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  Pharmaceutical Patents  

 

Title:  Chemical induced intracellular hyperthermia
United States Patent: 
7,635,722
Issued: 
December 22, 2009

Inventors:
 Bachynsky; Nicholas (Parkland, FL), Roy; Woodie (Parkland, FL)
Assignee:
  Saint Jude Pharmaceuticals, Inc. (Texarkana, TX)
Appl. No.:
 09/744,622
Filed:
 July 27, 1999
PCT Filed:
 July 27, 1999
PCT No.:
 PCT/US99/16940
371(c)(1),(2),(4) Date: 
May 07, 2002
PCT Pub. No.:
 WO00/06143
PCT Pub. Date:
 February 10, 2000


 

Training Courses -- Pharm/Biotech/etc.


Abstract

An invention relating to therapeutic pharmacological agents and methods to chemically induce intracellular hyperthermia and/or free radicals for the diagnosis and treatment of infections, malignancy and other medical conditions. The invention relates to a process and composition for the diagnosis or killing of cancer cells and inactivation of susceptible bacterial, parasitic, fungal, and viral pathogens by chemically generating heat, and/or free radicals and/or hyperthermia-inducible immunogenic determinants by using mitochondrial uncoupling agents, especially 2,4 dinitrophenol and, their conjugates, either alone or in combination with other drugs, hormones, cytokines and radiation.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention encompasses a composition and method using mitochondrial uncoupling agents, especially DNP, DNP with free radical producing drugs, DNP with liposomes, DNP conjugated to free radical formers, and DNP with other therapeutic pharmaceutical agents which are activated intracellularly by heat or reaction with mitochondrial electrons or free radicals to cause release of active medications for the treatment of cancer, HIV, other viruses, parasites, bacteria, fungi and other diseases. While not being bound by theory, it is submitted that the use of mitochondrial uncoupling agents, to increase intracellular heat and free radicals, as treatment for non-related cancers, viruses and other pathogens presupposes that the mechanism of action is non-specific for enzymes and receptors but is specific for interference with cellular and pathogen viability and induction of programmed cell death. The degree of intracellular heating, free radical formation, whole body hyperthermia and release of active drug molecules is controlled by the dose of DNP. Based on the quantity of oxygen consumed, the dose of DNP is adjusted to achieve the desired degree of hyperthermia. Safety and effectiveness is further controlled by manipulating metabolic rates of target tissues, duration of treatment and permissiveness of body cooling. In accordance with the present invention, intracellular, mitochondrial heat is generated by the use of DNP, other uncouplers, their conjugates, either alone or in combination with other drugs for the treatment of thermosensitive cancers such as non-Hodgkins lymphoma, prostate carcinoma, glioblastoma multiforme, Kaposi's sarcoma, etc; bacteria such as Borrelia burgdorferi, Mycobacterium leprae, Treponema pallidum, etc.; viruses such as HIV, hepatitis C, herpes viruses, papillomavirus, etc.; fungi such as Candida, Sporothrix schenkii, Histoplasma, Paracoccidiodes, Aspergillus, etc.; and, parasites such as Leishmania, malaria, acanthamoeba, cestodes, etc. 2,4-dinitrophenol was selected as the uncoupler of choice because it can be used at relatively high concentrations, permitting uniform distribution in organs and tissues. This invention also encompasses the use of DNP to selectively augment energy metabolism and heat production in inchoate malignant tumors for the purpose of increasing sensitivity of diagnostic positron emission tomography, temperature-sensitive magnetic resonance, and high-precision pixel temperature infrared imaging in differentiating normal from aberrant cell metabolisms. An additional object of the invention is the use of DNP to increase transcription of heat shock proteins, especially HSP 72, as a form of cellular pre-conditioning to decrease post-angioplasty restenosis, increase successful outcome of other surgeries, and facilitate antigen processing and presentation of immunogenic determinants on infectious agents, virally transformed cells and tumors so as to increase the natural or biologically activated immunological response.

In accordance with another aspect of the present invention, controlled thermogenesis with DNP is combined with other agents used to treat infectious, malignancy and other diseases. Examples of other agents include antifungal, antiviral, antibacterial, antiparasitic and antineoplastic drugs. Such drugs, including angiogenesis inhibitors and radiation have increased synergistic or additive activity when combined with hyperthermia in the treatment of cancer.

The method can be used for enhancing the sensitivity of positron emission tomography, nuclear magnetic resonance spectroscopy and infrared thermography in the diagnosis and monitoring of treatment of various diseases, including cancer. Similarly, the method can be used for enhancing the identification of unstable "hot" coronary and carotid artery plaques predisposed to rupture or undergo thrombosis. Such diagnostic and treatment monitoring methodology is based on the fact that most tumors have higher metabolic rates and generate more heat than normal tissues. Likewise, unstable atherosclerotic plaques are presumed to rupture because they have a dense infiltration of macrophages which have high metabolic rates and generate excessive enzymes and heat, causing the plaque to degrade and loosen. In both instances, controlled doses of DNP or other uncouplers can further increase metabolic rates and heat production to increase diagnostic sensitivity. Controlled heating with DNP and fibrinolytic recombinant tissue-type plasminogen activators can also be used therapeutically to accelerate fibrinolysis of clotted arteries.

In another aspect of the invention, DNP is administered in controlled and timed dosages to provide physiologic stress, "chemical exercise", so as to induce synthesis of autologous heat shock proteins (HSPs). Intracellular heat exposure associated with autologous HSP induction has a significant cytoprotective effect against ischemia and cellular trauma and acts as a form of cellular thermal preconditioning in patients about to undergo surgery. Induction of HSPs by DNP in patients some 8 to 24 hours prior to angioplasty, coronary bypass surgery, organ transplantation and other forms of high risk surgery, would provide for improved clinical outcome with decreased post-angioplasty intimal thickening or restenosis, increased myocardial protection from infarction, improved musculocutaneous flap survival in plastic reconstruction and reduced ischemia/reperfusion injury in organ transplantation cases.

Another aspect of the invention provides for controlled dosages of DNP to induce long duration (6 to 8 hour), mild whole body hyperthermia (39.0 to 40.0.degree. C.) to afford maximum expression of immunogenic HSPs or peptides associated with HSPs. The antigenic properties of HSPs and HSP-peptide complexes, induced by DNP in infectious agents, especially those located intracellularly, or on tumors can be exploited to enhance the immune response. This aspect of the present invention provides a process for modulating the immune system of a patient with other therapies, comprising the steps of: (1) increasing the expression of HSPs by the process described above, and (2) administering humanized monoclonal or polyclonal antibodies, or (3) administering recombinant cytokines, lymphokines, interferons, etc., or (4) administering standard anti-infectious or anti-neoplastic therapy.

DETAILED DESCRIPTION OF THE INVENTION

Electron transferring, transporting and energy converting elements are ubiquitous and are necessary for life. All eukaryotic and prokaryotic organisms depend on electron transferring and transporting elements such as metal containing hemes and nonmetal moieties such as flavins and adenine nucleotides. These biochemical entities convert the energy stored in chemical bonds of foodstuffs into cellular and organelle membrane potentials, high energy containing molecules such as adenosine triphosphate (ATP), creatinine phosphate, and other forms of chemical energy needed to maintain the highly negative entropic state of life.

