CN102056605A - Pharmaceutical composition - Google Patents

Abstract

A pharmaceutically-acceptable modulator of the regulation or perturbation of the structure, expression, and/or activity of at least one enzyme and/or enzyme complex, or subunit thereof, such as via the altered mitochondrial energy metabolism of the pyruvate dehydrogenase (PDH) complex of warm-blooded animals, including humans, and methods of use thereof, comprises an effective amount of at least one lipoic acid derivative and at least one pharmaceutically-acceptable carrier thereof to affect the complex's phosphorylation state. By increasing PDH kinase activity and/or decreasing PDH phosphatase activity, the modulator prevents the detoxification anaerobic glycolytic toxic metabolites through inhibition of the activity of the PDH complex's El a subunit, obliging increased mitochondrial oxidative phosphorylation activity. As cells characterized by hyperproliferation, such as tumor cells, cannot also generate acetyl-CoA and NADH because of the modulator's additional action in inhibiting the action of the PDH complex's E2 subunit, the mitochondrial membrane polarization is lost, facilitating cell death.

Description

Pharmaceutical composition

Invention field

The present invention relates to treatment and diagnosis composition, relate more particularly to pharmaceutical composition, and using method, it shows as by selectivity and absorbs in the cell (comprising cancerous cell) that is characterized as hyper-proliferative, and the structure of its regulatory enzyme, expression and/or active adjustment or interference, thereby be convenient to detection and treatment or destroy these cells.More particularly, these reagent targeting and activity or its adjustment of disturbing the mitochondrion energy metabolism of observed change in such as the site of pyruvic dehydrogenase (PDH) complex of the modification relevant with most of cancers.

Background of invention

Mitochondrion be Eukaryotic energy generate with cells survival-and-the major control center of death process.Though exist and the alternative various mechanism of other parts of cell, is but reversible phosphorylation to regulate important way (the Pagliarini D.J. and Dixon J.E. (2006) the .Mitochondrial modulation:reversible phosphorylation takes center stage of mitochondrial function? TRENDS in Biochem.Sci.31:26-34, passim is incorporated herein by reference) quantity of mitochondrion kinases, phosphatase and phosphoprotein of report stably increases and shows that phosphorylation occurs as the common theme in regulating the mitochondrion process probably.Regulate relevant pathological change or heredity and change and help to treat disease and may be the important target spot for the treatment of disease with active with the structure of cyclophorase, function.

Consistent with its effect as the regulator of centrostigma (point of convergence) and various kinds of cell function, mitochondrion is in the generation of apoptosis, reactive oxygen species (ROS) and comprise surpassing in many metabolic processes that 90% cell ATP generates to have decisive role.And, when cell growth and division, must produce new mitochondrion, this process itself needs to coordinate modestly transcribing and translating of nuclear DNA and mitochondrial DNA.At last, when the energy demand of cell changed, mitochondrion must be by adjusting its ATP output rapid answer.Therefore, mitochondrion need with the alternative complication system of cell function.As obviously as can be known, travel to and fro between mitochondrial signaling molecule and comprise ion, gas, metabolite, hormone, transcription factor and albumen from Fig. 1.Therefore, the mitochondrial identification as the center of reception, integration and transfer sell signal is the design of medicine and the impressive progress (advance) in the test.

In mitochondrion was adjusted, the basis of signal conduction was reversible phosphorylation.Yet, though confirmed the protein kinase incident first at report in 1954, and almost found to adjust by reversible phosphorylation the fact of core-wire plastochondria (core mitochondrial) function before 40 years, but the report of mitochondrion phosphorylation event seldom.Really so, although just discerned the cascade of a large amount of signal transduction phosphorylation in the eighties and the nineties, it crosses plasma membrane and passes cytosol and arrives nucleus.The shortage of this respect knowledge may partly be because the following fact: as if after most of mitochondrial protein machines were present in two double-layers of lipoid, it placed mitochondrial protein and has exceeded the zone that cytosolic signal cascade reaches.What under any circumstance, do not accept extensively is that mitochondrion is to control crucial mode by reversible phosphorylation conditioning signal for disease.Yet, as shown in table 1, by 2006, all surpass 60 protein in the mitochondrion compartments (be substrate, inner membrance, intermembrane space and adventitia, comprise the outer surface of kytoplasm-face) and be accredited as the phosphoprotein that participates in the wide spectrum mitochondrial function.Fixed data (Mounting data) has further confirmed the purposes of the composition for improved treatment of cancer of the importance of reversible phosphorylation of mitochondrion target spot and targeting mitochondrion target spot.

