Tablet composition for anti-tuberculosis antibiotics

Abstract

Bacterial resistance to antibiotics is increasing worldwide creating a global threat. Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, is a bacterial infectious disease that results in over one million deaths annually. The discovery outlined here involves a tablet composition for patient administration and subsequently a new paradigm in drug delivery vehicles in vivo and in vitro and is applied to existing TB antibiotics in order to increase their efficacy. The drug delivery system is a three component complex that is administered with the TB antibiotic or a combination of TB antibiotics. The components are a saccharide or saccharides, a transition metal ion or a combination of metal ions that can bind a nitrogen and/or oxygen atom(s), and a water soluble polymer capable of aggregating and enclosing the other constituents. The three component molecular delivery approach has demonstrated ability to overcome M. tuberculosis bacterial resistance to an existing antibiotic.

Claims

1. A pharmaceutical composition comprising i. one or more front line antibiotics used in the treatment of the bacterial infection Mycobacterium tuberculosis; ii. sucrose, wherein its mass percent is a minimum of about one percent and a maximum of fifty percent of the total composition mass; iii. copper (Cu), which is present in an amount from at least about one percent to about forty percent of the total mass of the composition; and iv. a polyethylene glycol (PEG) polymer wherein its mass percent is a minimum of at least five percent and a maximum of 90% of the total composition mass.

2. The pharmaceutical composition according to claim 1, wherein the antibiotic is an anti-tuberculosis antibiotic selected from the group consisting of ethambutol (EMB), isoniazid (INH), pyrazinamide (PZA), rifampicin (RIF), rifabutin (RBT), and rifapentine (RPT).

3. The pharmaceutical composition according to claim 1, wherein the copper is copper (I) or copper (II).

4. The pharmaceutical composition according to claim 1, wherein the copper is a combination of copper (I) and copper (II).

5. The pharmaceutical composition according to claim 1, wherein the polyethylene glycol comprises the repeating unit (C.sub.2H.sub.40).sub.n where n is between ten and two hundred.

6. The pharmaceutical composition according to claim 1, wherein the composition is compounded using wet granulation procedures or dry granulation procedures; and is dried using a tray-dryer or a fluid-bed dryer.

7. The pharmaceutical composition according to claim 1, wherein the composition has a mass less than two thousand milligrams.

8. A method of formulating the composition of claim 1 comprising binding the transition metal cation with the saccharide to form a metal ligand complex and combining the metal ligand complex with at least polyethylene glycol and one or more front line antibiotics.

9. A method of formulating the composition of claim 1 comprising mixing the front line antibiotic with polyethylene glycol, binding the transition metal cation with the saccharide to form a metal ligand complex, and then combining the metal ligand complex with the mixed front line antibiotic and polyethylene glycol.

10. The composition of claim 1, further comprising pharmaceutically acceptable binders; disintegrants; glidants; solvents; lubricants; coatings; and/or other excipients that have a total percent of at least five percent of the total composition mass.

11. The composition of claim 1, wherein the PEG is PEG-3350.

12. The pharmaceutical composition of claim 1, wherein the one or more front line antibiotics, sucrose, copper, and PEG polymer are in a molar ratio of 1:1:1:1.

13. The pharmaceutical composition according to claim 11, wherein the composition dissolves in water and forms aggregates; wherein the aggregates can enclose and transport the antibiotic in vivo.

Description

DETAILED DESCRIPTION

(1) This disclosure involves a tablet composition that includes three molecular components that improve the efficacy of known antibiotics used in the treatment of pulmonary and extra-pulmonary tuberculosis in a variety of forms including drug resistant infections (i.e. MDR-TB, XDR-TB, TDR-TB). These components include a transition metal cation, a saccharide and a water soluble polymer that contribute to the composition of an antibiotic tablet to serve as a delivery system in a physiological environment and enhance the efficacy of an existing anti-tuberculosis antibiotic.

(2) The first component is a transition metal ion that can bind a nitrogen atom (i.e. in an amine, amide), which are present in all major anti-tuberculosis antibiotics. The cation(s) may also bind to oxygen atoms (i.e. in an alcohol, ether, ester, and/or carbonyl) if present in the antibiotic structure, however binding to nitrogen atoms is thermodynamically favored. The transition metal cation may be a physiologically relevant species such as copper, zinc, iron, or nickel. As an example, copper is discussed because it serves a critical functional role in several proteins at low concentrations but also functions as a biocide at higher concentrations.

(3) Copper ions can form hexavalent complexes, allowing a single copper(II) ion to bind to both a saccharide molecule and an antibiotic molecule. The divalent copper cation is used as a copper(II) chloride dihydrate in the preliminary step(s) of tablet production for this technology. The chloride salt is a strong electrolyte, and the anion is ubiquitous in the physiological environment. Chloride has no other chemical properties (basicity, favorable redox potential, etc.) that negatively impact biochemical processes at the concentrations administered in drugs. The other copper salt anions used include sulfate (basicity), nitrate (may induce redox), hydroxide (cause copper(II) to precipitate), have some chemical characteristics that may induce a negative or undesired reaction. The copper ion can bind nitrogen, which all prominent TB drugs contain. This binding can increase the stability of the drug, perform independently as a biocide, increase log P values, make the molecule a polarity adaptive species, and serve as a delivery platform. When copper is drawn into the bacterium as a nutrient source, it potentially can aid in transporting the drug attached across the membrane. Copper(II) has a high affinity for nitrogen and oxygen atoms which are present in isoniazid (C.sub.6H.sub.7N.sub.3O). The cation can also act to minimize hydrogen bonds in the antibiotic by binding and subsequently blocking nitrogen's from unwanted interactions, a favorable trait outlined by Lipinski's Rule of Five.

(4) The second component is a water soluble polymer, which is demonstrated using polyethylene glycol (PEG-3350). A medium sized PEG molecule can form aggregates to enclose and transport drugs in vivo and in vitro, increasing residence times and decreasing the chances of the copper-saccharide-antibiotic complexes attaching to an unwanted target. PEG-3350 is a small enough polymer to have favorable water solubility at body temperature (37 degrees Celsius) but large enough to form aggregates. The polymer forms a loosely packed aggregate that encloses and transports the complex, minimizing or preventing the complex from being hindered by other processes in the body. PEG is widely used in medicinal applications and is considered a very mild antiseptic. It has also been used in numerous published clinical studies as a carrier for pharmaceutical applications.

(5) The third component is a saccharide, and may be a monosaccharide, disaccharide, an oligosaccharide or a combination of different saccharide molecules. This work focuses on using the disaccharide sucrose. Saccharides or sugars in these forms serve several key roles in a physiological environment including one of an energy source and a building block for a polymer. For example, sucrose can be hydrolyzed into two smaller molecules, glucose and fructose. Less complex saccharides are more readily absorbed into the body as they pass through the gastrointestinal tract. In Mycobacterium species, the sacB gene encodes for the enzyme levansucrose, which is responsible for the breakdown of sucrose and the production of levans. Levans are polysaccharides built from repeating units of fructose. There is a toxicity associated with sucrose in these bacteria which may be attributed to different effects such as (a) fructose residues blocking or adhering to receptor molecules which alter their function and/or (b) the levans that accumulate over time may impact the periplasm due to their molar mass and size. Sucrose is an energy source for many types of cells, but can be toxic to Mycobacteria, an aspect that provides a level of selectivity which may translate into lower side effects and lower dosages if complexed to an anti-tuberculosis drug.

