Abstract
A novel dual neurotransmitter nanoparticle composition is provided to store and transport protons and cations into neural cell membranes and to disassemble salt-bridge stabilized toxic protein plaques. These properties function to mitigate cognitive deficits in neurological diseases such as Parkinson's disease and Alzheimer's disease, as well as to reduce the severity of aging related reactive oxygen species damage by the sequestration and termination of free radicals and reactive oxygen species. The composition comprises C60 bonded to one or more glutathione molecules and one or more molecules of either levodopa or dopamine. The composition can be produced at low temperatures through reactive shear milling. This composition therapeutically improves and prophylactically preserves cognitive performance, memory, and mental acuity on aging to promote mental performance and health-span improvement.
Claims
1. A compound comprising: a buckminsterfullerene C60 bonded to a first neurotransmitter.
2. The compound of claim 1 further comprising a second neurotransmitter bonded to the buckminsterfullerene C60 and different than the first neurotransmitter.
3. The compound of claim 1 wherein the buckminsterfullerene C60 is bonded to the first neurotransmitter by a pi bond.
4. The compound of claim 2 wherein the buckminsterfullerene C60 is bonded to the first neurotransmitter by a first pi bond and the buckminsterfullerene C60 is bonded to the second neurotransmitter by a second pi bond.
5. The compound of claim 1 wherein the first neurotransmitter comprises glutathione.
6. The compound of claim 2 wherein the first neurotransmitter comprises glutathione and the second neurotransmitter comprises either levodopa or dopamine.
7. The compound of claim 1 disposed within a zeolite.
8. A method of curing, treating, or prophylactically avoiding motor neuron dysfunction related to oligomeric alpha-synuclein plaque formation in Parkinson's disease and Lewy Body Disease in a subject, or prophylactically avoiding motor neuron dysfunction related to oligomeric plaque formation in Alzheimer's disease or Amyotrophic Lateral Sclerosis (ALS) in the subject, comprising the step of: administering to the subject an effective amount of a compound including a buckminsterfullerene C60 bonded to a first neurotransmitter and a second neurotransmitter different than the first neurotransmitter.
9. The method of claim 8 wherein the first neurotransmitter comprises glutathione and the second neurotransmitter comprises either levodopa or dopamine.
10. The method of claim 8 wherein administering the compound comprises administering a composition containing the compound in a pharmaceutically acceptable carrier.
11. The method of claim 10 wherein the composition comprises a tablet, capsule, pill, powder, granule, or a form suitable for injection.
12. The method of claim 10 wherein the pharmaceutically acceptable carrier comprises a zeolite.
13. The method of claim 8 wherein administering the compound comprises administration by an intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal, topical, nasal, or oral route.
14. The method of claim 8 wherein an oral dosage comprises up to about 500 mg of the compound.
15. The method of claim 8 wherein administering the compound comprises intramuscular, intravenous, or subcutaneous administration in an amount of from about 0.1 mg/Kg to about 5 mg/Kg.
16. The method of claim 8 wherein administering the compound comprises administration by a nano aerosol, a vapor, a powder, a dust, or an aerosolized inhalant.
17. A method of making a C60 bonded to a neurotransmitter, the method comprising: bonding a glutathione to the C60; and bonding either a levodopa or a dopamine to the C60.
18. The method of claim 17 wherein bonding the glutathione to the C60 and bonding either the levodopa or the dopamine to the C60 are performed at no more than 40° C.
19. The method of claim 17 wherein bonding the glutathione to the C60 and bonding either the levodopa or the dopamine to the C60 is performed by reaction shear mixing.
20. The method of claim 17 wherein bonding the glutathione to the C60 and bonding either the levodopa or the dopamine to the C60 is performed in an oxygen-free environment.
21. The method of claim 17 wherein bonding the glutathione to the C60 and bonding either the levodopa or the dopamine to the C60 are performed together.
22. The method of claim 17 further comprising disposing the buckminsterfullerene C60 bonded to the glutathione and either the levodopa or the dopamine within channels of a zeolite.
23. The method of claim 17 further comprising combining the buckminsterfullerene C60 bonded to the glutathione and either the levodopa or the dopamine with a pharmaceutically acceptable carrier.
24. The method of claim 17 further comprising adding the buckminsterfullerene C60 bonded to the glutathione and either the levodopa or the dopamine to a mixture of glycerol and polypropylene glycol.
25. The method of claim 17 further comprising dissolving the buckminsterfullerene C60 bonded to the glutathione and either the levodopa or the dopamine, into a hyaluronic acid solution.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Preferred embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:
[0036] FIG. 1 is an illustration of some molecular structures of raw materials relevant to the teachings of the present invention.
[0037] FIG. 2 is an illustration of molecular structures of the reversible REDOX reaction of glutathione (GSH).
[0038] FIG. 3 is an illustration of molecular structures of the reactions of glutathione (GSH) with buckminsterfullerene (C60).
[0039] FIG. 4 is an illustration of the molecular structures of the reactions of levodopa (L-dopa) with buckminsterfullerene (C60).