Table 1. mitochondrion phosphoprotein

Abbreviation: AA, aminoacid (S, serine; T, threonine; Y, tyrosine); ANT, adenine nucleotide transfer protein; Axmito, the mitochondrion annexin; β-OX, beta oxidation; BCKAD, the branched-chain keto acids dehydrogenase; CI-CV. respiratory chain complex 1-5; CPT. Carnitine palmitoyltransferase; CREB.cAMP-response element (Cre)-conjugated protein; CYP, Cytochrome P450; DBP. 12 polymer are conjugated protein; EF. elongation factor; GST. glutathione S-transferase; HSP. heatshock protein; IM., inner membrance; IMS. intermembrane space; M. substrate; MnSOD, the manganese dioxide dismutase; MTERF, the mitochondrion transcription termination factor; MtGAT. mitochondrion glycerol-3-phosphate enzyme Acetylase; Mthsp. Mitochondrial H SP; MtTBP. mitochondrion telomer β-conjugated protein; NDK. nucloside-diphosphate kinase; OM. adventitia; OXPHOS. oxidative phosphorylation; The P site; Phosphorylation site; POC E1/3, pyruvate dehydrogenase complex E1/3 subunit; PDK, pyruvic dehydrogenase kinase; PLA, phospholipase A; ScIR P.e-immune-active peptides subunit, SSAT. spermidine/spermine Acetylase; StAR, steroid give birth to the sensitive albumen of regulating of hormone; TCA., tricarboxylic acid cycle; TRAP-1, tumor necrosis factor 1 receptor associated protein; VQAC., voltage dependence anion channel.

^bPhosphorylation is only observed recombinant protein external.

^cThe protein transposition is not to mitochondrion (that is, protein is inherent mitochondrial protein).

The kinases and the phosphatase family of maximum in the human genome, protein kinase (PK) and Protein Tyrosine Phosphatases (PTP) have respectively above 500 with above 100 members.With the kinases and the phosphatase of less family, these signaling molecules almost comprise 3 percent of all proteins of encoding in the human genome.Find that in the research of wonderful quantity similar with aforesaid phosphoprotein, kinases and phosphatase participate in mitochondrial function, have reported that so far at least 25 kinases and 8 phosphatases are positioned mitochondrion, as shown in Figure 2.These kinases and phosphatase obviously are not limited to a group or family; On the contrary, their representatives are each known mammal kinases and phosphatase subgroup almost, reflects the scope that may influence mitochondrial signal transduction pathway.These signaling molecules comprise substrate specificity aspect (for example, tyrosine kinase, classical PTP subgroup, serine/threonine kinase and bispecific PTP); Catalyst mechanism aspect (for example, based on the PTP of cysteine, based on the PTP and the metal-dependency phosphatase of aspartic acid); With different kinases and the phosphatases in evolution conservative aspect (for example, the relevant pyruvic dehydrogenase kinase (PDK) and phosphatase (PDP), branched-chain keto acids dehydrogenase kinase (BCKDK) and phosphatase (BCKDP) and many mammal enzyme-specifics of antibacterial).

These signaling molecules of great majority have other non-mitochondrion effects in cell, mainly find to be present in the mitochondrion outside.For most protein, they translocate to mitochondrial power or mechanism is known little about it.Yet, clearly be that kinases and phosphatase are present in mitochondrial all compartments as the phosphoprotein of listing before, as Fig. 3 clearly as seen, and the multiple mitochondrial function of their activity influence.