The most common form of biologic energy is adenosine triphosphate (ATP). ATP is produced either anaerobically through the Embden-Myerhoff Pathway (glycolysis) or through oxidative phosphorylation. The latter, an oxygen dependent chemical energy conversion process, is generally associated with the Tricarboxylic Acid Cycle [(TCA), Krebs Cycle or Citric Acid Cycle]. The TCA cycle links the products of glycolysis to a multi-enzyme coupled series of electron carriers called an electron transport chain (ETS). The electron transport chain is coupled to production of ATP. The entire TCA cycle and oxidative phosphorylation process is located in intracellular organelles known as mitochondria.

While release of energy from foodstuffs can come about through a variety of biochemical means, the most important means by which energy release is initiated is by splitting glucose into two molecules of pyruvic acid. This occurs through the non-oxygen dependent process of glycolysis in a series of ten chemical steps depicted in FIG. 1 (see Original Patent). The overall efficiency of trapping energy in the form of ATP through this anaerobic process is 43%. The remaining released energy (57%) is discharged in the form of heat.

Pyruvic acid molecules derived from glucose, as well as end products of fat and protein breakdown, are transported into the mitochondrial matrix where they are converted into 2 carbon fragments of acetylcoenzyme A, FIG. 2 (see Original Patent). As depicted, these acetyl fragments enter the TCA cycle were their hydrogen atoms are removed and released as either hydrogen ions (H+) or combined with nicotinamide and flavin adenine dinucleotides (NAD+ and FADH) to produce large quantities of usable reducing equivalents (NADH and FADH2). The carbon skeleton is converted to carbon dioxide (CO2) which becomes dissolved in body fluids. Ultimately the dissolved CO2 is transported to the lungs and expired from the body. As noted in FIG. 2, the flux of reactants in the TCA cycle is always in the same direction because NADH and FADH2 is constantly removed as hydrogen is oxidized by the mitochondrial electron transport chain.

It is the electron transport chain that provides approximately 90% of the total ATP formed by glucose catabolism. During this process, known as oxidative phosphorylation, hydrogen atoms that were released during glycolysis, the TCA cycle, and converted to NADH and FADH.sub.2, are oxidized by a series of enzymatic redox complexes (electron transport chain) located in the inner mitochondrial membrane, FIG. 3 (see Original Patent). Energy released in these steps is captured by a chemiosmotic mechanism that is dependent on the ultimate reduction of O.sub.2 to form H.sub.2O. As depicted in FIG. 4 (see Original Patent), oxidative phosphorylation is two distinct processes: (1) oxidation of NADH and FADH.sub.2; and, (2) formation of ATP. Both processes are interdependent or "coupled" by a high energy linked proton (H.sup.+, pH) gradient and membrane potential across the inner mitochondrial membrane provided by electrons as they pass through the electron transport chain. Energy released by the electrons pumps hydrogen ions (H.sup.+) from the inner matrix of the mitochondrion into the outer inter-membrane space, FIG. 5 (see Original Patent).  This process is known as chemiosmosis and creates a high concentration of H.sup.+ outside the inner mitochondrial membrane and a powerful negative electrical potential in the inner matrix. This transmembrane proton gradient (protonmotive force) causes hydrogen ions to flow back into the mitochondrial matrix through an integral membrane protein (ATP synthase) to form ATP from ADP and free ionic phosphate. The efficiency of oxidative phosphorylation in capturing energy as ATP is about 69%. The remaining (31%) liberated energy is dissipated as heat. The overall efficiency of energy transfer to ATP from glucose via glycolysis, the TCA cycle and oxidative phosphorylation is 66% with about 34% of the energy being released as heat.

Heat is continually produced by the body as a byproduct of metabolism and eventually all energy expended by the body is converted to heat. On a thermodynamic basis, total body heat production is the algebraic sum of the enthalpy changes of all biologic processes in the body. The pathways are irrelevant, even though in the body oxidation involves numerous enzyme catalyzed reactions taking place at 37.degree. C. Biochemically, approximately 95% of all the oxygen (O.sub.2) consumed is used by mitochondria to stoichiometrically couple oxygen reduction to ATP and heat production via oxidative phosphorylation. The rate of O.sub.2 consumption (VO.sub.2) can be measured by indirect calorimetry and thus related to body heat production. Although this method does not include anaerobic processes such as glycolysis, indirect calorimetry is in close agreement with direct body heat measurements and it is generally accepted that 1 liter of VO.sub.2 generates 4.825 Kcal (kilocalorie of energy), .sup.ths of which can be detected as heat.

In human adults, increased VO.sub.2 and endogenous heat production can occur via muscular (work or shivering) and/or chemical [(cathecholamines, thyroid, etc.) non-shivering] thermogenesis. Whereas muscular activity can increase heat production 4-10 fold, non-shivering thermogenesis can only increase heat production by a maximum of 15%. However, oxygen consumption and non-shivering thermogenesis can dramatically increase when even mild injury to the inner mitochondrial membrane occurs so that it is no longer intact and protons leak or reenter the mitochondrion, uncoupled to ATP synthesis. Heating, endotoxin, osmotic imbalance, etc., can cause such injury, i.e., loss of coupling, with resulting respiration and ATP metabolism proceeding independently and maximally--respiration forward, phosphorylation in reverse. FIG. 6 (see Original Patent) compares normal coupled respiration and ATP formation to that which occurs when there has been injury to the inner mitochondrial membrane. The increased reduction of oxygen results in increased heat production.

Additionally, certain chemicals, including biologicals, can selectively increase the transport of protons across uninjured, intact inner mitochondrial membranes and dramatically increase VO.sub.2 and heat production. These compounds dissipate the electrochemical-protonmotive transmembrane potential of mitochondria and uncouple the electron transport chain from ATP synthesis. FIG. 6(a) depicts one such uncoupling agent, DNP, cycling protons across an intact mitochondrial membrane. DNP and other uncouplers permit each of the two distinct processes involved in oxidative phosphorylation to "unlink" and increase their rates according to their own separate kinetic and thermodynamic signals, FIG. 6(b). Uncouplers increase respiratory rates, electron transport, VO.sub.2, heat production and increased utilization of foodstuff substrates through glycolysis and the TCA cycle. Controlled doses of an uncoupler will increase 0.sub.2 consumption and heat production with minimal or no decrease in ATP levels because of intracellular equilibrium shifts in creatinine phosphate, oxidative phosphorylation reactants and increased production of ATP through the anaerobic, glycolytic pathway. Excess or toxic doses of virtually all uncouplers however, will produce secondary untoward effects, including decreased respiration, decreased heat production and eventual cellular death.

In addition to heat being a byproduct of oxidative phosphorylation, reactive oxygen species are also continuously produced by the mitochondrial electron transport chain. Free radicals of oxygen are produced during aerobic oxidation as electrons are transported by the electron carriers to ultimately reduce O.sub.2 to H.sub.2O. As depicted in FIG. 7 (see Original Patent), superoxide (O.sub.2.sup.-) radicals are generated by leaked electrons through the univalent reduction of oxygen. FIG. 7(a) shows that superoxide dismutase then converts the superoxide radical to hydrogen peroxide. Additional hydrogen peroxide (H.sub.2O.sub.2) and hydroxyl (OH.) radicals are formed through the Haber-Weiss Reaction, the hydroxyl radical being the most reactive species, reacting with any biologic moiety instantly. FIG. 7(b) depicts the overall scheme of oxygen metabolism and free radical formation at the level of the mitochondrion.