[0040] FIG. 5 is an illustration of dopamine, glutathione, and C60 chemically reacting to synthesize C60-GSH-Dopa having multiple aryl pi-pi bond formation.
[0041] FIG. 6 is an illustration of C60-GSH-L-dopa conformations in which pi-carbonyl bonds, aromatic pi to aromatic-pi bonds, and zwitterionic hydrogen bonds create a molecular network structure.
[0042] FIG. 7 is an illustration of alpha-synuclein plaques being intercalated with and disassembled by clusters of C60-GSH-L-dopa and/or metabolites thereof comprising C60-GSH-DOPA.
[0043] FIG. 8 is an illustration of clusters of C60-GSH-L-dopa and/or metabolites thereof comprising C60-GSH-DOPA providing protection and treatment at the neural synapse and at neural membranes.
[0044] FIG. 9 is an illustration of a molecular structure for Transcarpathian zeolite (clinoptilolite) binder permeated or filled with C60-GSH-L-dopa.
[0045] FIG. 10 is a flowchart representation of a synthesis of C60-GSH-L-dopa with a formulation for use as a nano-aerosol inhalant.
[0046] FIG. 11 is a flowchart representation of a synthesis of C60-GSH-L-dopa with formulations for Oral Administration.
[0047] FIG. 12 is an illustration of personal administration of aspirated nano-aerosol C60-GSH-L-dopa.
[0048] FIG. 13 is an illustration of experimental FTIR data for levodopa (L-dopa).
[0049] FIG. 14 is an illustration of experimental FTIR data for buckminsterfullerene levodopa (C60-L-dopa).
[0050] FIG. 15 is an illustration of experimental FTIR data for reduced glutathione (GSH).
[0051] FIG. 16 is an illustration of experimental FTIR data for buckminsterfullerene glutathione (C60-GSH).
[0052] FIG. 17 is an illustration of experimental FTIR data for C60-GSH-L-dopa.
[0053] FIG. 18 is an illustration of an experimental negative mode mass spectrograph data for C60-L-dopa.
[0054] FIG. 19 is an illustration of an experimental negative mode mass spectrograph data for C60-GSH.
[0055] FIG. 20 is an illustration of an experimental negative mode mass spectrograph data for C60-GSH-L-dopa.
[0056] Some embodiments are described in detail with reference to the related drawings. Additional embodiments, features, and/or advantages will become apparent from the ensuing description or may be learned by practicing the invention. In the illustrations, which are not drawn to scale, like numerals refer to like features throughout the description. The following description is not to be taken in a limiting sense but is made merely for describing the general principles of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The following detailed description, taken in conjunction with the accompanying drawings, is merely exemplary in nature and is not intended to limit the described embodiments or the application and uses of the described embodiments. Any implementation described herein as “exemplary” or “illustrative” is not necessarily to be construed as preferred or advantageous over other implementations.
[0058] Furthermore, there is no intention to be bound by any expressed or implied theory presented in the preceding technical field, background, brief summary or the following detailed description. It is also understood that the specific devices, systems, methods, and processes illustrated in the attached drawings, and described in the following specification, are simply exemplary embodiments of the inventive concepts defined in the appended claims that there may be variations to the drawings, steps, methods, or processes, depicted therein without departing from the spirit of the invention. All these variations are within the scope of the present invention. Hence, specific structural and functional details disclosed in relation to the exemplary embodiments described herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present embodiments in virtually any appropriate form, and it will be apparent to those skilled in the art that the present invention may be practiced without these specific details.
[0059] Various terms used in the following detailed description are provided and included for giving a perspective understanding of the function, operation, and use of the present invention, and such terms are not intended to limit the embodiments, scope, claims, or use of the present invention.
[0060] FIG. 1 illustrates molecular structures 10 used or metabolized in the composition of the present invention. Dopamine (DOPA) 11 has chemical formula C.sub.8H.sub.11NO.sub.2 and is also known as the endogenous neurotransmitter 3,4-dihydroxyphenethylamine. Levodopa (L-dopa) 12 is an amino acid of chemical formula C.sub.9H.sub.11NO.sub.4 that is commercially available as a synthetic food supplement and is readily metabolized by decarboxylation to form the neurotransmitter dopamine (DOPA) 11 as well as other neurotransmitters. It is generally understood and recognized that L-dopa 12 is a chief chemical precursor to DOPA 11 and may be used in neuroprotective treatments for Parkinson's Disease and other neurological disorders. The molecular structure 17 is reduced glutathione and has the chemical formula C.sub.10H.sub.17N.sub.3O.sub.6S. GSH may function somewhat as a neurotransmitter in that it operates on GABAergic neurons to release gamma amino butyric acid (GABA) and may have other endogenous signaling functions. Buckminsterfullerene 16 is a single molecule comprised of 60 carbon atoms arranged as a sphere and has the chemical formula of C60. Substances 11, 12, 13, 17 may be used to help create, process, or deliver parts of the composition of C60-GSH-L-dopa.