As if yet some signaling molecules mainly are positioned mitochondrion.Except PDK and PDP, this group comprises the bispecific PTP of the inductive kinases PINK1 of PTEN-, targeting mitochondrion PTPMT1 and based on the phosphatase/adenosine triphosphatase Tim50 of aspartic acid.Although many these proteinic substrate are unknown at present, clear according to biology and data on genetics, they have conclusive effect in mitochondrion.For example, although its submitochondrial location still do not determine, PINK1 via N-terminus signal sequence targeting in mitochondrion.This kinases, itself and Ca ^{+ 2}As if the kinases family of/calmodulin, CaM-adjusting enjoys high sequence homology, participate in short survival (pro-survival) activity.Similarly, PTPMT1 is accredited as first PTP recently, and it mainly is positioned at mitochondrion inside, is similar to PINK1, in mitochondrion, and finds that the stromal surface of itself and mitochondrial inner membrane is closely related via the terminal signal peptide targeting of N-.PTPMT1 highly is expressed in the pancreatic beta cell, and its mitochondrion has the effect of important coupling glucose metabolism for secretion of insulin.At last, Tim50, a kind of TIM (translocase of inner membrance) complex has sequence homology with CTD family based on the phosphatase/adenosine triphosphatase of aspartic acid.Tim50, the same with other member of this family, may play adenosine triphosphatase, but its also demonstrate have external anti-phosphotyrosine analog right-phosphatase activity of α-nitrobenzophenone phosphate ester.Based on above-mentioned, as if it is not only other place from cell and adds to kinases and phosphatase in the mitochondrion as can be seen, and mitochondrion itself has part intrinsic signal molecule.

Although phosphorylation is not clear to the effect of most of known mitochondrion phosphoproteins, kinases of Fu Zeing and phosphatase are not still differentiated sometimes, have partly characterized some phosphorylation events.These examples, phosphoprotein and signaling molecule are not limited to a mitochondrial zone as previously discussed.

Phosphorylation on the mitochondrial outer membrane has conclusive effect in regulating apoptosis.A clear and definite especially incident is the phosphorylation of BAD, and BAD is the short apoptosis member of BCL-2 family.Show that after with short survivaling cell factor interleukin 3 treatment, the PKA transposition is to adventitia.In case A-kinases anchorin (AKAP) is anchored on the adventitia, and PKA is phosphorylation BAD on Ser 112, cause inactivation and disassociation from mitochondrial BAD, this is the process among a kind of Fig. 3 of being described in A.The p70S6 kinases is at the inactivation that on the Ser 136 phosphorylation and the unidentified kinases of BAD is also participated in BAD to the phosphorylation of BAD on Ser 155.

The example of knowing most that plays the reversible phosphorylation of regulatory mechanism effect in the cell mitochondrial of health is those of PDH complex in the substrate, and one it oversimplify sketch and be illustrated among Fig. 3 B.The deutero-acetone acid of this complex catalysis glycolysis changes into S-acetyl-coenzyme-A (CoA), and S-acetyl-coenzyme-A is the main precursor of tricarboxylic acids (TCA) circulation.Owing to the connection between these two the main energy-producing approach,, must suitably regulate the PDH complex in order to keep grape cell sugar homeostasis.

Because the PDH complex is accredited as first kind of mitochondrion phosphoprotein, at length studied the adjusting of this complex and reversible phosphorylation thereof.PDK and PDP carry out the phosphorylation and the dephosphorylation of PDH complex respectively.At least four PDK isoforms and two PDP isoforms are known, and all is all relevant with the E2 subunit of PDH complex.Phosphorylation event occurs on three isolating serine residues of E1 subunit, and each all causes the remarkable inactivation of this complex.Especially, exist at least PDK itself by the report of phosphorylation now.The phosphorylation that PKC carries out has demonstrated inactivation PDK, confirms that potentially reversible phosphorylation regulates the PDH complex of other level.Therefore, described PDH complex is a kind of mitochondrial main example that uses phosphorylation the adjusting level to be joined other conservative process.