As mitochondria become progressively heated, uncoupling occurs with increased flux of oxygen free radicals. The effects of heat on mitochondrial uncoupling and superoxide radical generation are depicted in FIG. 8 (see Original Patent). A linear correlation of 0.98 (P<0.01) is obtained for the relationship between percent uncoupling and percent superoxide generation. Similar to exercise increased body temperature and VO.sub.2, hyperthermia induced by uncoupling agents appears to inhibit electron transport at the level of cytochrome c in the redox chain. Normal rat liver, infused with DNP, increases formation of reactive oxygen species threefold upon cessation of uncoupling, FIG. 9 (see Original Patent).

Generally, uncouplers are agents that are hydrophobic ionophores which bind protons and traverse biologic membranes to dissipate transmembrane proton (pH) and membrane potential gradients (.DELTA..PSI., Delta Psim). In so doing, uncouplers increase the rate of metabolism (substrate utilization) in intact animals and isolated tissues by increasing the rate of oxygen reduction through increased availability of protons. 0.sub.2 consumption is increased and remains rapid as long as the mitochondrial respiratory (electron transport) chain attempts to overcome the effects of the uncoupler to maintain a pH gradient. Energy is still used to pump protons across the mitochondrial membrane, but the protons are carried back across the membrane by the uncoupler as depicted in FIG. 6(a). This creates a futile cycle and energy is released as heat. This chemical heat releasing process is comparable to heating that occurs when an electrical wire is "short circuited". Depending on the degree of external body heat dissipation, body temperature rises some 30 to 60 minutes after the increase in 0.sub.2 consumption. Onset of action is rapid after an intravenous injection of an uncoupler. Depending on the intravenous dosage, human oxygen consumption is increased in about 15-20 minutes and the intracellular heat production is increased proportionately. Metabolic rates as high as 10 times normal have been reported. Persistent increases in the metabolic rate can continue as long as 12 to 36 hours because of the long hydrophobic half-life of uncouplers in tissues. Temperature increases can be seen within 10 to 15 minutes in subjects whose heat dissipation mechanisms have been compromised. Heretofore, hyperthermia induced by uncoupling compounds has not been reported to have any therapeutic application.

While there are three general classes of uncoupling agents, each containing specific uncouplers of oxidative phosphorylation, the present invention utilizes 2,4-dinitrophenol (DNP) as the preferred embodiment. This is because DNP has been extensively studied. DNP was commonly used in food dyes in the late 1800's and in the munitions industry of World War I. Rapid increased respiration and hyperthermia, up to 49.degree. C., was noted in man and animals that were accidentally intoxicated. Such dramatic physiologic effects by the dinitro-aromatic dyes, especially DNP, caused them to be inextricably tied to early and later modern studies of metabolism and bioenergetics. In the 1930's DNP was introduced into clinical medicine for the purpose weight loss. It was, however, sold as an over the counter secret nostrum and seriously misused. Had its long half-life in tissues been recognized and physician supervision implemented, it might have become an accepted drug. DNP has been reported in countless, different enzyme, cellular and metabolic studies. Review of such vast published studies have documented DNP's very specific mechanism of action as a proton ionophore, with all other effects a direct pharmacologic extension thereof. DNP is not mutagenic by the Ames and modified Ames tests; it has not been found to be carcinogenic or teratogenic; and, DNP blood plasma levels can easily be determined. DNP can be used at pharmacologic doses that achieve therapeutic concentrations in tissues. Further, DNP is stable, inexpensive and commercially available in reagent grade purity. It is understood however, that other uncouplers and combinations of other uncouplers with other drugs, hormones, cytokines and radiation can potentially be used under appropriate clinical settings and dosages to induce intracellular hyperthermia and promote additive or synergistic effects.

FIG. 10 shows the overall intracellular mechanism of action of DNP (and other uncouplers). Intracellular foci of increased heat and oxygen free radical flux are highlighted. Circled numbers in the figure indicate both direct and indirect effects of DNP: circled 1 and 2 effects shows that upon its intercalation into the inner mitochondrial membrane, DNP shuttles H.sup.+ (hydrogen ions) across the membrane [see FIG. 6(a)]--this short circuits (de-energizes) the proton gradient established by the H.sup.+ pumping action of the mitochondrial electron transport system (see FIG. 5). As a consequence, the inner mitochondrial membrane potential is lowered from -180 to -145 mV. Circled 3, 4, 5 and 6 effects shows that normal oxygen consumption and flux of NADH and FADH.sub.2 (reducing equivalents) through the electron transport system is coupled to H.sup.+ re-entry via mitochondrial availability of ADP for re-synthesis of ATP (see FIG. 4). By freely returning protons into the mitochondrial matrix without concomitant dependency on ADP to ATP reformation, DNP increases oxygen consumption proportionately to the degree of uncoupling. The rate of oxygen consumption remains linked however, to the flux of electrons provided by NADH and FADH.sub.2 through the electron transport chain [see FIG. 6(a)]. NADH and FADH.sub.2 utilization (re-oxidation) is concomitantly increased. Circled 7, 8, 9, and 10 effects show that oxygen use and electron transfer proceed at increasing rates to accelerate proton pumping against the added hydrogen ion load introduced by DNP. As a result, NADH and FADH.sub.2 is continually depleted by re-oxidation to NAD.sup.+ and FAD.sup.++. The high "oxidation pressure" of NAD.sup.+ and FAD.sup.++ increases substrate oxidation and flux of 2 carbon segments through the tricarboxylic acid cycle (TCA). Augmented acetyl-CoA consumption in turn is maintained by an increased rate of glycolysis by depletion of pyruvate. If oxygen delivery is inadequate, or the dose of DNP excessive, the concentration of reduced NADH increases, pyruvate oxidation through acetyl-CoA and the TCA cycle is inhibited and lactic acid will accumulate. Lactate is also overproduced when cellular hypoxia is not present per se but glycolysis exceeds pyruvate oxidation. Such intracellular lactic acidosis exists in neoplastic cells, when there is lack of insulin, when fructose is infused and in other conditions or use of drugs which augment glycolysis and/or inhibit the mitochondrial electron transport system. While it is understood that the intracellular heat generated by DNP is the algebraic sum of the enthalpy changes from all the metabolic processes within the cell, effects circled as 11, 12 and 13 depict the most significant intracellular foci of heat generated by DNP. Intracellular and total body hyperthermia results when DNP releases energy at a rate faster than it can be dissipated. Heat is generated mainly at the inner mitochondrial membrane (electron transport system), the TCA cycle and sites of cytoplasmic glycolysis. Initially DNP generates heat at the inner mitochondrial membrane by discharging a portion of the energy stored in its electrochemical gradient. Operationally, such heat is from the "chemical short circuit" created by DNP shuttling protons to the negative (matrix) side of the polarized inner mitochondrial membrane [see FIG. 6(a)]. By usurping controlled proton re-entry and energy capture as ATP from availability of ADP through ATP-synthase, DNP causes NADH and FADH.sub.2 (higher concentrations of NAD.sup.+ and FAD.sup.++) reoxidation to occur at rates much higher than necessary for oxidative phosphorylation. This causes an increased fall of electrons through the electron transport chain with rapid reduction of oxygen to water (see FIG. 3). The resultant energy is released as heat within the mitochondrial membrane. The rate of heat production from the TCA cycle is increased as it operates at a higher flux to maintain depleting amounts of reduced NADH and FADH.sub.2 used to reduce molecular oxygen. Flux of acetyl-CoA and all metabolites through the TCA cycle (see FIG. 2) is increased by activation of enzymes which sequentially degrade the hydrogen containing two carbon fragments to CO.sub.2, NADH, FADH.sub.2 and heat.