(6) A full treatment with isoniazid can cost less than fifty USD for a complete regimen, but resistant strains of M. tuberculosis may render this regimen ineffective. Since this technology repurposes and improves existing anti-tuberculosis drugs rather than developing a new molecule, this technology not only provides a medical solution but also provides an economical solution to the global TB issue. The three components (water soluble polymer, saccharide, and a transition metal ion) comprising the tablet composition and subsequently a delivery system have a lower cost per gram than the front-line antibiotic used. This detailed description will describe how three component systems can be prepared for pharmaceutical applications, provide analytical data used for structural studies, provide cell line study data, and provide in-depth details of the pharmaceutical efficacy of the three component complex. A synopsis of manufacturing and administering the tablet(s) which deliver the three component-antibiotic complex to a patient is outlined as well.

(7) The actual dosage of the drug will depend upon the weight of the patient, the route of administration and the severity of the disease. Slight changes to amounts of components of the tablet will be determined by a health care provider depending on the diagnosis made. For example, isoniazid is used in the treatment of patients with latent and active tuberculosis and the recommended regimen changes with the type of TB present (latent vs. active, resistant vs. nonresistant). Dosage can be correlated with the efficacy of the medicinal agent within a certain toxicity threshold, as described in the context of the embodiment of this invention. Dosages can be correlated and determined, in some cases with the minimum inhibitory concentration, or MIC value, of the pharmaceutical agent. Parameters related to the pharmacokinetic and pharmacodynamic values such as ADME, or its absorption, distribution, metabolism, and excretion, are used in determining these dosages. For antibiotics, a value prescribed for a medicinally appropriate quantity is between five and ten times the MIC value.

(8) The lowering of MIC values, which will be demonstrated and discussed, against active and resistant strains of TB is desirable in drug development. It is well recognized in pharmaceutical practice that the dose given is proportional to a MIC value, and can be calculated using the following equation:
Dose=(C.sub.max*V.sub.d)/F(1)
Where C.sub.max is the highest concentration in blood plasma and is calculated from multiplying the MIC value times two, or:
Dose=(MIC*2*V.sub.d)/F(2)
Other parameters to define are V.sub.d which is the volume distribution, and F which is the systemic availability or the fraction of a drug that reaches the blood unaltered. Lowering the MIC value can translate into a lower dosage and lower side effects. When isoniazid is bound to copper and/or sucrose and enclosed by PEG, the F value increases, decreasing the dosage size.

(9) Currently, drug resistant forms of TB are more difficult to impossible to treat with the recommended front-line TB antibiotics, while second-line antibiotics are more expensive and can induce harsh side effects. Currently, there are ten drugs approved by the United States Food and Drug Administration for treating individuals infected with the M. tuberculosis. The four primary drugs that are recommended are isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA). There are variations on the effective administration of these drugs. For example, the primary treatment regimen involves eight weeks of initial treatment in which each drug is given daily totaling over two hundred and twenty pills. The second phase is the continuation phase, which involves daily administration of isoniazid and rifampin for eighteen weeks in which approximately two hundred and fifty pills are given to the patient. A total of over four hundred and fifty pills are given in this standard six month regimen. One issue found internationally is compliance to treatment due to length, number of pills and side effects among other reasons, which has aided in evolution of resistance mechanisms to these antibiotics by the bacterium.

(10) For applications in many medical situations globally it is important to recognize that delivery approach to the patient can be critical for compliance to the prescribed regimen. Combining this unique three component complex with the existing antibiotic and forming it into a tablet is described below for different dosages and different antibiotics. Examples of the three component delivery complexes composed of a saccharide, a copper ion, PEG and the antibiotic are described below, but should not be construed to be in any way limiting for the present invention. a. Isoniazid is a front-line TB drug that may be given as a tablet in dosages ranging from fifty to three hundred milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a fifty milligram dose of isoniazid, the antibiotic would be complexed with 124.8 milligrams of sucrose, 62.04 milligrams of copper (II) chloride dihydrate and 1222.62 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. Experimentally determined MIC values for active TB will be presented below but, as an approximation, if MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio, the adjusted quantities could be 10 milligrams of isoniazid, 24.96 milligrams of sucrose, 12.408 milligrams of copper (II) chloride dihydrate, and 244.52 milligrams of PEG-3350. b. Rifampin is a front-line TB drug that may be given as a capsule in dosages that are one hundred and fifty or three hundred milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a one hundred and fifty milligram dose of rifampin, the antibiotic would be complexed with 62.41 milligrams of sucrose, 31.02 milligrams of copper (II) chloride dihydrate and 611.3 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 30 milligrams of rifampin, 12.482 milligrams of sucrose, 6.204 milligrams of copper (II) chloride dihydrate, and 122.26 milligrams of PEG-3350. c. Rifabutin is a TB drug that may be given as a capsule in dosages that are one hundred and fifty milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a one hundred and fifty milligram dose of rifabutin, the antibiotic would be complexed with 60.56 milligrams of sucrose, 30.10 milligrams of copper (II) chloride dihydrate and 593.3 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If the MIC values decrease between five and ten fold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 30 milligrams of rifabutin, 12.12 milligrams of sucrose, 6.02 milligrams of copper (II) chloride dihydrate, and 118.66 milligrams of PEG-3350. d. Rifapentine is a TB drug that may be given as a tablet in dosages that are one hundred and fifty milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a one hundred and fifty milligram dose of rifapentine, the antibiotic would be compounded with 58.49 milligrams of sucrose, 29.076 milligrams of copper (II) chloride dihydrate and 573 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 30 milligrams of rifapentine, 11.71 milligrams of sucrose, 5.81 milligrams of copper (II) chloride dihydrate, and 114.6 milligrams of PEG-3350. e. Pyrazinamide is a TB drug that may be given as a tablet in dosages that are one hundred and fifty or five hundred milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a one hundred and fifty milligram dose of pyrazinamide, the antibiotic would be complexed with 417.07 milligrams of sucrose, 207.32 milligrams of copper chloride dihydrate and 40,850 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 30 milligrams of pyrazinamide, 83.41 milligrams of sucrose, 41.46 milligrams of copper (II) chloride dihydrate, and 8,170 milligrams of PEG-3350. f. Ethambutol is a TB drug that may be given as a tablet in dosages that are one-hundred or four-hundred milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a 100 milligram dose of ethambutol, the antibiotic would be complexed with 167.40 milligrams of sucrose, 83.21 milligrams of copper (II) chloride dihydrate and 1,640 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 20 milligrams of ethambutol, 33.48 milligrams of sucrose, 16.66 milligrams of copper (II) chloride dihydrate, and 328 milligrams of PEG-3350. g. Cycloserine is a TB drug that may be given as a capsule in dosages that are two hundred and fifty milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a two hundred and fifty milligram dose of cycloserine, the antibiotic would be complexed with 838.2 milligrams of sucrose, 416.66 milligrams of copper (II) chloride dihydrate and 8211 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. Experimentally determined MIC values for active TB will be presented below but, as an approximation, if MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 50 milligrams of cycloserine, 166.44 milligrams of sucrose, 83.33 milligrams of copper (II) chloride dihydrate, and 1642.2 milligrams of PEG-3350. h. Ethionamide is a TB drug that may be given as a tablet in dosages that are two hundred and fifty milligrams. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a two hundred and fifty milligram dose of ethionamide, the antibiotic would be complexed with 515.04 milligrams of sucrose, 256.02 milligrams of copper (II) chloride dihydrate and 5045 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 50 milligrams of ethionamide, 103.008 milligrams of sucrose, 51.204 milligrams of copper (II) chloride dihydrate, and 1009 milligrams of PEG-3350. i. Streptomycin is a TB drug that may be given as an aqueous solution for intravenous or intramuscular administration in dosages that are one gram vials. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a one thousand milligram dose of streptomycin, the antibiotic would be compounded with 588.64 milligrams of sucrose, 292.59 milligrams of copper chloride dihydrate and 5766 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 200 milligrams of streptomycin, 117.72 milligrams grams of sucrose, 58.52 milligrams of copper (II) chloride dihydrate, and 1153.2 milligrams of PEG-3350. j. Amikacin is a TB drug that may be given as an aqueous solution for intravenous or intramuscular administration in dosages that are one gram. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a 1000 milligram dose of amikacin, the antibiotic would be complexed with 584.61 milligrams of sucrose, 290.59 milligrams of copper (II) chloride dihydrate and 5726 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 200 milligrams of streptomycin, 117.72 milligrams grams of sucrose, 58.52 milligrams of copper (II) chloride dihydrate, and 1153.2 milligrams of PEG-3350. k. Capreomycin is a TB drug that may be given as an aqueous solution for intravenous or intramuscular administration in dosages that are one gram vials. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a 1000 milligram dose of capreomycin, the antibiotic could be compounded with 511.97 milligrams of sucrose, 254.49 milligrams of copper (II) chloride dihydrate and 5015 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 200 milligrams of capreomycin, 102.39 milligrams grams of sucrose, 50.89 milligrams of copper (II) chloride dihydrate, and 1003.0 milligrams of PEG-3350. l. p-Aminosalicylic acid (PAS) is a TB drug that may be given as an aqueous solution for intravenous administration. A four gram dose, given twice a day is often prescribed. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation for a 4,000 milligram dose of p-aminosalicyclic, the antibiotic would be compounded with 8,941 milligrams of sucrose, 4,444.4 milligrams of copper chloride dehydrate and 87,582 milligrams of PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 800 milligrams of PAS, 1,788.2 milligrams grams of sucrose, 888.8 milligrams of copper (II) chloride dihydrate, and 17,516 milligrams of PEG-3350. m. Penicillin-G, representing the beta-lactams, is rarely used as a tuberculosis drug but its performance may be enhanced using this three component approach. A one hundred milligram dose is proposed as a starting point considering published preclinical trial data. As an example of a formulation that represents a 1:1:1:1 molar ratio of the anti-tuberculosis drug as part of the formulation, the antibiotic would be compounded with 99.11 milligrams of sucrose, 50.56 milligrams of copper (II) chloride dihydrate and 994.06 milligrams PEG-3350. Lowering the MIC values for a known drug can result in lower dosages. If MIC values decrease between five and tenfold, the dose or quantity of the antibiotic administered may decrease five to tenfold. Maintaining the 1:1:1:1 molar ratio the adjusted quantities could be 20 milligrams of penicillin, 19.82 milligrams of sucrose, 10.11 milligrams of copper (II) chloride dihydrate, and 198.81 milligrams of PEG-3350. n. A combination of antibiotics including isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB) are considered the front-line TB drugs and are used together as the standard regimen to treat TB. 75 milligrams or 0.00054 moles of isoniazid, 400 milligrams or 0.00031 moles of pyrazinamide, 275 milligrams or 0.00074 moles of ethambutol, and 150 milligrams or 0.000180 moles of rifampicin are often the dosages prescribed to treat TB patients. Considering the combined number of moles for the four antibiotics, 0.00177 moles, the following is a molar ratio formulation that includes four antibiotics and the three additional drug delivery components (INH:PYR:ETH:RIF:Cu:SUC:PEG) would be (0.54:0.31:0.74:0.180:10.62:7.08:0.885). The total molar amount of sucrose is a 1:1 molar ratio with the total number of moles for the four antibiotics, while the copper ion is a 3:1 molar ratio because some of the antibiotics have more than one nitrogen atom. This ensures that all of the antibiotics can have at least one copper ion bound to an amine, while a larger species with several nitrogen atoms (i.e. RIF) would likely have two copper ions. This will also increase aggregation between the molecules via ion dipole interactions and hydrogen bonds. The PEG-3350 component is added at a 1:2 ratio (moles of PEG:moles antibiotics) for the moles of polymer to total moles of antibiotics. Because the copper ions, antibiotics and disaccharide are bound together and entrapped in a loose aggregate, less PEG is required, lowering its mass percentage.