[0061] FIG. 2 illustrates the molecular structures of the reversible biochemical oxidation reaction of glutathione (GSH) 20. Two of the reduced form of glutathione molecules 22 become oxidized into a dimeric form of glutathione having a characteristic sulfur to sulfur bond 24. In the biochemical process of cellular respiration, the oxidized form of glutathione 24 (also known by the abbreviation GSSH), is reduced by two hydrogen protons 26 to reform GSSH into two discrete GSH molecules 22. It is understood that this is a reversible biological oxidation and reduction (redox) process as indicated by the directions of the upward and the downward solid black arrows. Reversible REDOX reactions will likewise take place with the GSH functional groups of the various derivatives of glutathione specified herein.
[0062] FIG. 3 illustrates molecular structures of two chemical reaction pathways 30 of glutathione (GSH) 32 with buckminsterfullerene (C60) 31. Hydrogen bonds are indicated by dotted lines and pi-bonding is indicated by dashed lines herein and throughout this specification. At elevated temperatures being above about 50° C., the direction of the reaction pathway increasingly follows the white arrow 33 to produce at least one covalent bond 38 between the at least one GSH nitrogen functional group and the C60 functional group to form a covalently bonded GSH-C60 34. This high temperature reaction pathway is undesirable because it removes the neuroprotective and antioxidant effect of the nitrogen amine functional group. At room temperature being below at most 40° C., the direction of the reaction pathway under high pressure shear conditions, substantially follows the solid black arrow 35 to produce at least one sulfide bond 36 between the at least one GSH and the C60 functional group, forming the configurational isomer of GSH-C60, 37 having the preferred geometry in which the amine nitrogen of GSH is free to act as a reducing agent against oxidants in a neuroprotective manner.
[0063] FIG. 4 illustrates molecular structures of two chemical reaction pathways 40 of L-levodopa (L-dopa), 42 with buckminsterfullerene (C60), 41. At elevated temperatures being above 120° C., the direction of the reaction pathway follows the white arrow to produce at least one covalent bond 44 between the at least one L-dopa nitrogen functional group, or at the carboxylic acid functional group, to react with a carbon atom in C60 43; this type of reaction is to be avoided at the C60 functional group, because these two covalently bonded configurational isomers of L-dopa do not ensure the preservation of a labile and neurologically available amine adduct in accordance with the molecular design specified herein. The pi bonded L-dopa with C60 is capable of being achieved under shear mixing conditions and at room temperature or below at most about 40° C. Pi bonds are stronger than hydrogen bonds, but much weaker than covalent bonds; this also means that they can form with less energy. The low temperature and high shear pressure reaction is in the direction of the reaction pathway that follows the solid black arrow to produce aromatic pi to carbonyl bond 45 and/or an aromatic-pi to aromatic-pi bond 46 between the at least one GSH functional group and the C60 functional group, being C60-Ldopa 47, having the preferred adduct geometry in which the amine nitrogen of the L-dopa functional group 49 is free to attract a hydrogen proton to act as a reducing agent against oxidants in a neuroprotective manner.
[0064] FIG. 5 illustrates the chemical reaction 500 of dopamine (Dopa) 530 and glutathione (GSH) 520 with buckminsterfullerene (C60) 510, to generate the products shown in the direction of the large black arrow. It is understood that metabolic conversion of L-dopa to dopamine 530 occurs by loss of the carboxyl (—COOH) functional group from levodopa, therefore the use of dopamine as a starting material is interchangeable with, and functionally equivalent to, the use of L-dopa in the present invention and is hereby explicitly specified. The multiplicity of functional groups of GSH denoted by the subscript letter x after the molecular structure within the bracketed region 540, is shown covalently bonded through sulfur to C60 595. The multiplicity of Dopa functional groups 570 denoted by the subscript letter y after the molecular structure within the bracketed region, may become reversibly hydrogen bonded to any GSH 540, 520 though a hydrogen bond 550. Nominally, x is 1 and y is 2, where it is understood that the neurotransmitter Dopa functional groups 570 can be replaced by levodopa as these will become metabolized to Dopa. Aromatic pi-to aromatic-pi bond represented by dashed line 560 and aromatic pi to carbonyl bond 590 each have more molecular structural strength than a hydrogen bond 550 but are less strong than a covalent bond such as bond 580. Temperatures above 55° C. tend to form the sulfur covalent bond 580, however the formation of pi-carbonyl bonds 590 may form in preponderance without covalent bonding 580 at reaction temperatures below about 40° C. Both GSH 520 and dopamine 530 function as independent neurotransmitters, however it is the design of the present composition 500 to promote these as a dual neurotransmitter function of functional groups GSH 540 and Dopa 570, thereby conferring oxidation resistance to those regions of the neuron such as the post-synaptic bouton where the absorption of functional group dopamine 570 coupled with GSH acts to promote neuronal healing and recovery from the neurological damage of Parkinson's disease and other neurological pathology.