Glycolysis and its associated heat production in the cytoplasm is also increased by DNP. Glycolytic activity is increased by reduced concentration ratios of ATP to ADP, activating pyruvate dehydrogenase and phosphofructokinase respectively (see FIG. 1). These enzymes increase the rate of glucose catabolism to pyruvate and its conversion to acetyl-CoA for entry into the TCA cycle. Glycolysis is very "energy inefficient" in making up the energy equilibrium shortfall created by DNP. Uncaptured energy from the glycolytic exergonic reactions accelerated by DNP is released as heat in the cytoplasm DNP stimulated anaerobic heat production through glycolysis can oftentimes be greater than that produced by the mitochondria. By example, many tumors and normal fibroblasts treated with DNP increase heat production by 83%, with only a 36% increase in oxygen consumption. Glycolysis is known to contribute greater than 62% of the total heat produced by human lymphocytes. Circled effect 14 shows that the mitochondrial electron transport chain normally produces reactive oxygen species through the univalent reduction of oxygen [see FIG. 7, 7(a) & 7(b)]. Under physiologic conditions, 2 to 4% of mitochondrial oxygen is converted to superoxide. DNP induced partial uncoupling and mitochondrial heating increases reactive oxygen species production manifold. Cytochrome oxidase and reductase is known to be inhibited by heating of the electron transport system. As a result, heated mitochondrial membranes produce increased amount of oxygen free radicals when DNP induced uncoupling is stopped and oxygen consumption is normalized (see FIG. 9). Reactive oxygen species act in synergy with beat to alter proteins, induce membrane changes and initiate apoptosis in susceptible cells. Circled effects 15 and 16 shows the effects of DNP on intracellular calcium homeostasis. Normally calcium is stored in the mitochondrial matrix, being pumped by the energized mitochondrial membrane. By DNP directly de-energizing mitochondria, and indirectly inducing membrane heating and prooxidant stress, inner mitochondrial membrane permeability is non-specifically increased with calcium efflux and cycling. This activates intramitochondrial dehydrogenases to produce more reducing equivalents in the form of NADH and FADH2 to match increased energy demands. Heat production is increased as a byproduct from the augmented TCA cycle.

Other known uncouplers that are considered to be "classic", in the same category and act as DNP include clofazimine, albendazole, cambendazole, oxibendazole, triclabendazole (TCZ), 6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole and their sulfoxide and sulfone metabolites, thiobendazole, rafoxanide, bithionol, niclosamide, eutypine, various lichen acids (hydroxybenzoic acids) such as (+)usnic acid, vulpinic acid and atranorin, 2',5-dichloro-3-t-butyl-4'-nitrosalicylanilide (S-13), 3,4',5-trichlorosalicylanilide (DCC), platanetin, 2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799, AU-1421, 3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradec- in-1,7(8H)-dione (zearalenone), N,N.sup.1-bis-(4-trifluoromethylphenyl)-urea, resorcylic acid lactones and their derivatives, 3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847), 2,2-bis (hexafluoroacetonyl)acetone, triphenyl boron, carbonylcyanide 4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA), carbonyl cyanide 3-chlorophenylhydrazone (CICCP), 1,3,6,8-tetranitrocarbazole, tetrachlorobenzotriazole, 4-iso-octyl-2,6-dinitrophenol (Octyl-DNP), 4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxal bisguanylhydrazone), pentachlorophenol (PCP), 5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI), N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid), 4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol, 2-azido-4-nitrophenol, 5-nitrobenzotriazole, 5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole, methyl-o-phenylhydrazone, N-phenylanthranilic acid, N-(3-nitrophenyl)anthranilic acid, N-(2,3-dimethylphenyl)anthranilic acid, mefenamic acid, diflunisal, flufenamix acid, N-(3-chlorophenyl)anthranilic acid, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), SR-4233 (Tirapazamine), atovaquone, carbonyl cyanide 4-(6'-methyl-2'-benzothiazyl)-phenylhydrazone (BT-CCP), ellipticine, olivacine, ellipticinium, isoellipticine and related isomers, methyl-0-phenylhydrazonocyanoacetic acid, methyl-0-(3-chlorophenylhydrazono) cyanoacetic acid, 2-(3'-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic acid, 2-(2',4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile, relanium, melipramine, and other diverse chemical entities including unsaturated fatty acids (up to C.sub.14 optimum), sulflaramid and its metabolite perfluorooctane sulfonamide (DESFA), perfluorooctanoate, clofibrate, Wy-14, 643, ciprofibrate, and fluoroalcohols. Additional unnamed classic uncouplers can include any analog which generally has a weakly acidic, removable proton and an electron withdrawing, lipophilic molecular body that is capable of charge delocalization. Hydrophobicity and capacity to exchange proton equivalents are integral features of classic DNP types of uncouplers.

A second class of uncouplers are ionophorous antibiotics. These molecules uncouple oxidative phosphorylation by inducing cation or anion influx across the mitochondrial membranes and diffusing back in a protonated form. As a result, chemical futile cycling ensues to reestablish the initial membrane potential. Liberated energy is dissipated as heat. Examples of ionophores that shuttle potassium ions (K.sup.+) across membranes includes the antibiotics gramicidin, nigericin, tyrothricin, tyrocidin, and valinomycin. Nystatin shuttle sodium ions. The calcium ionophore, compound A23187, is a lipid soluble ionophore which mediates the electroneutral exchange of divalent cations for protons. Alamethicins, harzianin HA V, saturnisporin SA IV, zervamicins, magainin, cecropins, melittin, hypelcins, suzukacillins, monensins, trichotoxins, antiamoebins, crystal violet, cyanine dyes, cadmium ion, trichosporin-B and their derivatives are examples of uncoupling ionophores that depend on shuttling inorganic phosphate (P0.sub.4.sup.=) across the mitochondrial membrane.

A third class of uncouplers is a group of heterogeneous compounds that dissipate the proton gradient by attaching or interacting with specific proteins in the inner mitchondrial membrane. Examples of such compounds include desaspidin, ionized calcium (Ca.sup.++), uncoupling proteins such as UCPI-1, UCP-2, UCP-3, PUMP (Plant Uncoupling Mitochondrial Protein) histones, polylysines, and A206668-a protein antibiotic that ties up phosphoryl-transfer proteins. Examples and a potency comparison of a few uncouplers are depicted in FIG. 11.