(11) While the sample formulations utilize different molar ratios of the three components, the antibiotic(s), and the other components, specific values are varied to optimize the treatment procedure. For example, PEG can retain water at a ratio of up to three water molecules per ethanol unit (CH.sub.2CH.sub.2O), so the molar ratio of PEG may decrease to 0.1 to 0.2 per antibiotic molecule in some cases. Likewise, copper is a hexavalent species and can bind two or three antibiotic molecules and/or saccharides in some cases per ion (i.e. Cu(INH).sub.2, Cu(SUC).sub.2) or form an antibiotic-saccharide complex (i.e. Cu.sub.1(INH).sub.1(SUC).sub.1) so its molar ratio may, in some cases, be lowered. Likewise, some antibiotics contain multiple nitrogen atoms that can bind more than a single copper ion, and therefore may create instances in which the moles of copper ion need to be greater than the moles of the antibiotic. Antibiotics with more than one or two nitrogen and/or oxygen atoms in their structure, such as capreomycin (C.sub.25H.sub.14H.sub.14O.sub.8) may require additional copper ions to minimize unwanted hydrogen bonding to stabilize its structure. Decreasing or increasing the moles of the components of the drug delivery system may decrease or increase the quantities of the components added, therefore altering the total milligrams of the tablet.

(12) Computational work associated with this disclosure has shown that important values outlined by Lipinski's Rule of Five are maintained or improved in these complexes. For example, log P (log of the partition coefficient for the distribution of a species between water and octanol layers in a glass vessel) for [Cu(INH).sub.2(H.sub.2O].sub.2].sup.+ is similar to that of the monodentate ligand complex (Cu(INH).sub.1). Likewise, the Total Polar Surface Area (TPSA) remains below the threshold set by Lipinski's rules for bi and tridentate complexes for many of the antibiotic and/or saccharide complexes.

(13) In addition, the preparation of a tablet would include other inert compounds such as microcrystalline cellulose, colloidal silica, hydroyxypropyl methylcellulose, magnesium stearate, sodium ascorbate, suspended silicon dioxide, purified water and a basic coating premix yellow and/or red. These excipients and others may serve as binders, disintegrates, glidants, lubricants, solvents, and/or coatings. The coating procedure may be performed below fifty degrees Celsius and with a specific spray varying between twenty and one hundred and twenty grams per kilogram of the compressed phases. Once the mixture is prepared, it can be made into tablets with masses equal to or less than 1.5 grams but may be as low as three hundred milligrams.

(14) The present invention includes a more detailed process for the tablet preparation, that should not be construed in any way as to limit the present invention, is described as: (i) Mixing the active ingredient, an antibiotic, with a specified molar ratio of copper (II) chloride dihydrate, a molar ratio of a saccharide such as sucrose, and a molar ratio of a water soluble polymer such as polyethylene glycol in a high shear mixer. (ii) Adding one or more binders such as crystalline cellulose in the same high shear mixer. (iii) Adding a solvent such as highly purified water, to expose the composition to a dissolving environment in the same high shear mixer. (iv) Adding a lubricant such as magnesium stearate in the same high shear mixer. (v) Adding a glidant such as suspended silicon dioxide in the same high shear mixer. (vi) Adding sodium ascorbate or ascorbic acid as an anti-oxidant in the same high shear mixer. (vii) Excess water would be removed in a dehydration step. (viii) A tablet would be made by compression in a tablet press to a size not to exceed eleven millimeters in diameter and a mass not to exceed two thousand milligrams but preferably below twelve hundred milligrams. (ix) Coating would take place in a pan coater such as those manufactured by Glatt.