[0065] FIG. 6 illustrates the molecular structures leading to formation of a networked C60-GSH-L-Dopa 60. It is understood that dopamine will be formed after decarboxylative metabolism of some or all the L-dopa functional groups 64. Administering L-dopa alone can lead to excessive undesirable neural signaling and may also cause many of the adverse side effects associated with Dyskinesia under conditions of oxidative stress by means of the networked molecular structure 60 promoting neuroprotection by means of the antioxidant buckminsterfullerene (C60) group 69 and the antioxidant reduced glutathione (GSH) groups 61, 67. A multiplicity of hydrogen bonds is represented by dotted lines 63, 66 in these structures. A multiplicity of pi-bonds is illustrated as dashed lines 62, 65 extending outwards from the C60 group, by representative pi-carbonyl 65, and pi-pi bonds 62. Levodopa (L-dopa) 64 is an amino acid of chemical formula C.sub.9H.sub.11NO.sub.4 that is commercially available as a synthetic food supplement. Each levodopa (L-dopa) 64 functional group on C60, 69 is readily metabolized by decarboxylation to form dopamine (DOPA). Glutathione 61, 67 is considered a neurotransmitter as well as an antioxidant. It can reversibly bond through the sulfur atom to C60 at 68, or it can bond by pi-carbonyl bond to C60 at 61, where the latter pi-carbonyl molecular configuration 61 allows the reducing power of the sulfur group (sulfhydryl) to perform as a reducing agent.
[0066] At least one glutathione (GSH) molecule 61 has formula C.sub.4H.sub.9NO.sub.2 and reacts with a multiplicity (z) with C60 69. At least one and as many as about 8 (y=1 to 8) of the levodopa functional groups 64 reacts with C60, 69. Sometimes, GSH 67 can react through the sulfur group 68 to form the derivative C60-GSH providing a multiplicity of GSH functional groups. Both GSH and L-dopa form zwitterions at physiological pH of 7.3, and as C60 is normally considered anionic when it collects as many as six negative charges, the association of C60 with these zwitterions has the properties of being an organic salt, in which both hydrogen bonding as well as aromatic pi bonding contribute to the stability of these structures. Composition variations may be tuned by the number but not the type of functional groups, depending on penetrating and trafficking function, and may be from at least one L-dopa to about 6 L-Dopa, in which C60 bonded with 2 DOPA and 1 GSH functional groups promote adequate and sufficient medical improvement in human Parkinson's disease case studies. The fully decarboxylated metabolite C60-GSH-DOPA promotes therapeutic neuroprotective and neurogenesis functions, according to the teachings of the present invention.
[0067] FIG. 7 illustrates the role of metabolized C60-GSH-DOPA to disassemble the toxic oligomeric plaque of alpha-synuclein 70. A substantially one-dimensional fibril of alpha-synuclein 71 tends to form lengthwise abutting bonds with a multiplicity of other alpha-synuclein fibrils termed more generally an oligomeric plaque 72. The type of bonding along adjacent fibril lengths can include van-der-Waals induced charges, however salt cations such as sodium 74 may also intercalate or squeeze between these fibrils to create tangles that increase in size with time; oxidative species may additionally interpose cross-links and protein functional groups into random locations of the alpha-synuclein fibrils to include aldehydes or carboxylic acids under oxidative conditions. Free radical additions may also form bonds between fibrils when free radicals are present.
[0068] The introduction of clusters containing C60-GSH-DOPA 72, 73 into and among alpha-synuclein plaques 72 allows the quenching of free radicals and provides anti-oxidant functionality. Clusters containing C60-GSH-DOPA 72, 73 also store and then release hydrogen protons 76 carried at the amine nitrogen of dopamine functional groups, in which up to about five additional hydrogen protons may be carried by the fullerene C60 functional group. Fullerenes are also known for their ability to store as many as six (6) negative charges, whereby the high negative charge concentration in the clusters of C60-GSH-DOPA 72, 73 can extract sodium cations 74 from plaque 72, thereby freely releasing individual alpha-synuclein fibrils 71 from the collective plaque tangle 72. The combination of free-radical quenching, anti-oxidant function, cationic extraction, and free proton release enables the proper function of dopamine neurotransmitter. The targeting of reductive DOPA functional groups from C60-GSH-DOPA to those oxidative locations at the post-synaptic terminal counteracts oxidative stress, and is accomplished by the chemical affinity of the dopamine ligands within the C60-GSH-DOPA clusters 72, 73. The provision of C60 fullerene as a multifunctional center for these chemistries, in addition to the role of C60 as a hydrogen storage functional group, helps to create local reducing conditions suitable for prophylactic neural protection from oxidative stress induced degradation.