Various conjugates, adducts, analogs and derivatives of the above mentioned agents can be formulated and synthesized to enhance intracellular uncoupling and heat production. Further, various covalent compounds of uncouplers may be synthesized as prodrugs, which upon, redox or reaction with free radicals within the cell will become activated to induce uncoupling, heat production and free radical cycling. Such derivatives and formulations may be desirable in the treatment of many tumors with higher mitochondrial membrane potentials and increased total bioreductive capacity. Uncoupling-free radical prodrug compounds may thus exert greater selective killing of transformed cells by undergoing a higher flux of reduction or electron acceptance in tumor cells. In this regard, the contents of U.S. Pat. No. 5,428,163 and the published methods of C-Alkylation of phenols and their derivatives by Hudgens, T. L. and Tumbull, K. D. are hereby incorporated by reference

From a physico-chemical and thermodynamic standpoint, the amount of heat produced by uncoupling is proportional to the density and rate of flux of electrons through the mitochondrial electron transport chains. Such electron flux is initially reflected by the magnitude of the electrochemical proton gradient across the inner mitochondrial membrane. Those cells, tissues, organs and organisms that are metabolically more active will generally have an increased membrane potential and will respond with a greater amount of heat production for a given dose and type of uncoupler. FIG. 12 lists the six most "hottest" organs in the human body along with their rates of blood flow and rates of heat production. The actual amount of intracellular hyperthermia produced by an uncoupler is dependent on the uncoupler dose, its relative potency and availability of substrate such as glucose, glutamine, fatty acids or other substances that produce NADH or FADH.sub.2. Oxygen and magnitude of the mitochondrial proton electrochemical gradient (.DELTA..mu.H.sup.+) are additional factors that determine the amount of heat that can potentially be released by an uncoupler. Among all the constituents, .DELTA..mu.H.sup.+ is the most clinically important. .DELTA..mu.H.sup.+ is composed of the transmitochondrial membrane potential [.DELTA..PSI. (charge difference)] and pH gradient [.DELTA. pH (H.sup.+ concentration difference)], .DELTA..mu.H.sup.+=F.DELTA..PSI.-2.3RT.DELTA.pH, where, F=Faraday Constant, R=Gas Constant, and T=degrees Kelvin. Thus, .DELTA..mu.H.sup.+ represents the potential amount of heat that can be liberated by an uncoupler when 1 mole of H.sup.+ is dissipated through the inner mitochondrial membrane. This potential heat energy is normally expressed in units of millivolts (mV) and is called the protonmotive force, .DELTA.p=.DELTA..mu.H.sup.+/F=.DELTA..PSI.-2.3(RT/F).DELTA.pH. In vivo, .DELTA.pH is generally 1 unit or less so that 75% or more of the total .DELTA.p is comprised of .DELTA..PSI.. Consequently, the intracellular heat produced by an uncoupler can be estimated by the mitochondrial membrane potential (.DELTA..PSI.) alone.

Knowing the .DELTA..PSI. is of practical importance because biopsy specimens may be incubated with cationic organic probes to estimate the .DELTA..PSI. and the degree of differential heating that will occur between normal and transformed tissues. Dyes such as rhodamine 123, mitotracker green, calcein plus Co.sup.++, 3,3.sup.1-dihexyloxacarbocyanine, triphenylmethylphosphonium, JC-1,5,5.sup.1,6,6.sup.1-tetrachloro-1,1.sup.1,3,3.sup.1-tetraethylbenzim- idazolocarbocyanine, etc., all have an affinity for a negative mitochondrial .DELTA..PSI.. Based on the amount of cationic dye uptake, the membrane potential of specific tissue, tumors, and cells may be determined through the Nernst equation: .DELTA..PSI.=-(RT/F) ln(C.sub.in/C.sub.out). Which at physiologic conditions and 37.degree. C. is =-61 log(C.sub.in/C.sub.out), where C.sub.in/out is the concentration of the probe inside or outside the mitochondria and plasma membrane. By example, a 10 to 1 gradient=-60 mV, 100 to 1=-120 mV. Uncouplers dissipate the .DELTA..PSI., generate heat and release or prevent uptake of cationic dyes. Six years of systematic measurement of mitochondrial membrane potentials have been performed on human and mammalian cells, including some 200 cell types derived from human malignant tumors of kidney, ovary, pancreas, lung, adrenal cortex, skin, breast, prostate, cervix, vulva, colon, liver, testis, esophagus, trachea and tongue. Based on this exhaustive study, a .DELTA..PSI. difference of at least 60 mV is known to exist between normal epithelial cells and carcinoma cells. This is significant for the present invention in that uncoupling or "short circuiting" a 60 mV potential across a 5-nm mitochondrial membrane would be equivalent to the amount of heat generated by short circuiting 120,000 V across 1 centimeter. By exploiting or increasing the membrane potential between normal and transformed cells the rate of intracellular heat production by an uncoupler can be selectively increased in target tissues.

In order for uncoupler induced intracellular hyperthermia to be of therapeutic benefit, the development of thermotolerance is also taken into account in practicing this invention. Mammalian cells and prokaryotes acclimate and acquire transient resistance or thermotolerance to gradual or non-lethal hyperthermia. Such adaptation is believed to occur through increased synthesis of highly conserved groups of proteins known as heat shock proteins (HSP). The amount of HSP present in tissues, cells and organisms subjected to non-lethal heat, or other forms of prolonged metabolic stress, is proportional to their survival at higher temperatures. In general, thermotolerance develops after 3 to 4 hours of continuous hyperthermia, peaks in 1 to 2 days and decays back to normal thermosensitivity within 3 to 4 days. Thermotolerance is known to alter lethality of hyperthermia by as much as 2.degree. C. increase or double the heating time required to achieve the same temperature-cytotoxic effect. Such adaptive thermoresistance by human tumors is problematic for continuous or fractionated cytotoxic treatment with hyperthermia. Induction heating times with the present invention are therefore kept to a minimum of 1 to 2 hours. Further, the uncoupler induced cytotoxic hyperthermia in the present invention induces relative tissue hypoxia, lowers intracellular pH and limits the production of ATP, all of which repress the development of thermotolerance. Low doses of uncoupler, which produce gradual heating can be used to induce HSP synthesis and promote thermotolerance.

Determining the amount of DNP in mg/kg of body weight required to produce the desired level of cytotoxic hyperthermia in a safe and efficacious manner is established from the thermal equivalents (Kcal) of oxygen consumed (V0.sub.2), and the known average specific heat capacity of the human body. It is known that at standard temperature and barometric pressure, 1 liter of oxygen consumed per minute (VO.sub.2) generates approximately 4.862 Kcal. It is also known that the average specific heat capacity of humans is about 0.83 of that required to raise 1 gm of H.sub.20 1.degree. K4.184 J, a heat capacity of 3.47 J g K.sup.-1. An initial estimate of the total energy required to be generated by DNP to induce 41.0.degree. C. hyperthermia in 1 hour may be very simply determined from the above and customized for a specific patient as outlined below:

Patient Characteristics

Body weight 70 kg

Resting V0.sub.2 0.25 L/min

Basal energy expenditure 73.1 Kcal/hr (1754.4 Kcal/24 hrs.)

Basal core temperature 37.0.degree. C.

Target temperature 41.0.degree. C.

Required Energy to Raise Temperature to Target Level in 1 Hour

(Weight in grams=70.times.10.sup.3) (human specific heat=3.47 J g K.sup.-1) (Temperature increase=41.0.degree.-37.0.degree. C.).about.0.97.times.10.sup.6 J. Since 1 J=4.184.times.10.sup.-4 Kcal, a total power input of about 232 Kcal would be required to raise the temperature of the patient to the objective level in 1 hour less that amount of heat generated by a heated metabolism outlined below.