(15) Another example would include the manufacturing of a tablet that contains more than one antibiotic. An example of this production, that should not be construed in any way as to limit the present invention, is outlined: (i) Mixing the active ingredients, the antibiotics, with a specified molar ratio of copper (II) chloride, a molar ratio of a saccharide such as sucrose, and a molar ratio of a water soluble polymer such as polyethylene glycol in a high shear mixer. The antibiotics would consist of a mixture of isoniazid, rifampicin, pyrazinamide and/or ethambutol. (ii) Adding one or more binders such as crystalline cellulose in the same high shear mixer.

(16) Analytical studies such as NMR help provide a model that includes the following structural features: (a.) the copper ion binds the isoniazid via nitrogen, oxygen and pi bonds in the isoniazid molecule (b.) the copper ion binds sucrose via various oxygen atoms (c.) when copper, isoniazid and sucrose are aggregated by PEG there is a complexation effect between copper, isoniazid and sucrose while there is only a weak interaction between the copper, isoniazid, sucrose and the PEG aggregate.

(17) As outlined previously in this discovery two component (i.e. copper(II), PEG) systems involving first and second-line antibiotics (i.e. rifampicin, capreomycin, amikacin) demonstrated that in some cases these components, as either individual chemical species or as a combination, can increase the efficacy of the antibiotics against active and resistant strains of TB. Further testing consists of the three component delivery system composed of copper(II), PEG, and sucrose is described below.

(18) Four different combinations of compounds including the frontline antibiotic isoniazid, sucrose, copper(II) ion, and the biopolymer PEG-3350 were tested against the M. tuberculosis H37Rv strain, through a program sponsored by the National Institutes of HealthNational Institute of Allergy and Infectious Disease (NIH-NIAID). Minimum inhibition concentrations (MIC), inhibition concentrations (IC), minimum bactericidal activity (MBC), and intracellular activity were found in aerobic and low oxygen conditions.

(19) In order to understand the significance of this invention, it is important to outline experimental procedures in which the compounds in different variations were tested against M. tuberculosis. Compounds were removed from a negative twenty degrees Celsius system, warmed to room temperature, and reweighed before solubilization in diemthylsulfoxide (DMSO). Compounds were solubilized in DMSO to a concentration of ten micromolar. Assays were initiated within seven days of solubilization. Compounds were dispensed into ninety-six-well plates using a BioMek 3000 and serial dilutions were prepared. Dilutions were dispensed into final assay plates for each task.

(20) The MIC of each compound was determined by measuring bacterial growth after five days in the presence of test compounds. Compounds were prepared as twenty-point two-fold serial dilutions in DMSO and diluted into 7H9-Tw-OADC medium in ninety-six-well plates with a final DMSO concentration of two percent. The highest concentration of compound was two hundred micromolars where compounds were soluble in DMSO at ten millimolar. For compounds with limited solubility, the highest concentration was fifty times less than the stock concentration (e.g. one hundred micromolar for five millimolar DMSO stock, twenty micromolar for one millimolar DMSO stock). Control compounds were prepared as ten-point two-fold dilution series. Each plate included assay controls for background (medium/DMSO only, no bacterial cells), zero growth (one hundred micromolar isoniazid) and maximum growth (DMSO only), as well as an isoniazid dose response curve. Plates were inoculated with Mycobacterium tuberculosis and incubated for five days: growth was measured by optical density at five hundred and ninety nanometers and fluorescence (Excitation at 560 nm/Emission at 590 nm) using a plate reader. Growth values were calculated separately for OD.sub.590 and relative fluorescence units (RFU). To calculate the MIC value, the dose response curve was plotted as percent (%) growth and fitted to the Gompertz model.

(21) The MIC value is defined as the minimum concentration at which growth was completely inhibited and was calculated from the inflection point of the fitted curve to the lower asymptote (zero growth). In addition, dose response curves were generated using the Levenberg-Marquardt algorithm and the concentrations that resulted in fifty percent and ninety percent inhibition of growth were determined (IC.sub.50 and IC.sub.90 respectively). As an example, raw data provided in table two can be used to plot either type of curve.

(22) TABLE-US-00001 TABLE 2 Sample RFU and OD measurements used to determine MIC values for various complexes. Concentration % Growth % Growth (M) (RFU) (OD) 200 3 5 100 3 4 50 3 5 25 3 5 12.5 3 5 6.3 3 5 3.1 3 5 1.6 4 6 0.78 24 31 0.39 79 95 0.20 91 109 0.10 94 108 0.050 95 107 0.025 96 106 0.013 94 108 0.0063 98 105 0.0031 96 106 0.0016 99 104 0.00078 97 106 0.00039 96 105 200 3 5 100 3 4 50 3 5 25 3 4 12.5 3 4 6.3 3 4 3.1 3 5 1.6 4 5 0.78 38 44 0.39 72 90 0.20 93 103 0.10 98 105 0.050 99 104 0.025 96 105 0.013 98 102 0.0063 99 103 0.0031 98 101 0.0016 98 100 0.00078 99 103 0.00039 99 106 200 1 2 100 2 2 50 3 4 25 3 4 12.5 3 5

(23) The antimicrobial activity of compounds against M. tuberculosis H37Rv grown under hypoxic conditions was assessed using the low oxygen recovery assay (LORA) (see table three). Bacteria are first adapted to low oxygen conditions and then exposed to compounds under hypoxia; this is followed by a period of outgrowth in aerobic conditions and growth is measured using luminescence. Most antibiotic screening involves bacteria that are replicating. M. tuberculosis can exist in an inactive state referred to as nonreplicating persistence (NRP). There is now substantial evidence reported in the scientific literature that this state may be responsible for the long pharmaceutical regimens patients endure in the treatment of tuberculosis. The LORA protocol was developed and is now implemented on a wide scale to test compounds for activity against bacteria in the NRP state. (iii) Adding a solvent such as highly purified water, to expose the composition to a dissolving environment in the same high shear mixer. (iv) Adding a lubricant such as magnesium stearate in the same high shear mixer. (v) Adding a glidant such as suspended silicon dioxide in the same high shear mixer. (vi) Add sodium ascorbate or ascorbic acid as an anti-oxidant in the same high shear mixer. (vii) Excess water would be removed in a dehydration step. (viii) A tablet composed of all the components would be made by compression in a tablet press in a size not to exceed eleven millimeters and a mass not to exceed two thousand milligrams but preferably below twelve hundred milligrams. (ix) Coating would take place in a pan coater such as those manufactured by Glatt.

(24) PEG-3350 is a water soluble polymer that is widely used as a laxative. A typical dosage is seventeen grams dissolved in water. The pharmaceutical quantities outlined above all lie significantly below this value giving an example of its lack of toxicity. Additionally, the sucrose quantities do not approach toxicity levels. For example, the LD.sub.50 (mg/kg) level of sucrose is 29,700 which is much higher than ethyl alcohol at 14,000, sodium chloride (common table salt) at 3,000, vitamin A at 2,000, vanillin at 1,580 and aspirin at 1,000 which are all consumed on a regular basis. There may be concerns about the toxicity of copper being used in a medicine. The LD.sub.50 for copper gluconate, which is used to treat copper deficiency, is 1710 mg/kg for rats (oral) whereas the LD.sub.50 for copper (II) chloride is 140 mg/kg for rats (oral) demonstrating the impact that binding the copper ion to a chelating agent can have on its toxicity. In most cases a chelating agent is a species that binds the cation via covalent, ionic, and/or ion dipole interactions. For further explanation, the species EDTA or ethylenediaminetetraacetic acid is a well-known chelating agent that can bind an ionic species such as copper(II), nickel(II), iron(II) or zinc(II) via interactions involving its amines (i.e. copper-nitrogen) and/or its carboxylates (COO.sup.-copper(II)).