As shown in the mitochondrion tyrosine kinase listed before and the phosphatase, the phosphorylation in this organelle is not limited to serine and threonine residues.Influence in the adjusting of example cytochrome c oxidase (COX) in inner membrance of tyrosine phosphorylation of mitochondrion energy and can see, be described among Fig. 3 C.COX, as the terminal transferase in the respiratory chain, with oxygen reduction Cheng Shui, synchronous pump goes out proton and passes inner membrance.Similar with the PDH complex, COX is subjected to ATP and ADP and thyroxin T2 and possible Ca ^{+ 2}Ionic allosteric is regulated.Except these adjusting forms, shown in vitro and in vivo in the HepG2 cell, COX in cAMP dependency mode by phosphorylation.COX comprises 13 subunits, and crystallization is dimer.Identified that its phosphorylation site is the Tyr 304 of subunit 1, it is positioned at the dimer interface on the inner membrance.Described phosphorylation event may suppress the COX activity significantly by destroying dimer formation.

In second example of the tyrosine phosphorylation of COX, be similar to the Lyn tyrosine kinase, a part of nonreceptor tyrosine kinase c-Src is positioned at mitochondrion inside, causes the COX tyrosine phosphorylation on the unidentified subunit II site in the osteoclast.The result of described phosphorylation event with seen for subunit I opposite, cause the COX increased activity.

An importance of mitochondrion signal conduction is that how self regulates for kinases and phosphatase.Mainly be arranged in other place of cell but as if the mitochondrial many kinases of targeting only just can be like this in its activated state such as AbI, Akt, GSK3I3 and PKC δ.Therefore, the degree of some kinase activity may be only by the quantity decision that is incorporated into the enzyme in the organelle in the mitochondrion.

Yet for the intrinsic signal molecule, different regulative modes must be suitable.Although these methods do not determine that still the second message,second messenger plays a crucial role probably.The activity of known PDK and PDP isoform is subjected to ion and micromolecule such as Mg ²⁺, Ca ²⁺, K ⁺Control with ADP.The sign of mitochondrion nitricoxide synthase and the recent findings of the soluble adenylate cyclase in the mitochondrion provide the second message,second messenger to help to regulate the further chance of mitochondrion signaling molecule.At last, ROS has been confirmed as a kind of mode of regulating other local signaling molecule in the cell, almost participates in regulating kinases and phosphatase in the mitochondrion inevitably, and most of reactive oxygen specieses produce in mitochondrion.Relative expression's level of the isoform of kinases and phosphatase may play an important role in pathology, and relevant with other signal transduction incident of disease association.The change of gene and the expression also variation with such is relevant.

Even after the investigation of some deep proteomics, only known 2/3rds mammal mitochondrial protein group according to estimates.The major part of residue 1/3rd may be made up of low-abundance protein, and described low-abundance protein is such as signal-proteins, and it is under the detection level of these mass spectral analyses.From these researchs, also can be clear that high variations from protein content between the mitochondrion of different tissues.For example, have been found that in the proteomics research that protein only-50% all guards in four tissue examinations (that is, brain, heart, liver and skeletal muscle).Different probably mitochondrion signal transduction pathway is not only different between tissue and tissue in the same way, still may all very well help this observed mitochondrion multiformity.

Yet exist very competent evidence to reach a conclusion: reversible phosphorylation participates in the adjusting of mitochondrion process.Have the phosphoprotein that surpasses 60 reports, 30 kinases and phosphatase and multiple auxiliary signal protein, the site that thinks poorly of of mitochondrion yes reversible phosphorylation signal conduction, in fact such adjusting can be used for treating hyperproliferation disease such as cancer.

With respect to unconverted cell, most growing tumors cells fast demonstrate significant heredity, biochemistry and histology's difference.Compare with tissue of origin, the many and energy metabolism in these changes relevant.Changes in energy metabolism in foremost, the tumor cell known in addition at high O ₂Under the situation of concentration, the glycolysis ability increases, and this is a kind of phenomenon of the Warburg of being called effect.

It is the energy deficiency that is caused by irreversible mitochondrial function damage that Warburg proposes the driving force that glycolysis improves in the tumor cell at first, and wherein similar with anaerobism muscle, glucose changes into slower excretory lactic acid via glycolysis.Propose, this increase of glycolysis flow is a kind ofly to have low O in the tumor cell ₂Guarantee the metabolic strategy of survival and growth in the environment of concentration, described low O ₂Concentration ratio is as observed local anoxia in the solid tumor of oxygenate difference.Especially, because O in the hypoxic tumor of many mankind ₂Concentration ratio 20 μ M low, so wherein oxidative phosphorylation is limited.Therefore, the glycolysis main energy pathway of solid tumor (for example, slow growing melanoma and breast carcinoma) seemingly.