Increase in Metabolic Rate/Heat Production with Increase in Body Temperature

The basal metabolic rate (BMR) is known to increase in patients with endogenous fevers by approximately 7% for each 0.5.degree. C. rise in temperature. This is graphically depicted in FIG. 11a. As a result, the increase in BMR relative to the temperature will in itself assist in achieving the objective level during the induction phase by the following equation: BMR.sub.Tcore=73.1.times.1.07.sup.(Tcore-37)/0.5

Thus, at 41.0.degree. C. the metabolic rate will be 134.4 Kcal/hr, 61.3 Kcal/hr above the basal energy expenditure level. This increase in metabolic rate will therefore reduce the initial energy required to heat the patient by approximately 61 Kcal over the 1 hour timeframe.

Initial Net Energy Input Required to Reach Target Temperature in 1 Hour

232 Kcal-61 Kcal (by increased BMR)=171 Kcal

Required Increase in Initial V0.sub.2 to Obtain 171 Kcal Heat Input

Since the Kcal equivalent for 1 liter of oxygen consumed per minute is 4.862, then the initial increase in VO.sub.2 required to generate 171 Kcal can be calculated as follows: Heat in Kcal/min=V0.sub.2.times.4.862. Since the individual patient has a resting V0.sub.2 of 0.25 l/min which =73.1 Kcal/hour BMR, then X(V0.sub.2)=171 Kcal, or X=0.25.times.171/73.1 An initial minimal increase in V0.sub.2 to approximately 0.60 l/min is required. DNP Dosage Required to Increase V0.sub.2 to 0.60 l/min

The individual DNP dosage (mg/kg) required to produce an increase in oxygen consumption to 0.60 l/min so as to achieve a 171 K/cal heat output is accomplished in the following fashion: (1) DNP is prepared in a 200 mg/100 ml sterile aqueous solution. If not fully dissolved, it can be brought into solution by buffering with 1% NaHCO.sub.3, the pH must be kept below 8 to avoid hydrolysis; (2) the dose of DNP for each intravenous infusion can vary from 0.5 to 4 mg/kg and will depend on the clinical situation, as well as the initial and subsequent increases in the metabolic rate (V0.sub.2). In an especially preferred embodiment, the patient is given an initial dose of DNP no greater than 1 mg/kg intravenously, infused over no less than a 2 minute period. Within approximately 10-15 minutes, a minimum of a 15% increase in V0.sub.2 will occur. The V0.sub.2 will continue to increase until a plateau is reached within an additional 5 to 10 minutes. After a 5 minute plateau in V0.sub.2, a subsequent dose of either 0.5, 1, 2, 2.5, or 3.0 mg/kg DNP is administered and V0.sub.2 is again increased until a desired plateau is reached. Additional infusions of DNP or other medications are administered under clinical parameters of V0.sub.2, respiratory rate, pulse rate, blood pressure, urine output, cardiac output, core temperature, and clinical status of the patient so as to maintain safe and effective control of heating. If heat dissipating mechanisms are neutralized, measurable increases in core temperature will occur approximately 20 to 30 minutes after an increase in the V0.sub.2. FIG. 13 illustrates the increases in V0.sub.2 associated with repeated infusions of DNP.

Medications which increase the overall metabolic rate, or that of specific target tissues, and have short half-lifes can be utilized to increase the relative activity of DNP or other uncouplers to further adjust V0.sub.2 and heat production. Examples of such medications are almost limitless because any drug, hormone or biologic response modifier that causes changes in enthalpy (heat content) during the course of its intracellular chemical and biophysical activity and interaction in the life cycle of biological cells can be utilized. A few illustrative examples include glucagon (half-life of 9 minutes in plasma), arbutamine (half-life 10 minutes), dobutamine (half-life 2 minutes), and vasopressin (half-life 5 minutes). Various amino acids and fatty acids, e.g., glutamine, proline, octanoate, etc., increase V0.sub.2 by translocating reducing equivalents into the mitochondrial matrix via the malate-aspartate shuttle, B-oxidation or proline metabolism. Agents such as methylene blue (tetramethylthionine), ubiquinone, menadione, hematoporphyrin, phenazine methosulfate, 2,6-dichlorophenolindophenol, coenzyme Q1, CoQ2, or their analogs duroquinone and decylubiquinone, etc., can increase heat and/or free radical production by acting as artificial electron acceptors. Such agents, and numerous others, can be co-administered with DNP or other uncouplers to effectively increase the enthalpy changes in the entire organism or specific targeted tissues.

Minimizing Heat Loss and Temperature Control

Increased radiative and evaporative heat loss from man are the two most dominant thermoregulatory mechanisms for cooling the body. The body's methods of adjusting heat loss are vasoconstriction and vasodilation in the skins blood vessels. Radiation can account for 60% of the heat loss generated by the body, while evaporation by sweating at 1.0 liter/hour can represent a potential heat loss of about 1,000 Kcal/hour. By far, sweating and evaporation is the principal mechanism that dissipates heat under conditions that induce large heat gains. Depending on the clinical circumstances, heat loss due to evaporation, as well as radiation, can be managed and controlled by a variety of methods including, but not limited to, using vasoconstricting agents, placing the patient in a scuba diving wet suit, humidified survival suit, or enveloping the patient in a water soaked blanket covered or containing a polyethylene lining to prevent evaporative heat losses. Use of room ultrasonic nebulizers to induce continuous mist and high humidity is also known to prevent evaporative heat losses. Evaporative and radiant heat loss from the cranium is controlled by appropriate head gear, shower caps and/or wet towels. Control of local air velocities and management of surroundings as to temperature, emissivity, drafts, and convection currents are important to avoid large heat losses. In those clinical circumstances where total body hyperthermia is required, failure to adequately control body heat loss will necessitate using higher doses of DNP and induce a greater metabolic stress upon the patient.

If the core target temperature is exceeded or continues to rise after the target temperature is achieved, exposure of an extremity or body surface for a brief interval will permit sufficient heat loss to lower the core temperature to the target range. At target temperatures of 39-41.degree. C., residual uncoupling by DNP will continue for approximately 3 hours. Heat production as a byproduct of glycolysis, and heated metabolism further maintains body heat content and compensates for any heat loss. Therefore, target plateau temperatures can be regulated with a large margin of safety and with little to no additional use of uncoupler. Therapy is terminated by removing the vapor barrier from the patient. Evaporative and radiant heat loss from the patient generally produces a fall in core temperature of about 2-2.5.degree. C. in about 20-30 minutes. Obese patients and those with compromised thermoregulatory systems experience a slower falloff in temperatures.

Patient Monitoring, Fluid Support and Evaluation During Treatment

Placement of physiologic monitoring sensors, intravenous fluids, supplemental oxygen (4 l/min) and optional oral diazepam sedation (5-10 mg) is initiated prior to treatment. Patients receive 0.85 to 1.0 liter of intravenous (IV) 5% dextrose in 0.25 normal saline per hour alternated with 5% dextrose in 0.5 normal saline plus 7.5 to 10 meq of KCl per liter to insure a urinary output of no less than 1 ml/kg/hr. Oxygen consumption, caloric expenditure, rectal core temperature, cardiac rhythm, blood pressure, heart rate and respiratory rate are continuously displayed, monitored by a trained member of the treatment staff. The data is automatically downloaded into a computer every 20 seconds to 3 minutes for the entire procedure and immediately re-displayed on computerized graphs and charts. Two hours after treatment and 48 hours post-treatment, serum chemistries and hematologic profiles are repeated. A typical patient flow chart is depicted in FIG. 14.