(25) In this embodiment the copper ion binds to the nitrogen (and/or oxygen atoms if present, e.g. isoniazid) in the antibiotic and to the oxygen present in the sucrose molecule while being enclosed in the PEG aggregate. Two milligrams of copper per day is the minimum requirement for an adult. The value of copper intake of 0.5 milligrams per kilogram body weight, or fifty milligrams for a one hundred kilogram person is recommended. Animal toxicity with copper salts was observed only with quantities several orders of magnitude greater than that used as food supplements. In addition, most copper in the body is bound to proteins and is critical for physiological processes. Unbound copper ions are the potentially toxic species, while bound or trapped copper have a significantly lower toxicity in the human body.

(26) The geometry of the tablet is an important parameter in producing an oral medication. A recent document entitled Size, Shape, and Other Physical Attributes of Generic Tablets and Capsules. Guidance for Industry released by U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER) in June 2015, provides an outline for tablet dimensions. The three component delivery complex and the antibiotic described here would have a typical density range between 1.2 and 1.7 grams per centimeter cubed. Currently, isoniazid tablets have several forms. For example, the isoniazid 100 Ver are round, white tablets imprinted with Westward and 260 on opposite sides. The isoniazid 100 mg-BAR is a round, white tablet and the isoniazid 300 mg-BAR is an oval, white tablet. Isoniazid 300 mg-VER is round, white and imprinted with WestWard and 261 on opposite sides. Using the proposed formulation of a fifty milligram dose of isoniazid complexed with 124.8 milligrams of sucrose, 62.04 milligrams of copper (II) chloride dihydrate and 1222.62 milligrams of PEG-3350, results in a total mass of approximately 1460 milligrams and an approximate density of 1.5 grams per centimeter cubed, depending on the exact conditions under which the tablet was formed. If the correction outlined above taking into account the lower MIC values and subsequently a lower dosage is utilized, this value may decrease to 0.3 centimeters cubed. If additional species are added such as the lubricant magnesium stearate, and a glidant such as silicon dioxide, the tablet would have a total volume of 1.0 centimeter cubed and a mass of 1.5 grams. Considering the FDA recommended size of eight millimeters (0.8 cm), this mass may be divided over five equal mass and volume shaped sized tablets for a single dose, as an example of the tablet production that should not be construed to be limiting in any way to the present invention.

(27) Proton (.sup.1H) and carbon (.sup.13C) NMR was used to study the copper-glucose-PEG-isoniazid and the copper-sucrose-PEG-isoniazid complexes and their subcomponents to understand the types of interactions and bonding that exists between the different components (see table one). This structural knowledge helps illustrate the manner in which the aggregate and its contents can perform as a delivery agent, perform as a nutrient source, how the toxicity of the copper(II) ion might be minimized, how isoniazid functions as a prodrug, how the copper(II) ion binds isoniazid or sucrose, and the interactions between the four components. First, the NMR spectral features of the pure organics (glucose, sucrose, isoniazid, PEG) were measured by proton (.sup.1H) and carbon (.sup.13C) NMR. These studies were followed by the NMR measurements of copper(II) complexed to each of the organics (i.e. copper-isoniazid, copper-sucrose, copper-PEG, and isoniazid-copper-sucrose). Copper(II) chloride is paramagnetic and the binding of the cation to nitrogen and/or oxygen atoms can impact the NMR spectral line position, its full width half maximum and in some cases, whether it can be observed at all. For example, figure seven shows the .sup.13C NMR for pure isoniazid, which shows the four peaks for the four carbon species in different environments. Figure eight provides the .sup.13C NMR spectra for the copper-isoniazid complex and illustrates the impact that the paramagnetic species has on the .sup.13C spectra showing a loss of spectral features. The fact that the spectral features associated with all four carbon atoms in the isoniazid molecule were impacted by the binding to the copper ion indicates the complex is a polarity adaptive molecule or the copper ion moves around the molecule, binding to pi bonds, oxygen and nitrogen molecules.

(28) TABLE-US-00002 TABLE 1 The spectral positions (ppm) for the proton (.sup.1H) and carbon (.sup.13C) NMR datum of isoniazid complexes and their subcomponents. (ppm is parts per million and represents chemical shifts of the resonant frequency in the magnetic field of the nucleus). .sup.1H NMR .sup.13C NMR Compounds (ppm) (ppm) Isoniazid 4.8 ppm, 7.5 ppm, 8.5 167 ppm, 149 ppm, ppm 140 ppm, 121 ppm Glucose 3.1 ppm, 3.3 ppm, 3.65 61 ppm, 69 ppm, 72 ppm, 4.55 ppm, 5.2 ppm ppm, 73 ppm, 74 ppm, 76 ppm, 92 ppm, 96 ppm PEG-3350 3.65 ppm 69 ppm Cu(II)-PEG- 3.65 ppm, 4.8 ppm 69 ppm 3350 Cu(II)-Glucose Spectral feature stretching 61 ppm, 71 ppm, 72 from 4 ppm to 5.5 ppm, ppm, 73 ppm, 74 and other features at 3.2 ppm, 76 ppm, 96 ppm and 3.6 ppm. ppm Cu(II)-Isoniazid 4.8 ppm No observable spectral features Cu(II)-PEG- 3.65 ppm, 4.65 ppm 69 ppm Glucose- Isoniazid

(29) TABLE-US-00003 TABLE 3 Raw data used for LORA measurements (RLU is relative light units or relative luminescence units). Concentration Growth Oxygen Tension (M) (RLU) Low Oxygen 200 13 100 18 50 21 25 15 12.5 19 6.3 26 3.1 42 1.6 82 0.78 112 0.39 1950 0.20 6980 0.10 6840 0.05 6920 0.025 7120 0.013 7150 0.0063 7100 0.0031 7000 0.0016 7250 0.00078 7020 0.00039 7120 Normal Oxygen 200 75 100 40 50 30 25 41 12.5 74 6.3 53 3.1 51 1.6 62 0.78 74 0.39 74 0.20 22300 0.10 23200 0.05 23600 0.025 24400 0.013 25400 0.0063 23600 0.0031 25100 0.0016 22100 0.00078 25800 0.00039 25500

(30) The MIC values measured under low oxygen were prepared as twenty-point two-fold serial dilutions in DMSO and diluted into DTA medium in ninety-six well plates with a final DMSO concentration of two percent. The highest concentration of compound was two hundred micromolar where compounds were soluble in DMSO at ten millimolar. For compounds with limited solubility, the highest concentration was fifty times less than the stock concentration, for example one-hundred micromolar for a five millimolar DMSO stock solution, twenty micromolar for a one millimolar DMSO stock solution. Control compounds were prepared as ten-point, two-fold serial dilutions in DMSO and diluted into DTA medium in ninety-six-well plates with a final DMSO concentration of two percent.

(31) M. tuberculosis constitutively expressing the luxABCDE operon was inoculated into DTA medium in gas-impermeable glass tubes and incubated for eighteen days to generate hypoxic conditions (follows the Wayne model of hypoxia). At this point, bacteria are in a non-replicating state (NRP stage two) induced by oxygen depletion.

(32) Oxygen-deprived bacteria were inoculated into compound assay plates and incubated under anaerobic conditions for ten days followed by incubation under aerobic conditions (outgrowth) for five days. Growth was measured by luminescence. Oxygen-deprived bacteria were also inoculated into compound assay plates and incubated under aerobic conditions for five days. Growth was measured by luminescence. Rifampicin was included in each plate and metronidazole was included in each run as positive controls for aerobic and anaerobic killing of M. tuberculosis, respectively.

(33) The bactericidal activity of compounds was assessed against M. tuberculosis H37Rv grown in aerobic conditions in 7H9-Tw-OADC medium. Viable cell counts were measured over three weeks of exposure to determine the rate of kill.