[0069] In the cellular lysosomes and other cellular vesicles, hydrogen protons 76 are exchanged with sodium 74, potassium 75, and other cations by various endogenous early endosome sodium-hydrogen exchangers such as NHE6. Medical evidence of genetic defects in the proteins used to traffic these cations are associated with the oligomeric agglomeration of plaques such as found in Alzheimer's Disease, some forms of autism, and Christianson syndrome; alpha synuclein is yet another oligomer that has confirmed salt bridges in Parkinson's disease. Alpha-synuclein needs to be present as individual fibrils to transport cations to biological membranes. A multiplicity of salt bridge hydrogen bonds are represented by the dotted lines 77 to bind the oligomer fibrils together so that they may no longer perform their cation shuttling function. C60-GSH-DOPA functions to artificially accelerate the trafficking of cations for proton exchange using a prosthetic pathway that prevents salt accumulation among the oligomeric fibrils, disassembles the oligomeric plaques formed by salt cations, and extracts the salt cations 74, 75 from alpha synuclein so that cations may not serve as salt bridges. The clusters of C60-GSH-DOPA 72, 73 constitute a prosthetic dual neurotransmitter having properties of both GSH and DOPA to enable the neural disease treatment of this composition according to the teachings of the present invention.
[0070] FIG. 8 illustrates the role of alpha-synuclein at a synapse and at some of the organelles of a neuron 800. It is well understood that alpha-synuclein binds to and regulates the transfer of calcium ions, especially those that are pooled and clustered within the synaptic vesicles 864 during neurotransmitter release 867 at the synaptic junction 860 between two neurons 810, 850. Alpha synuclein also influences the regulation of the vesicle trafficking from the endoplasmic reticulum 842 to the cell membrane at dendrites 844, and in vesicle adhesion to the Golgi complex 835 and neural cell nucleus 830. Alpha-synuclein localizes at the mitochondrial membranes 837, where it mitigates the effects of oxidative stress. These functions are enabled by the free radical, antioxidant, hydrogen proton storage, and cation trafficking composition of the C60-GSH-DOPA clusters 846, 868 that complement endogenous cation porter molecules in the manner of neurotransmitters, and thereby act to maintain the non-plaque independent fibril form of alpha-synuclein, as well as to establish cellular homeostasis among neurons.
[0071] Filopodia 820 are slender cytoplasmic neural projections that extend beyond a first neuron 810 and may have at least one synaptic junction 860 with a second neuron illustrated as a partial section of another filopodium extension 850. At least one metabolized C60-GSH-DOPA cluster 868 has been reduced in size to about less than 35 nanometers as part of the metabolic process, which enables it to enter the synaptic cleft between 864 and 862. Cluster 868 provides multifunctional roles to stabilize the membrane lipid interaction at the synaptic junction 860 where neurotransmitter 866 accumulates within the presynaptic terminal as neural bouton 864 for release into the synaptic gap 867 to be received by neural receptors at the proximal neuron providing the post synaptic terminal 862. Vesicles such as 864 may detach and travel with neurotransmitter 867 while carrying charged cations such as Na+ and Ca+2, wherein independent alpha-synuclein fibrils are critical to maintain the multiplicity of cations as adducts. The redox chemistry homeostasis provided by C60-GSH-DOPA clusters 846, 868 destabilizes plaques by the prevention of the free-radical and oxidative kinetics of alpha-synuclein aggregation, and by extracting cations from between alpha-synuclein fibrils, thereby halting or reversing the formation of oligomeric protein aggregates and their associated toxicity, according to the teachings of the present invention.
[0072] FIG. 9 illustrates a zeolite impregnated with a C60-GSH-L-dopa 90. Transcarpathian zeolite (clinoptilolite) 91 is a type of mineral provided with a highly negative charged network structure achieving a system of reproducible and well-defined pores and channels. Clinoptilolite zeolite 91 is well known to adsorb oppositely charged nitrogen containing compounds including protonated ammonia and protonated amino acids which serve as positive counter-ion and hydrogen bonding adducts with the composition of C60-GSH-L-dopa in the form of clusters 92, 93, 94, 95, 96, and 97 having sizes sufficiently small to fit within the mineral scaffold, where the channels therein can typically range from greater than 100 nanometers to less than about 5 microns in size. It is also known that at pH greater than 7, as well as under saline or physiological ionic salt conditions, clinoptilolite zeolite displaces and expresses the positively charged nitrogen compounds and counterions stored within the channels of zeolite 91. The salt and pH moderated regenerant property of clinoptilolite 91 towards reversible expression and delivery or release of positive charged nitrogen compounds has led to the widespread economic commercial adoption of clinoptilolite Transcarpathian zeolite 91 as a dietary supplement. It is therefore specified to utilize this ion-exchange property of zeolite 91 as one exemplary way to perform timed release of the C60-GSH-L-dopa composition of the present invention as a practical and cost-effective method of delivering this composition by oral administration, according to the teachings of the present invention.