Treatment of Excessive Heating and Antidotes

In those rare instances when too much uncoupler is administered or the metabolic rate of the patient unexpectedly increases and V0.sub.2, hyperthermia, pulse rate and patient fatigue ensue, appropriate supportive measures of cooling, intravenous hydration and administration of specific medication should be instituted. Cooling should be instituted by uncovering the patient, spraying with tepid water and fanning with an industrial grade fan. If cooling is inadequate, surface, axillary and groin ice packs and intravenous cold glucose solutions should immediately be considered. Bicarbonate, 1-2 mEq/kg should be administered in the absence of blood gas analysis. Urine output of >1 ml/kg/hour should always be maintained to avoid pre-renal azotemia and oliguria secondary to possible rhabdomyolysis and myoglobinuria. Mannitol should be administered if urine output is inadequate. Hypoglycemia should immediately be corrected with 50% saturated intravenous glucose. If severe or persistent hypermetabolism ensues, rectal propylthiouracil-1,000 mg, hydrocortisone (100 mg q 6 h) or dexamethasone 2 mg q 6 h intravenously and/or sodium iodide as 1 g sodium ipodate (contrast agent) should be administered intravenously to induce iatrogenic hypothyroidism. The decreased metabolic rate will dramatically reduce the physiologic response to DNP. Patient agitation and restlessness can be avoided by appropriate IV or IM dose of diazepam. Salicylates are of no value and may contribute to further uncoupling. Medications that reduce sweating, e.g., tricyclic antidepressants, antihistamines, anticholinergics, phenothiazines, or decrease vasodilation, e.g., sympathomimetics, .alpha.-agonists, or decrease cardiac output, e.g., diuretics, beta-blockers or induce hypothalamic depression, e.g., neuroleptics, .alpha.-blockers, opioids, etc., should be avoided prior, during and immediately after treatment with uncouplers.

The hypermetabolic and hyperthermic activity of DNP can further specifically be reduced by using calcium channel blockers such as nifedipine, verapamil and others, in intravenous doses that do not cause a drop in blood pressure or induce cardiac arrhythmias. Dihydrobenzperidol (a neuroleptic drug with .alpha..sub.1-adrenergic properties) can also be used to cause similar, significant reductions in DNP induced hypermetabolism and hyperthermia. Dosages of these anti-DNP agents are titrated in 5 mg to 30 mg increments and can be given either by mouth or intravenously. In those cases where DNP appears to decrease electrical conduction or cause EKG conduction abnormalities, Coenzyme Q10, in doses of 50 mg/kg, can be used to restore normal electrical activity.

Patient Selection and Pretreatment Evaluation

It is imperative that in the practice of this invention, patients be selected and evaluated prior to treatment. Recommended patient inclusion and exclusion criteria includes: (1) patients have a definitive histopathologic or other laboratory confirmed diagnosis of their disease; (2) the disease or condition should be responsive to intracellular hyperthermia treatment; (3) patients should have a Karnofsky score of 70% or greater; (4) not be pregnant; (5) weight should be within 45% (+/-) of ideal body weight and patients must weigh at least 35 kg; (6) there should be no history or findings of anhidrosis, scleroderma, ectodermal dysplasia, Riley-Day Syndrome, arthrogryposis multiplex, extensive psoriasis, serious dysrhythmias, malignant hyperthermia or neuroleptic malignant syndrome, pheochromocytoma, hypocalcemia, repeated episodes of hypoglycemia, chronic or recurrent venous thrombosis, alcoholism, renal failure, cirrhosis, untreated hyperthyroidism, anaphylaxis associated with heat or exercise-induced cholinergic type urticaria, exercise or heat induced angioedema, schizophrenia, catatonia, seizure disorders, emotional instability, Parkinson's disease, brain irradiation, cystic fibrosis, unstable angina pectoris, congestive heart failure, patients with cardiac pacemakers, severe cerebrovascular disease, spinal cord injury, severe pulmonary impairment, hereditary muscle disease such as Duchenne type muscular disease, central core disease of muscle, myotonia congenita, King-Denborough syndrome, Scwanry-Jampol syndrome, or osteogenesis imperfecta; (6) no immediate use of drugs that impair the body's heat dissipation mechanisms such as phenothiazines, anticholinergics, antihistamines, antiparkinsonians, glutethimide, hallucinogens, lithium, cocaine or other illicit drug use, monamine oxidase inhibitors, sympathomimetics, phencyclidine, opioids, phenylephrine, INH, tricyclic antidepressants, withdrawal from dopamine agonists, or cardiovascular drugs that clinically impair cardiac output or thermoregulatory vasodilation such as high doses of .beta.-blockers, vasodilators, or calcium channel blockers; and, (7) the patient should not be anemic or otherwise have a reduced oxygen absorbing, carrying or utilizing capacity.

Pretreatment evaluation should include a complete medical history and physical examination focused on the selection criteria listed above. Laboratory evaluation should include pulmonary function tests-if indicated, full hematological survey with hemostatic profile, EKG, liver function tests, serum biochemical profile, thyroid panel, serum creatinine, calcium, phosphate, and stress-EKG or exercise-multigated radionucleotide ejection scan on patients whose cardiac ejection fraction is suspect not to be greater than 45% with probable deterioration on exercise. While clinical exceptions to entry laboratory values may exist, the following laboratory data should be a benchmark guide for initiation of treatment: hemoglobin>=11.0 g/dl for men and >=10.0 g/dl for women, platelet count>=75.00 platelets/mm.sup.3, bilirubin<=2.times.ULN (ULN=upper limit of normal), ALT (SGPT)<=2.times.ULN, AST (SGOT)<=2.times.ULN, pancreatic amylase<1.5.times.ULN, neutrophil count>=1,000 cells/mm.sup.3. Serum electrolytes and K.sup.+ should be well within normal limits, as hypokalemia decreases muscle blood flow, cardiovascular performance, and sweat gland function.

More generally, the method outlined above is to be tailored to an individual patient. As set forth above, the DNP may be administered by intravenous infusion. Alternatively, the route of administration may also be orally, rectally or topically. The frequency and optimal time interval between administrations is individualized and determined by measuring V0.sub.2, as well as other parameters. For example, various laboratory, x-ray, CAT scan, MRI, PET scan, HIV load, CD4+ lymphocyte counts, HSP expression, prostatic specific antigen (PSA) and other surrogate markers of clinical outcome can establish the VO.sub.2, frequency and duration of therapy. One treatment, or treatments as frequent as every day, or every other day, as far apart as 1 year or longer may be required for sustained beneficial results.

The optimal VO.sub.2, temperature, duration, and frequency between treatments will probably vary from patient to patient and the specific disease or condition being treated. One skilled in the art would be able to modify a protocol within the present invention, in accordance with standard clinical practice, to obtain optimal results. For example, the HIV relationships between viral load, CD4.sup.+ lymphocyte counts, presence of opportunistic infections and clinical status of the patient can be used to develop more optimal regimes of DNP administration. Applicants' studies have revealed that the methods of the present invention can be effective in the diagnosis and treatment of a wide range of disease states and conditions in which uncoupler induced hypermetabolism, hyperthermia, oxidative stress and their sequela, play a beneficial role. To those skilled in the art, it is also encompassed that a variety of different veterinary, as well as medical, applications for treatment and diagnosis can be practiced with the present invention.