(34) M. tuberculosis was grown aerobically to logarithmic phase and inoculated into the liquid medium containing four different compound concentrations with a final maximum concentration of two percent DMSO. For compounds with an MIC value (from group assay), the concentrations selected were ten times the MIC value, five times the MIC value, one time the MIC value, and one-quarter the MIC value. Cultures were exposed to compounds for twenty-one days and cell viability is measured by enumerating colony forming units (CFU's) on agar plates on day zero, seven, fourteen and twenty-one. MIC values were calculated as the average of the MIC value derived from the Relative Fluorescence Unit and OD from assay group one.

(35) The general definition of the minimum bactericidal concentration, or MBC, is the minimum concentration of an antibacterial chemical species required to kill a specific bacterium. In this study or evaluation of anti-tuberculosis complexes, the MBC is defined as the minimum concentration required achieving a two-log kill in twenty-one days. For compounds with a greater than one-log kill, an assessment of time and/or concentration-dependence was determined from the kill kinetic measurements. A DMSO solution was used as a positive control for growth.

(36) The cytotoxicity of the compounds and controls towards eukaryotic cells was determined using the Vero African green monkey kidney cell line. Vero cells were incubated with compounds for two days, and the cell viability was measured. The IC.sub.50 is determined as the concentration of compound causing a fifty percent loss in viability. The intracellular activity of compounds is measured using a macrophage cell line infected with M. tuberculosis. Murine macrophages are infected with bacteria and viable bacterial counts measured over four days using luminescence as a measure of growtha linear relationship between the colony forming units and luminescence was established.

(37) The cytotoxicity of both the novel medicinal compounds and the controls was determined by measuring the Vero cell viability growth after two days in the presence of test compounds. Compounds were prepared as ten-point three-fold serial dilutions in DMSO. Vero cells were cultured in DMEM containing high glucose and GlutaMAX, ten percent FBS, and a penicillin-streptomycin solution. Cells were inoculated into assay plates and cultured for twenty four hours before compound dilutions were added to a final DMSO concentration of one percent. The highest concentration of compound tested was one hundred micromolar where compounds were soluble in DMSO at ten millimolar. For compounds with limited solubility, the highest concentration was fifty times less than the stock concentration; for example a one hundred micromolar solution from a five millimolar DMSO stock, twenty micromolar for a one millimolar DMSO stock. Each plate included staurosporine as a control. Staurosporine is an antibiotic that is a natural product extracted from the microbe Streptomyces staurosporeus. The pharmaceutical activity of staurosporine involves the inactivation of protein kinases by preventing ATP from interacting with the kinase. Staurosporine binds the site stronger than ATP does. Staurosporine is not very selective as to which kinase it binds, but it does bind them with a strong bond.

(38) Assay plates were incubated for two days at thirty-seven degrees Celsius in a five percent carbon dioxide atmosphere, growth was measured using a Luminescent Cell Viability Assay which uses ATP as an indicator of cell viability. Relative luminescent units (RLU) were measured using a plate reader. The dose response curve was fitted using the Levenberg-Marquardt algorithm. The Levenberg-Marquardt algorithm is also called the damped least-squares approach, and is utilized to solve problems that involve non-linear least squares which arrive in treating data that involves curve fitting. The TC.sub.50 was defined as the compound concentration that produced fifty percent inhibition of growth. M. tuberculosis resides in macrophages, making treating the infection difficult. This encapsulation can protect the bacterium from both antibiotic and natural treatment. The protection of the macrophage can provide a long term reservoir of the microbe in a patient. The following is the protocol used to measure intracellular activity of isoniazid.

(39) Murine J774 macrophages were infected with a luminescent strain of H37Rv (which constitutively expresses luxABCDE) at a multiplicity of infection of one. After eighteen hours, extracellular bacteria were removed by washing and the compound was added. The infected macrophages were incubated in the presence of compound for four days at one time and ten times the MIC (as determined in aerobic culture in liquid medium from Task 1 outlined above).

(40) Bacteria were harvested from macrophages by lysis with one tenth of a percent sodium dodecyl sulfate (SDS), inoculated into growth media and allowed to grow aerobically for five days, at which time the quantity of bacteria present was determined by luminescence. All assays were conducted in triplicate; each assay included a positive control (four micromolar isoniazid) and a negative control (two percent DMSO). The intracellular activity was expressed as a log reduction of M. tuberculosis using the formula:
[Log (RLU Day 4 Compound)][Log (RLU Day 4 DMSO)](3)
As a control for each assay, the inoculum was plated into ninety-six-well plates, grown for five days and luminescence measured; correlation of RLU and CFU was confirmed for each run. The baseline of infection was determined by harvesting bacteria from macrophages at day zero before compound addition and plating for colony formation units determination in triplicate.

(41) The activity of the three component carrier and its subcomponents were tested against five resistant isolates of M. tuberculosis strains under aerobic conditions and was assessed by determining the minimum inhibitory concentration (MIC) of each compound. Strains tested were two isoniazid resistant strains (INH-R1 and INH-R2), two rifampicin resistant strains (RIF-R1 and RIF-R2) and a fluoroquinolone resistant strain (FQ-R1). The assay is based on measurement of growth in liquid medium of each strain where the readout is optical density (OD). INH-R1 was derived from H37Rv and is a katG mutant (Y155*=truncation). INH-R2 is strain ATCC35822. RIF-R1 was derived from H37Rv and is an rpoB mutant (S522L). RIF-R2 is strain ATCC35828. FQ-R1 is a fluoroquinolone-resistant strain derived from H37Rv and is a gyrB mutant (D94N).

(42) The activity of the three component carrier and its subcomponents, referred to as compounds, was demonstrated with MIC values of the compound and were determined by measuring bacterial growth after five days in the presence of compounds. Compounds were prepared as twenty-point two-fold serial dilutions in DMSO and diluted into 7H9-Tw-OADC medium in ninety-six-well plates with a final DMSO concentration of two percent. The highest concentration of compound was two hundred micromolar where compounds were soluble in DMSO at ten millimolar. For compounds with limited solubility, the highest concentration was fifty times less than the stock concentration; for example one hundred micromolar for a five millimolar DMSO stock, twenty micromolar for one millimolar DMSO stock. Plates were inoculated with M. tuberculosis and incubated for five days; growth was measured by OD.sub.590. To calculate the MIC, the ten-point dose response curve was plotted as percent growth and fitted to the Gompertz model. The Gompertz growth model is frequently utilized when analyzing data plotted on a two dimensional graph, and functions best when the plotted data set is a smooth curve.

(43) From a graphical perspective, the MIC is the minimum concentration at which growth was completely inhibited and is calculated from the inflection point of the fitted curve to the lower asymptote (zero growth). The dose response curves are generated using the Levenberg-Marquardt algorithm and the concentrations that resulted in fifty percent and ninety percent inhibition of growth were determined (IC.sub.50 and IC.sub.90 respectively). Raw data is provided and can be used to plot either type of curve.

(44) Data resulted from testing various compounds including isoniazid, copper(II), sucrose, and PEG-3350 against resistant and nonresistant stains of M. tuberculosis are now presented and discussed. The front-line anti-tuberculosis drug isoniazid was used to demonstrate the three component system in a number of experiments. Figure nine shows graphical data for the determination of the MIC, IC.sub.50 and IC.sub.90 values for pure isoniazid, isoniazid-sucrose, isoniazid-copper-sucrose, and isoniazid-copper-PEG-sucrose, all in equal molar ratios. The x-axis provides the various concentrations added to the broth culture (micromolar), while the y-axis measures the percent growth of the microbe.

