rats.
The ability of NDGA derivatives to reduce systolic blood pressure can be measured in a rat model of fructose-induced hypertension, such as that described by Gowri, M. S., et al. (1999). In this system, male Sprague-Dawley rats are placed on a diet that provides 60% of their total calories as fructose. The fructose-enriched diet is given for a period of time, such as 11 days, during which time the rats are acclimated to the procedure of blood pressure measurement.
At the end of this dietary period, blood pressure of these rats can be determined and the rats can be divided into two groups, one being treated with one or more NDGA derivatives, and the other used as control. Both groups are maintained on the fructose-rich diet. One group is gavaged with the NDGA derivatives (at bout 80 mg/kg, twice daily), dissolved in a suitable vehicle, such as gelucire. The other group is treated in the same manner, but with vehicle alone. The systolic blood pressure is measured before and 4 days after treatment by the tail-cuff method without external preheating, in the conscious state, a method that has been shown to be similar to that obtained by direct arterial cannulation. Measurement is taken at ambient temperature, kept at 30° C. The mean of consecutive readings is used as the measurement of the systolic blood pressure of each rate for that day. The final blood pressure determinations are performed on the afternoon after the last morning dose of NDGA derivative or vehicle.
For some rats in the study, the tail vein blood can be removed about 4 hr after removal of food, centrifuged, frozen and later assayed for plasma glucose, insulin, and triglyceride concentrations. Plasma free fatty acid concentration is also assayed enzymatically by the ACS-ACOD method using a commercial kit (Waro Chemicals, Inc., Richmond, Va., USA). Results are expressed as mean values and the significance of differences between the NDGA derivative treated group and the controls can be determined by one-way analysis of variance (ANOVA) analysis.
Results show that while the baseline blood pressures in the NDGA derivative-treated group and the control group are similar, they diverge dramatically after NDGA derivative-treatment began. While the blood pressure of the control group continues to increase, the blood pressure of the NDGA derivative-treated group decline to below baseline.
Results also show that while the plasma glucose of the treated and control groups are similar, the insulin, free fatty acid, and triglyceride levels of the treated rats are significantly lower than the control rats.
The ability of the NDGA derivatives to reduce serum triglycerides and LDL cholesterol in a population of hypercholesterolaemic patients is determined through the employment of a method such as that described in Branchi, A. et al. (1999). In this study, hypercholesterolaemic patients, such as those with LDL cholesterol levels of 160 mg/dl or greater and with serum triglyceride levels of less than 400 mg/dl at two consecutive examinations are selected for the research. Patients with diabetes, hypothyroidism, renal failure, and liver disease are excluded. All patients have to follow a low-fat low-cholesterol diet for more than three months before entering the study, and are required to refrain from the use of drugs affecting lipid metabolism.
The selected patients are given daily treatments of NDGA derivatives for two months, for example, at a dose selected from the group consisting of about 1, 5, 10, 20, 40, and 50 mg per day, and blood samples are collected in the mornings following an overnight fast. The blood is then centrifuged and high-density lipoprotein (HDL) is separated by precipitating apo-B containing lipoproteins with phosphotungstate/Mg2+ (Boehringer Mannheim Italia S.p.A., Milano, Italy). Total and HDL cholesterol are measured using a cholesterol oxidase method such as that produced by Ames-Bayer Diagnostics (ORCQ-Tournai, Belgium) and serum triglyceride levels are determined using a glycerol phosphate technique (Kallestad-Pasteur Diagnostici, Milano, Italy). NDGA derivatives at a therapeutic dose can be seen to significantly reduce serum triglyceride concentration.
Guidelines for the prevention of coronary heart disease (“CHD”) have called for reducing the level of plasma low-density lipoprotein cholesterol (“LDL-C”) as the primary target of treatment. Furthermore, recent evidence indicates that elevated level of triglycerides (“TG”) constitutes an additional independent risk factor for CHD. Therefore, to achieve the greatest possible reduction in risk of CHD, it is useful to target treatment to reduce both LDL-C and TG levels.
The reduction of LDL-C and TG by NDGA derivatives as a preventative action of coronary heart disease in humans can be assessed by a method similar to that described in A. G. Olsson et al. (2003). Briefly, adult patients, such as those between ages of 35 and 75 years with cardiovascular disease and dyslipidemia (defined as having plasma LDL-C concentration of greater than or equal to 4 mmol/L, or greater than or equal to 155 mg/dL on 2 consecutive occasions) are treated with NDGA derivatives.
Treated patients receive once-daily oral treatment of an NDGA derivative in a suitable vehicle. The patients' progress is monitored by determining fasting (12-hour) blood lipid levels and clinical laboratory values after 4, 8, 12, 26, and 52 weeks. The dose of the NDGA derivative may be increased after 12 weeks if a predetermined level of LDL-C and/or TG is not reached at 8 weeks. The LDL-C concentrations in the blood are calculated using the Friedewald formula. TG concentration is measured enzymatically with a Technicon DAX-96 analyzer. NDGA derivatives can be seen to lower plasma LDL-C and serum TG.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
This application is a continuation-in-part of International Patent Application No. PCT/US2004/016117, filed May 20, 2004, and published in the English language as International Publication No. WO 2004/112696 Dec. 29, 2004; which claims priority to U.S. provisional application No. 60/472,008, filed May 20, 2003; U.S. provisional application No. 60/472,144, filed May 20, 2003; U.S. provisional application No. 60/472,188, filed May 20, 2003; U.S. provisional application No. 60/472,282, filed May 20, 2003; and U.S. provisional application No. 60/472,299, filed May 20, 2003; the contents of all of which are incorporated herein by reference in their entireties. This application is designated as a continuation-in-part out of an abundance of caution, since some language has changed compared to the parent International Application No. PCT/US2004/016117; however the applicants believe that this application is fully supported by and within the scope of the parent International Application.
| Number | Date | Country | |
|---|---|---|---|
| 60472008 | May 2003 | US | |
| 60472144 | May 2003 | US | |
| 60472188 | May 2003 | US | |
| 60472282 | May 2003 | US | |
| 60472299 | May 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US04/16117 | May 2004 | US |
| Child | 11284110 | Nov 2005 | US |