For quick growth tumor cell, determined the proportionate relationship between the speed of the speed of cell proliferation and ATP supply.It is active relevant with the grade malignancy of tumor that some scholars propose glycolysis, therefore bigger in slow growing tumors or normal cell of the glycolysis speed ratio in highly differentiation and quick growing tumors.The predictive factors of high-caliber lactate as malignant tumor in fact, proposed.These incidents may be associated with other signal transduction incident and heredity variation, and example comprises the generation and the release of hypoxic inducement and angiogenesis factor.

Described in Fig. 4, for many years, it is important that tricarboxylic acids (TCA) circulation is regarded as merely biology, only is because it is producing as the effect among the ATP of the organism energy.Yet current research demonstrates TCA circulation activity also influences the signal transduction pathway function, comprise cell growth and apoptotic decision-making, and relevant glycolysis and tricarboxylic acid cycle enzyme can be upward or downward.Exist directly related between the activity of tumor development and glycolytic enzyme hexokinase that in the tumor cell of growing fast, greatly increases and phosphofructokinase (PFK) 1.Therefore, suppose and demonstrate insufficient tumor cell ratio by its oxidation capacity to have those of active oxidation phosphorylation more harmful.No matter be in hypoxic condition or under aerobic conditions, cancerous tissue is all relevant with virulent increase to glucolytic dependence.

Studied the effect of thioctic acid in the PDH of healthy cell complex well.The PDH complex has three center subunit E1, E2 and E3 (being respectively pyruvic dehydrogenase, dihydro sulfur caprylyl Acetylase and dihydrolipoamide dehydrogenase).These complex all have center E2 core, and other subunit forms complex around this core.In the gap between two subunits, described sulfur decoyl district transports the intermediate between the avtive spot.Described sulfur decoyl district itself is connected to the E2 core by flexible joint.When acetone acid and phosphorylated thiamine reaction formation hemimercaptol, this anion is attacked the S1 of the sad thing class of sulfur oxide that is connected to lysine residue.Therefore, described thioctic acid S2 is replaced by sulfide or sulfydryl part, and thiazole is emitted in breaking of tetrahedron hemimercaptol subsequently, discharges the TPP cofactor and produce thiacetate on this thioctic acid S1.In this, described thioctic acid-thioesters functional group translocates to the E2 avtive spot, and wherein the acyl group transfer reaction will be transferred to the mercaptan of coenzyme A from the acetyl group of " swing arm " of thioctic acid.This can produce S-acetyl-coenzyme-A, and it discharges from described multienzyme complex, then enters tricarboxylic acid cycle.Described dihydrolipoic acid is still in conjunction with the lysine residue of described complex; move to the E3 avtive spot then; wherein dihydrolipoic acid carries out the oxidation of flavin mediation; be converted back into its thioctic acid resting state; produce FADH2 (with final NADH), and make described thioctic acid be converted back into effective acyl acceptor.If this thioctic acid thing class is ended, then will there be electronics to flow into FADH ₂Or the generation S-acetyl-coenzyme-A, the result is that the toxin of acetone acid in the cell is assembled.In cancerous cell, the generation of acetoin is shown it is the needs of cell detoxifcation and survival.

As previously mentioned, the activity of PDH complex is subjected to the altitude mixture control of multiple allosteric effector and covalent modification in the mitochondrion.The PDH activity is subjected to the adjusting of its phosphorylation state, its active maximum under the dephosphorylation state.The PDH phosphorylation is subjected to the catalysis of PDK.The PDK activity is subjected to the increase of ATP, NADH and acetyl coa levels and improves.The negative effector of PDK is ADP, NAD ⁺, CoA-SH and acetone acid, its level increases when the ATP level reduces.When regulating PDP, activate the enzyme of PDH via dephosphorylation and not exclusively understood, well-known Mg ^{+ 2}And Ca ^{+ 2}Activate PDP.