(45) Table four provides the MIC, IC.sub.50, and IC.sub.90 values of all four complexes under aerobic conditions. IC.sub.50 and IC.sub.90 values were determined from the drugs concentrations (x-axis, log values) plotted against the experimentally determined inhibition values. These values are the determination of the efficacy of a drug in inhibiting a biochemical cycle that impacts the ability of an organism to function. These values are often used for in vitro measurements while the EC.sub.50 methods are used for in vivo measurements. Isoniazid had lower MIC (0.97 micromolar), IC.sub.50 (0.63 micromolar), and IC.sub.90 (1.0 micromolar) values than when complexed with sucrose and when complexed with both copper(II) and sucrose. However, the three component complex sucrose-PEG-copper-isoniazid had lower MIC (0.21 micromolar), IC.sub.50 (0.16 micromolar), and IC.sub.90 (0.21 micromolar) values compared to isoniazid alone. Combining the copper(II) ion with the isoniazid-sucrose complex increased the MIC and both IC values. This indicates that the three component complex deliver system can potentially have four additional medicinal benefits when compared to isoniazid alone. First, lower dosages of the antibiotic could be used when treating a tuberculosis infection. Second, if lower dosages are used to obtain the same level of pharmaceutical activity then the extent of the side effects, which can be significant with anti-tuberculosis drugs, could be lessened. Third, depending on dosage manipulation, a lower MIC value for a drug could indicate a shorter treatment length. Currently, treatment length for tuberculosis typically lasts six months. Fourth, the PEG-copper-isoniazid-sucrose aggregate may be perceived as biological waste to the human immune system and the macrophage may engulf it via the process of phagocytosis.

(46) Table five provides the MIC, IC.sub.50, and IC.sub.90 values of all four complexes under low oxygen conditions using a low oxygen recovery assay (LORA). Isoniazid had the lowest MIC (0.62 micromolar) and IC.sub.90 (0.46 micromolar) values compared to isoniazid complexed with copper, sucrose, and PEG. However, the copper-isoniazid-sucrose-PEG complex had a slightly lower IC.sub.50 (0.3 micromolar) value compared to isoniazid alone (0.35 micromolar).

(47) Table six provides the minimum bactericidal concentration (MBC), or the minimum concentration of an antibiotic that is needed to kill the bacteria, values which demonstrate that all four complexes tested were considered to be bactericidal. MBC values are measured by a broth dilution and subculturing to agent-free agar plates and are calculated by determining the lowest compound concentration needed to reduce the starting bacterial inoculation by greater than ninety-nine percent (99.9%). Antibacterial agents are considered bactericidal if the MBC/MIC value is less than four. Pure isoniazid exhibited the lowest MBC (0.3 micromolar) value compared to the other three complexes; while copper-isoniazid-sucrose and copper-isonaizid-sucrose-PEG had the same MBC value (1.4 micromolar). Adding the copper ion and PEG reduced the MBC value of isoniazid-sucrose by about half

(48) Table seven provides data that demonstrates two of the four complexes were considered cytotoxic towards eukaryotic cells and both contained sucrose: copper-isoniazid-sucrose (IC.sub.50 53 micromolar) and copper-isoniazid-sucrose-PEG (IC.sub.50 32 micromolar). Eukaryotic cells are characteristic of all life except bacteria, blue-green algae, and some primitive microbes, compared to a prokaryote cell which is characteristic to bacteria and other single-celled organisms. Cytotoxicity is the ability of a compound to be toxic to cells. Isoniazid exhibits cytotoxicity in vivo and results with clinical side effects. The lower MIC values for isoniazid against active tuberculosis (see table four) suggest that a lower dose could be possible which could lower the cytotoxicity. While PEG, copper(II), sucrose and isoniazid are not cytotoxic at these concentrations, the results of the combination indicate a chemical reaction or reactions that impact one or more cellular processes. The cytotoxic and MIC values indicate there is a concentration range lower than the cytotoxicity limit and above the MIC values allowing additional testing for dosage determination.

(49) Table eight shows all four isoniazid based complexes had intracellular activity against M. tuberculosis. M. tuberculosis can survive in host macrophages for long periods of time (i.e. weeks). A published study entitled Intracellular Activity of Tedizolid Phosphate and ACH-702 versus Mycobacterium Tuberculosis Infected Macrophages, demonstrated intracellular activity for two new compounds, Tedizolid and ACH-702 which resulted in a drop of activity of approximately 1.4 and 1.3 log(kill) units. As the data in table eight demonstrates, the average log(kill) value for isoniazid is 2.5. None of the three components of the drug carrier interfere with the medicinal activity of isoniazid in this process. A drug with intracellular activity can impact pharmacokinetic parameters such as penetration and retention, accumulation, subcellular disposition, bioavailability and metabolism. The drug can also impact pharmacodynamic parameters such as expression of activity and bacterial responsiveness. While PEG, copper, sucrose and isoniazid are not independently cytotoxic at these concentrations, the results of the combination indicate a chemical reaction or reactions that impact one or more cellular processes. The catalase-peroxidase enzyme in M. tuberculosis katG is responsible for activation of the drug. KatG causes isonicotinic acyl-NADH to form, which inhibits the production of different forms of mycolic acids, a major component of the cell wall of M. tuberculosis. Isoniazid is bactericidal to mycobacteria that reproduce rapidly, but it is bacteriostatic if the bacteria are slow-growing.

(50) Table nine provides data that illustrates four of the eight complexes exhibited increased efficacy to isoniazid resistant isolates (INH-R1, INH-R2). The history and concepts of bacterial resistance is critical in recognizing the significance of these results. Bacterial resistance occurs when an isolate evolves a mutation to avoid the mechanism of action of an antibiotic. These bacteria then replicate and cause an infection that cannot be affected by the antibiotic to which the isolate has developed a resistance to.

(51) The isoniazid resistant isolates tested were derived from the H37Rv strain and contained a mutation in the katG gene. The copper-isoniazid-sucrose-PEG complex clearly exhibited the lowest MIC values compared to the other four compounds at 25 and 28 micromolar, followed by the copper-isoniazid-sucrose complex at 89 and 92 micromolar. The isoniazid and isoniazid-sucrose complexes exhibited higher MIC values at >200 micromolar. The isolates showed the highest resistance to isoniazid and isoniazid-sucrose and the lowest resistance to the complex containing all four compounds: copper, sucrose, PEG, and isoniazid. Pure isoniazid and the isoniazid-sucrose complexes had measured MIC values that were greater than two hundred micromolar, values that would be toxic if administered at these high concentrations. Considering the total compilation of measurements (tables four, five, six, seven, eight, nine), the three component aggregate has unique medicinal features of being toxic against M. tuberculosis and having the ability to kill its host. As the controls in various studies demonstrate, none of the individual components have these properties but the sum total of the three combined with the antibiotic possess these pharmaceutical traits. In presenting a novel aspect of this work, there is no record in the scientific literature of repurposing a known first or second-line antibiotic making it active again against a bacterium that has developed a resistance mechanism to it using an altered molecular delivery system.

(52) Another series of experiments involving M. tuberculosis cell line measurements that replaced the disaccharide sucrose molecule with the monosaccharide glucose molecule were performed. A total of four complex combinations, including the frontline antibiotic isoniazid, the monosaccharide glucose, the copper(II) ion, and PEG-3350 were tested against the M. tuberculosis H37Rv strain at the National Institutes of HealthNational Institute of Allergy and Infectious Disease (NIH-NIAID). These complexes contained different ratios of glucose and PEG, and the MIC and IC values were found in aerobic conditions.