[0073] FIG. 10 is a flowchart representation of a synthesis and nano-aerosol formulation of C60-GSH-L-dopa 100. In step 101 at least about one molar equivalents of pure glutathione (GSH) is combined with one molar equivalent of vacuum purified buckminsterfullerene (C60) and at least about one and nominally 2 molar equivalents of pure levodopa (L-dopa). In step 102, the dry powder mixture is reactive shear milled at greater than 1000 per second shear rate at a processing temperature maintained below 40° C. to minimize the covalent bonding of amine groups from the GSH onto the C60, while maximizing the pi-carbonyl and pi-aromatic bonding with C60. One way that low temperature processing can be accelerated at higher shear rates for less time, is to provide an oxygen free processing atmosphere. In step 103 the sheared C60-GSH-L-dopa product is added to polypropylene glycol (PPG) solvent in a 1:10 mass ratio of dry powder to solvent for liquid shear at about 1000 per second shear rate to full product dissolution. In step 104, the desired concentration of C60-GSH-L-dopa is created by dissolving a volumetric amount of the C60-GSH-L-dopa solution into a solvent mixture of glycerol with polypropylene glycol to achieve the desired final concentration of between about 20 ppm and 2000 ppm to obtain a suitable vaporized inhalant or a dosage for nano-aerosol inhalant delivery. This final dilution solvent mixture comprises about 70% glycerol and 30% polypropylene glycol by volume. All solvated components for dispensing are to be kept free of moisture in a quality-controlled process. In step 105, a metered amount of the nano aerosol is generated by a commercially available electronic dispensing device, such as by heating the formulated fluid at from about 255° C. up to about 300° C., but no greater than about 300° C. to avoid oxidation or breakdown of the nano-aerosol, and to maintain temperatures suitable for client aspiration, according to the teachings of the present invention.
[0074] FIG. 11 is a flowchart representation of a synthesis of C60-GSH-L-dopa and a formulation for Oral Administration 110. In step 111 at least about one molar equivalents of pure glutathione (GSH) is combined with one molar equivalent of vacuum purified buckminsterfullerene (C60) and at least about one and nominally 2 molar equivalents of pure levodopa (L-dopa). In step 112 the dry powder mixture is shear milled at greater than 1000 per second shear rate, the processing maintained at a temperature below 40° C. to minimize the covalent bonding of amine groups from the GSH onto the C60, while maximizing the pi-carbonyl and pi-aromatic bonding with C60. One way that low temperature processing can be accelerated at higher shear rates for less time, is to provide an oxygen free processing atmosphere. In a first alternative step 114, a desired quantity of hydrogen bonded C60-GSH-L-dopa powder product obtained from step 113 is dissolved into aqueous 0.1% to 0.3% hyaluronic acid, then desired colors, flavors, and preservatives such as potassium sorbate or sodium benzoate are added for oral administration or beverage servings. In a second alternative step 115, the C60-GSH-L-dopa powder product is combined with one or more pharmaceutically acceptable carriers like suitable USP food grade binders as delivery materials in any combination. These carriers and delivery materials are generally known as excipients and fillers, of which non-limiting examples include commercially available calcium carbonate, zeolite, methyl cellulose, and gel peptides for placement into a compressed tablet or a gel capsule as desired for oral administration, according to the teachings of the present invention.
[0075] FIG. 12 illustrates a personal administration method 120 for an aspirated nano-aerosol delivery system containing an C60-GSH-L-dopa composition. The nano-aerosol generating device filled with C60-GSH-L-dopa dispensing solution 128 is provided for dispersing the inhalant gas wherein the nano-particles are and nebulized. The dispensing method of commercially available device 128 may also be more commonly known as a nebulizer, or an electronic vaporizing device, or an electronic cigarette, or the functional part of a hookah to be shared among several users. In all cases these systems serve to carry the C60-GSH-L-dopa in a carrier fluid dispenser 128, move that composition in nebulized form along with an aerosolized solvent, and transfer this composition in substantially gaseous dispersion to the nose, mouth, trachea, and airways of a patient or user 127. One intended use of the C60-GSH-L-dopa composition is to treat, delay or arrest the incidence of Parkinson's disease (PD), Alzheimer's disease (AD), and other cognitive disorders wherein the nano-aerosol can expedite targeted delivery to the brain by avoiding a passage through the digestive system.
[0076] Some of the nano-aerosolized composition is exhaled and shown as particulate clusters 121, 122, 123 within exhaled smoke puffs 124 and 125 emitted on exhalation as indicated by the direction of thin line arrows radiating away from the nose of the subject 127. Delivery of the C60-GSH-L-dopa nano-aerosol composition from dispenser 128 provides antioxidant properties to the mucus airway tissues wherein destruction of free radicals and oxidants associated with motor neuron disease and Parkinson's disease are part of the treatment and alpha-synuclein plaque mitigation is provided using this method. Systems that may be used for the method of dispersion of the C60-GSH-L-dopa represented by exemplary device 128, include, without limitation, any of the electronic cigarette devices produced internationally and listed in Appendix 4.1, “Major E-cigarette Manufacturers” of the “2016 Surgeon General's Report: E-Cigarette Use Among Youth and Young Adults” published by the Center for Disease Control and Prevention (CDC), Office of Smoking and Health (OSH) freely available at the CDC.GOV website, and/or any combination of piezoelectric, resistively heated, or inductively heated vaporized fluid delivery methods that can be utilized to deliver the composition of the present invention, especially when approved as a medical drug delivery device. Each embodied variation of such methods without limit are intended to aspirate aerosols as the method of therapeutic substance delivery of the composition of the present invention directed into the nasal cavities, mouth, tracheal breathing orifice, or intubated trachea of a patient. The supply direction of nebulized feed of C60-GSH-L-dopa on inhalation and exhalation are delivered into the airways and lungs of the intended patient by the flow of supplied air as indicated by the direction of upward and downward facing large white arrows 126, when used according to the teachings of the present invention.