It is envisioned that DNP, or other uncouplers, may also be administered with other compounds used to treat infectious, malignant or other diseases. Examples of other agents include antifungal, antibacterial, antiviral or anti-neoplastic drugs, cell differentiating agents, and, various biologic response modifiers. Examples of anti-fungal agents include Amphotericin B, Griseofulvin, Fluconazole (Diflucan), Intraconazole, 5 fluoro-cytosine (Flutocytosine, 5-FC), Ketatoconazole and Miconazole. Examples of anti-bacterial agents include antibiotics, such as those represented from the following classifications: beta lactam rings (penicillins), macrocyclic lactone rings (macrolides), polycyclic derivatives of naphthacenecarboxamide (tetracyclines), amino sugars in glycosidic linkages (aminoglycosides), peptides (bacitracin, gramicedin, polymixins, etc.), nitrobenzene derivatives of dichloroacedic acid, large ring compounds with conjugated double bond systems (polyenes), various sulfa drugs including those derived from sulfanilamide (sulfonamides, 5-nitro-2-furanyl compounds (nitrofurans), quinolone carboxylic acids (nalidixic acid), fluorinated quinilones (ciprofloxan, enoxacin, ofloxacin, etc.), nitroimidazoles (metroindazole) and numerous others. These antibiotic groups are examples of preferred antibiotics, and examples within such groups include: peptide antibiotics, such as bacitracin, bleomycin, cactinomycin, capreomycin, colistin, dactinomycin, gramicidin A, enduracitin, amphomycin, gramicidin J, mikamycins, polymyxins, stendomycin, actinomycin; aminoglycosides represented by streptomycin, neomycin, paromycin, gentamycin ribostamycin, tobramycin, amikacin; lividomycin beta lactams represented by benzylpenicillin, methicillin, oxacillin, hetacillin, piperacillin, amoxicillin and carbenacillin; lincosaminides represented by clindamycin, lincomycin, celesticetin, desalicetin; chloramphenicol; macrolides represented by erythromycins, lankamycin, leucomycin, picromycin; nucleosides such as 5-azacytidine, puromycin, septacidin and amicetin; phenazines represented by myxin, lomofungin, iodin; oligosaccharides represented by curamycin and everninomycin; sulfonamides represented by sulfathiazole, sulfadiazine, sulfanilimide, sulfapyrazine; polyenes represented by amphotericins, candicidin and nystatin; polyethers; tetracyclines represented by doxycyclines, minocyclines, methacylcines, chlortetracyclines, oxytetracylcines, demeclocylcines; nitrofurans represented by nitrofurazone, furazolidone, nitrofurantoin, furium, nitrovin and nifuroxime; quinolone carboxylic acids represented by nalidixic acid, piromidic acid, pipemidic acid and oxolinic acid. The Encyclopedia of Chemical Technology, 3rd Edition, Kirk-Othmer, editors, Volume 2 (1978), which is hereby incorporated by reference in its entirety.

Antiviral agents that can be used with DNP include: interferons .alpha., .beta. and .gamma., amantadine, rimantadine, arildone, ribaviran, acyclovir, abacavir, vidarabine (ARA-A) 9-1,3-dihydroxy-2-propoxy methylguanine (DHPG), ganciclovir, enviroxime, foscarnet, ampligen, podophyllotoxin, 2,3-dideoxytidine (ddC), iododeoxyuridine (IDU), trifluorothymidine (TFT), dideoxyinosine (ddi), d4T, 3TC, zidovudine, efavirenz, protease inhibitors such as indinavir, saquinavir, ritonavir, nelfinavir, amprenavir, etc., and specific antiviral antibodies.

Anti-cancer drugs that can be used with DNP include, but are not limited to, various cell cycle-specific agents represented by structural analogs or antimetabolites of methotrexate, mercaptopurine, fluorouracil, cytarabine, thioguanine, azacitidine; bleomycin peptide antibiotics, such as podophyllin alkaloids including etoposide (VP-16) and teniposide (VM-26); and various plant alkaloids such as vincristine, vinblastine, and paclitaxel. Anti-neoplastic cell cycle-nonspecific agents such as various alkylating compounds such as busulfan, cyclophosphamide, mechlorethamine, melphalan, altretamine, ifosfamide, cisplatin, dacarbazine, procarbazine, lomustine, carmustine, lomustine, semustine, chlorambucil, thiotepa and carboplatin. Anticancer antibiotics and various natural products and miscellaneous agents that can be used with DNP include: dactinomycin, daunorubicin, doxorubicin, plicamycin, mitomycin, idarubicin, amsacrine, asparaginase, quinacrine, retinoic acid derivatives (etretinate), phenylacetate, suramin, taxotere, tenizolamide, gencytabine, amonafide, streptozocin, mitoxanthrone, mitotane, fludarabine, cytarabine, cladribine, paclitaxel (taxol), tamoxifen, and hydroxyurea, etc.

DNP can also be administered with various hormones, hormone agonists and biologic response modifying agents which include, but are not limited to: flutamide, prednisone, ethinyl estradiol, diethylstilbestrol, hydroxyprogesterone caproate, medroxyprogesterone, megestrolacetate, testosterone, fluoxymesterone and thyroid hormones such as di-, tri- and tetraiodothyroidine. The aromatase inhibitor, amino glutethimide, the peptide hormone inhibitor octreotide and gonadotropin-releasing hormone agonists such as goserilin acetate and leuprolide can also be used with DNP. Biologic response modifiers such as various cytokines, interferon alpha-2a, interferon alpha-2b, interferon-gamma, interferon-beta, interleukin-1, interleukin-2, interleukin-4, interleukin-10, monoclonal antibodies (anti-HER-2/neu humanized antibody), tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, macrophage-colony-stimulating factor, various prostaglandins, phenylacetates, retinoic acids, leukotrines, thromboxanes and other fatty acid derivatives can also be used with DNP.

The use of this invention should be under the strict direction of a qualified and specialized treatment team to insure safety and effectiveness. The treatment team remains with the patient throughout the procedure to insure that safe and controlled dosages of an uncoupler are administered by monitoring real time changes in V0.sub.2, metabolic rate, temperature, respiratory rate, heart rate, urine output and clinical status of the patient. This invention is practiced in controlled steps so as to attain a predetermined V0.sub.2 and plateau of heating time for a particular disease or condition. For example, in cases were heat dissipation mechanisms do not have to be blocked, the specialized team will periodically recheck V0.sub.2, heart rate, blood pressure, CAT scan, MRI, etc., and other laboratory and clinical parameters to insure continued safety and efficacy of DNP therapy. It is preferred that the specialized team undergo a training period in the use of this invention prior its administration to human patients.
 

Claim 1 of 9 Claims

1. A method for inducing intracellular hyperthermia in a subject comprising the step of administering to a subject having an infection of Borrelia burgdorferi, Mycobacterium leprae, Treponema pallidum, HIV, hepatitis C, or herpes virus, an amount of 2,4-dinitrophenol sufficient to induce whole body intracellular hyperthermia in the subject, wherein the whole body intracellular hyperthermia is sufficient to treat the Borrelia burgdorferi, Mycobacterium leprae, Treponema pallidum, HIV, hepatitis C, or herpes virus infection in the subject.

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