(53) Table ten provides the MIC, IC.sub.50, and IC.sub.90 values of the five complex variations. Each complex exhibited activity against the bacterium under aerobic conditions. Isoniazid had the lowest MIC (0.64 micromolar), IC.sub.50 (0.47 micromolar), and IC.sub.90 (0.62 micromolar) values compared to the complexes that contained glucose, copper, and PEG. When the ratios of glucose increased by four and sixteen fold in the complexes, the MIC, IC.sub.50, and IC.sub.90 values increased, indicating decreased pharmaceutical efficacy or that glucose inhibited isoniazid. When the quantity and concentration of PEG was increased by four fold in the complexes, the MIC, IC.sub.50, and IC.sub.90 values increased, also demonstrating that PEG lessened the pharmaceutical efficacy of isoniazid in the presence of glucose.

(54) The scientific hypothesis related to the development of this embodiment outlines four unique features related to the tablet composition and how its effectiveness compares to existing drug delivery systems in vivo and in vitro; (a) sucrose and copper would serve as biocides and/or toxic species or aid in enhancing these activities of the antibiotic (b) a water soluble polymer would produce an aggregate that could assimilate and enclose the components and antibiotic and this aggregate would be identified as foreign by the macrophage and be consumed, providing an entry method to the protected M. tuberculosis (c) copper and sucrose would serve as nutrient based components, would be delivered with the antibiotic in the PEG aggregate because they are similar in size to the drug, and serve a role in potentially accelerating some cellular processes, (d) the water soluble polymer forms a loosely shaped aggregate that could increase residence time in the patient and minimize hydrogen bonding to the drug and unwanted species.

(55) The tablet composition and the pharmaceutical results of this composition are significant for two reasons. One is that it is the first known demonstration of an existing front-line antibiotic exhibiting a reversal of resistance making the highly utilized antibiotic effective again against resistant strains of M. tuberculosis. Since many aspects of the front-line antibiotics, such as isoniazid, are already known, this development allows for the same medicinal agents to continue being used, rather than developing a new compound with anti-tuberculosis properties. This technology provides not only a medical advantage but also an economic one, since these drugs are already manufactured, distributed and stored worldwide. Second, this technology is the first positive demonstration of a nutrient based drug delivery system focused on being masked from the immune system and altering cellular processes with anti-tuberculosis properties.

(56) TABLE-US-00004 TABLE 4 All four complexes had activity against M. tuberculosis under aerobic conditions. MIC IC.sub.90 IC.sub.50 Complexes (M) (M) (M) Isoniazid (INH) 0.97 1.0 0.63 INH-SUC 1.3 1.3 0.71 Cu-INH-SUC 2.3 2.7 1.6 Cu-INH-SUC-PEG 0.21 0.21 0.16

(57) TABLE-US-00005 TABLE 5 All four complexes had activity against M. Tuberculosis under low oxygen concentrations. Low Oxygen Low Oxygen Low Oxygen Complexes MIC (M) IC.sub.50 (M) IC.sub.90 (M) Isoniazid (INH) 0.62 0.35 0.46 INH-SUC 3.7 2.0 2.7 Cu-INH-SUC 41 1.1 6.3 Cu-INH-SUC- 69 0.3 4.2 PEG

BRIEF DESCRIPTION OF FIGURES

(58) FIG. 1: (A, top) 500 MHz .sup.1H NMR spectra of the second line TB drug capreomycin and the (B, bottom) copper-capreomycin complex. The paramagnetic copper ion causes shifts and broadening to the entire NMR structure indicating the ion is moving around the structure and not bound to a single location when in the solvent.

(59) FIG. 2. A 500 MHz proton (H) NMR spectra of the copper-amikacin complex between 2.5 and 6 ppm. The broadened spectral features indicate the paramagnetic copper dication is interacting with the entire molecule.

(60) FIG. 3. A MALDI-TOF-MS spectrum of the copper-amikacin complex. The isotopic peaks and their natural abundance for the complex are 648.2 m/z (51.6%), 649.2 (13.1%), 650.2 (26.1%), 651.2 (6.6%) and 652.2 (1.47%). This is experimental evidence that the copper ion is bound to the antibiotic.

(61) FIG. 4. A proton (.sup.1H) NMR spectra of rifampicin (top) and of the copper-rifampicin complex (bottom). The paramagnetic copper ion can cause a spectral feature in NMR to be so broadened so it raises the baseline and is not observed by the analyst.

(62) FIG. 5 (A-D). FT-ICR provides the best resolution and mass accuracy available for molecular analysis. (A) Provides spectral data for rifampicin C.sub.43H.sub.58N.sub.4O.sub.12H.sup.+ (B) Provides spectral data for the presence of the ubiquitous sodium adduct of rifampicin (C) Provides spectral evidence for the presence of the copper-rifampicin complex minus water (D) Provides spectral evidence for the presence of the copper-rifampicin complex.

(63) FIGS. 6A and 6B are logP graphs of 187 antibiotic complexes. (A) The logP (i.e. the log.sub.10) value of the water-octanol partition coefficient is plotted against the number of hydrogen bond donors for 187 antibiotic complexes (B) the logP is plotted against the total polar surface area (TPSA) for 187 antibiotic complexes.

(64) FIG. 7. The 500 MHz .sup.13C NMR of isoniazid shows the four spectral features corresponding to the four carbons species.

(65) FIG. 8. The .sup.13C NMR of the copper(II)-isoniazid complex illustrates how the paramagnetic copper causes the spectral features to disappear.

(66) FIG. 9. Dose response curves for (from top) isoniazid, isoniazid-sucrose, isoniazid-sucrose-copper; and isoniazid-sucrose-copper-PEG, all in equal molar ratios. Dose response curves were used to calculate the MIC, IC.sub.50 and IC.sub.90 values.

(67) TABLE-US-00006 TABLE 6 All four complexes had bactericidal properties. Compound MBC (M) Isoniazid (INH) 0.3 INH-SUC 3.1 Cu-INH-SUC 1.4 Cu-INH-SUC-PEG 1.4

(68) TABLE-US-00007 TABLE 7 Two complexes have cytotoxic effects against eukaryotic cells. This data suggest that the compound(s) can affect the macrophages that harbor M. tuberculosis. Compound IC.sub.50 (M) INH >100 INH-SUC >100 Cu-INH-SUC 53 Cu-INH-SUC-PEG 32

(69) TABLE-US-00008 TABLE 8 Four complexes had intracellular activity against M. tuberculosis. Concentration Complexes (M) Log Kill Isoniazid (INH) 1.2 2.5 Isoniazid (INH) 12 2.5 INH-SUC 0.62 2.2 INH-SUC 6.2 2.7 Cu-INH-SUC 14 2.7 Cu-INH-SUC-PEG 1.4 2.4 Cu-INH-SUC-PEG 14 2.5

(70) TABLE-US-00009 TABLE 9 M. tuberculosis isoniazid resistant isolates showed increased resistance to four of eight complexes. Resistant Complexes MIC (M) Isolate Isoniazid (INH) >200 INH-R1 Isoniazid (INH) >200 INH-R2 INH-SUC >200 INH-R1 INH-SUC >200 INH-R2 Cu-INH-SUC 89.0 INH-R1 Cu-INH-SUC 92.0 INH-R2 Cu-INH-SUC-PEG 25 INH-R1 Cu-INH-SUC-PEG 28 INH-R2

(71) TABLE-US-00010 TABLE 10 All five complexes, including four that incorporated glucose, had activity against M. tuberculosis under aerobic conditions. MIC IC.sub.50 IC.sub.90 Complexes (M) (M) (M) Isoniazid (INH) 0.64 0.47 0.62 Cu-INH-GLU-PEG 2.7 1.7 2.5 Cu-INH-GLU.sub.4-PEG 1.8 1.1 1.8 Cu-INH-GLU.sub.16-PEG 12 8.8 11 Cu-INH-GLU-PEG.sub.4 4.2 2.8 4.7