[0077] FIG. 13 illustrates experimental FTIR data for levodopa. All the Fourier transform infra-red (FTIR) spectrographs hereinafter were measured by transmittance using the potassium bromide (KBr) compressed flow solid pellet compact preparation method. The material used for analysis was obtained by the method of mixing, crushing, and consolidating under 7 metric tons of pressure, about 0.001 grams of the analyte substance with 1 gram of a diluent solid KBr that is substantially transparent to infrared light, and which flows under pressure to form a translucent pellet of about 0.4 mm thickness. Spectral background subtraction in air using a control pellet of the same mass and thickness having pure KBr was used to obtain a baseline instrument infrared spectral response. This method is generally referred to as the ‘KBr pellet’ sample preparation method, and it is used hereinafter throughout for each FTIR experimental data collection and spectral analysis. The Fourier transform infrared spectrophotometer used herein to obtain FTIR spectra throughout, is a model RF6000 FTIR instrument manufactured by Shimadzu of Japan. Each FTIR data graph hereinafter is provided with a numeric scale ranging from 400 to 4000 to represent reciprocal centimeters or (cm−1) in wavenumbers.
[0078] The numeric scale ranging from 10 to 90 represents percentage transmittance and has units of %. The FTIR absorbance peak at 3359 cm−1 is attributed to the amine nitrogen-hydrogen vibration (N—H). At 3200 cm−1 appears an oxygen-hydrogen (O—H) stretching vibration, and at 3046 cm−1 is an aromatic hydrogen stretching vibration. The primary amine functional group is indicated by the two (N—H) bending absorbance vibration bands at 1653 cm−1 and at1567 cm−1. The peaks between 1064 cm−1 and 1200 cm−1 are due to (C—N) stretching vibrations. The sharp and intense peak at 817 cm−1 indicates the N—H bending vibration. There is evidence of a band at about 1500 cm−1 that can be attributed to the C═C bond in the benzene ring structure. Comparison of the illustrated experimental FTIR data for levodopa 1200 indicates similarity to the FTIR absorbances reported for levodopa that are available from the scientific literature, and may be used for confirmation of the raw material composition according to the teachings of the present invention;
[0079] FIG. 14 illustrates experimental FTIR data for fullerene C60 reacted with levodopa, being C60-Ldopa. The numeric scale ranging from 30 to 100 represents percentage transmittance and has units of %. The characteristic strong and sharp buckminsterfullerene (C60) aromatic carbon-carbon stretching band is present at 526 cm−1. The FTIR absorbance peak at 3373 cm−1 is attributed to the amine nitrogen-hydrogen vibration (N—H). At 3192 cm−1 appears an oxygen-hydrogen (O—H) stretching vibration, and at 3062 cm−1 is an aromatic hydrogen stretching vibration. The two bands arising from the primary amine functional group are indicated by the (N—H) bending absorbance vibrations and remain unchanged at 1653 cm−1 and at1567 cm−1, confirming that there was no chemical reaction to alter the amine functional group. The peaks between 1064 cm−1 and 1200 cm−1 are due to (C—N) stretching vibrations. The sharp and intense peak at 821 cm−1 indicates the N—H bending vibration. The observed reduction of absorbance intensity in the region of 1450 cm−1 to 1500 cm−1 can be attributed to the attenuation of the C═C bond vibrations in the levodopa benzene ring that has become sterically constrained by aromatic-pi bonding with the C60 functional group, wherein the spatially confined geometry impacts the bond mobility in bending and stretching modes associated with the formation of the aromatic pi to aromatic pi bonds.
[0080] FIG. 15 illustrates experimental FTIR data for GSH raw material that was used to synthesize the compositions of the present invention. The numeric scale ranging from 0 to 100 represents percentage transmittance and has units of %. The characteristic reduced glutathione sulfhydryl (—S—H) peak is observed at 2523 cm−1. The peaks at 2980 cm−1 and 1455 cm−1 arise from the stretching and bending vibrations of aliphatic C—H group in glutathione. The peak at 1276 cm−1 is a tertiary amide peak, and the very sharp absorbance peak at 1073 cm−1 provides a characteristic carbon nitrogen (C—N) stretch. The strong and sharp peak observed at 1713 cm−1 and the one at 1393 cm−1 are attributed to a deprotonated carboxylic acid (—COO), where the former is a symmetric vibration, and the latter is an asymmetric vibration mode of this functional group. The overall infrared absorbance spectral features are consistent with and indicate chemical similarity to reduced glutathione as may be found in published public FTIR spectra, according to the teachings of the present invention.
[0081] FIG. 16 illustrates experimental FTIR data for the intermediate compound fullerene glutathione (C60-GSH). The numeric scale ranging from 50 to 100 represents percentage transmittance and has units of %. In comparison with FIG. 15, it is notable that the reduced glutathione sulfhydryl (S—H) peak is no longer observed at 2523 cm−1, indicating here that the sulfur-hydrogen stretch has disappeared because of the chemical reaction of GSH with C60. This observation supports the molecular sulfur binding reaction of FIG. 3. Notable also is the very strong and sharp C60 fullerene aromatic carbon-carbon stretching bands at 576 cm−1 and 526 cm−1.
[0082] FIG. 17 illustrates experimental FTIR data for the final product C60-GSH-L-dopa. The numeric scale ranging from 0 to 100 represents percentage transmittance and has units of %. The absorption peak at 526 cm−1 is characteristic for C60 fullerene carbon-carbon (C═C) bonds. The sulfhydryl (S—H) vibration absorbance previously observed at 2523 cm−1 for the free glutathione in FIG. 16 was not observed for C60-GSH-DOPA experimental results. This indicates that the glutathione was probably deprotonated and coordinated to the surface of the fullerene (C60) nanoparticle through the sulfur and suggests that glutathione caps the fullerene nanoparticles through the thiol from the cysteine portion of the glutathione ligand. The characteristic primary amine group of levodopa or dopamine obtains a clearly resolved absorbance peak at 3389 cm−1; this indicates that these molecular groups are pi-bonded to the aryl regions of fullerene, leaving their primary amine function available for the intended interaction with cellular components. This confirms the absorbances of levodopa (L-dopa) and GSH interacting as functional groups in combination with C60 by FTIR as a distinguishable material having clearly recognizable chemical signatures that are characteristic of the composition of the present invention.
[0083] FIG. 18 illustrates experimental negative mode MALDI-TOF mass spectrograph data for C60-L-dopa material 1800. This sample, as well as each of the subsequent MALDI-TOF experimental test results hereinafter, was introduced for test by laser vaporization into a Voyager Mass Spectrograph from Applied Biosystems (Foster City, Calif., USA). Negative mode bombardment was by fast moving electrons at about 70 eV energy. This resulted in molecular fragmentation and electron removal from the highest molecular orbital energy as molecular ions were formed. The ratio of mass to charge (m/z) is used to determine the molecular ion fragments to help determine the pieces of the original molecule in this assay. The mass peak at 723 m/z corresponds to the molecular ion fragment of fullerene C60 of mass 720 having three adducted hydrogen atoms. The very broad mass peaks at 1370, 2042, and 2641 are attributed to indicate predominantly dimeric and some trimeric C60 chains appended to each other and to interstitial levodopa by pi-pi bonding. The rider peaks on the broader peaks indicate the loss of small ion fragments such as those having a mass of 17 from (—OH) hydroxyl groups. The overall experimental test results characterize the molecular ion breakdown products of C60-L-dopa, where C60-L-dopa may be used to further synthesize the composition of the present invention.
[0084] FIG. 19 illustrates experimental negative mode MALDI-TOF mass spectrograph data for C60-GSH material 1900. This test sample resulted from reacting an equivalent molar quantity of glutathione to the molar equivalent of pristine fullerene C60. The largest peak observed was the primary and core molecular ion, this being a fullerene ion as indicated by the numeric peak label at mass to charge ratio of 720. The primary molecular ion was subsequently verified using a pristine pure reference material of C60 tested immediately after this test, under both negative mode and positive mode test conditions (results are not shown here). The observed molecular fragment at 866 is characteristic for a fullerene C60 obtaining a residual spallation fragment from glutathione that was incompletely removed. The cluster of peaks with a maximum at 1454 is attributed to C60-GSH, wherein one molecular mass of glutathione is bonded to one molecular mass of buckminsterfullerene, and characterizes the C60-GSH component that may be used to further synthesize the composition of the present invention.
[0085] FIG. 20 illustrates experimental negative mode MALDI-TOF mass spectrograph data for C60-GSH-DOPA 2000. The largest peak observed is the molecular ion fragment for C60 fullerene as indicated by the mass to charge ratio of 719. The characteristic glutathione ion spallation fragment of 866 in FIG. 19 is also seen illustrated here at 865 mass to charge ratio. The first broad cluster of peaks present at 1368 m/z is like the result of 1370 m/z found for C60-DOPA in FIG. 20. The broad cluster of peaks at 2015 and 2637 m/z are attributed to dimeric and trimeric molecular ion fragments of C60-GSH-DOPA, where a loss of mass attributed to the decarboxylation or removal of some of the carboxylic acid functional group from the dimer or trimer can explain the mass reduction in these fragments. The overall illustrated mass spectral fingerprint of molecular ion fragments 2000 characterizes C60-GSH-DOPA according to the teachings and composition of the present invention.
[0086] As variations, combinations and modifications may be made in the construction and methods herein described and illustrated without departing from the scope of the invention, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative rather than limiting. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments but defined in accordance with the foregoing claims appended hereto and their equivalents.