Two the product NADH and the S-acetyl-coenzyme-A of described complex all are the negative allosteric effectors of PDH-a, and PDH-a is the dephosphorylation activity form of PDH.These effectors have reduced the affinity of enzyme to acetone acid, and therefore, the carbon that has limited via the PDH complex flows.In addition, NADH and S-acetyl-coenzyme-A are the strong positive effectors of PDK, and PDK is the enzyme of inactivation PDH by the PDH-b form that PDH is changed into its phosphorylation.Because NADH and S-acetyl-coenzyme-A are assembled when described cell energy charge is high, not making us surprised is that high ATP level also raises the PDK activity, the active downward modulation of PDH in the enhancing energy enrichment of cell.Yet, because acetone acid is effective negative effector of PDK, when the acetone acid level rises, PDH-a even be favourable with high-caliber NADH and S-acetyl-coenzyme-A.

The concentration of the acetone acid of maintenance PDH-a is enough high, makes in being rich in the cell of ATP the high K of allosteric effect downward modulation _mThe PDH of form still can change into S-acetyl-coenzyme-A with acetone acid.Under a large amount of acetone acid in the cell with high ATP and NADH level, the carbon of acetone acid will be directed to two main carbon storage forms (via the glycogen of glyconeogenesis with via the synthetic lipogenesis of fatty acid), and wherein S-acetyl-coenzyme-A is main carbon donor.Although the adjusting of PDP-b can not thoroughly be understood, it is conditioned with maximization pyruvate oxidation under the ATP concentration that reduces probably and minimizes the PDH activity under high ATP concentration.

Tumor cell can not ad infinitum be assembled acetone acid and associating aldehyde and free radical, and such as acetaldehyde, superoxides, hydrogen peroxide and hydroxyl radical free radical, because these molecules are that cytotoxicity is arranged under high level, its mechanism is as reducing cellular pH tempestuously.Therefore, for AS-30D and Ehrlich hepatocarcinoma, the mitochondrion acetone acid of having described signal portion by the E1 component of PDH complex via becoming 2-in conjunction with the decarboxylation of beta-hydroxyethyl thiamine pyrophosphate salt.This 2-is transferred and second acetaldehyde condensation, finally by using aminoacid glutamine or thioctic acid deacidification or the initial acetone acid that reduces, produce acetoin (3-hydroxyl-butanone), a kind of competitive inhibition PDH and for the cell chemical compound littler (for example, keeping intracellular pH homeostasis) than the toxicity of its acetone acid precursor.Although the importance of acetoin in the tumor cell detoxification pathways is to generate the accumulative result of acetone acid that the source causes because tumor cell depends on glycolysis as ATP, yet prior art does not almost have document to mention the influence of the generation of blocking-up acetoin to tumor cell existence.

Current research proposes to impel cancerous cell to enter more aerobic metabolisies inhibition tumor growths.Therefore, need block the PDH complex to the metabolic transformation of Warburg.In this transformation, the conduction of the signal of the anoxia-inducible factor in the cancerous cell (HIF) increases, and does not give HIF the remarkable effect in glucose metabolism astoundingly, as shown in Figure 5.The sudden change that directly or indirectly causes HIF signal conduction is the common mechanism in the cancer development in fact seemingly.HIF causes that PDK1 crosses expression, and then, it works to reduce the PDH complex activity.The phosphorylation of PDK1 may be effective especially for keeping inactive PDH complex, because this isoform three serine residues in the α subunit of phosphorylation E1 uniquely, the α subunit of E1 is first subunit in the PDH complex.The reactivate of E1 need be removed all three phosphates.And the activation of PDH complex can cause ROS generate to increase, its can then cause apoptosis.Yet, the change of observed PDK1 may be not only because the variation of its concentration in cancer, also because the variation of its its active and possible aminoacid sequence, between above-mentioned variation or even a kind of tumor type or the variation between a patient and another patient.In addition, PDK1 can form different complex with the multiple molecule relevant with tumor, depends on tumor type.Therefore, suppress PDK and produce apoptotic potential target spot in the tumor.Yet, up to now, the inhibition of this isozyme of known PDK1 inhibitor confirmation can causing at most only 60%.

Though traditional chemotherapy targeting division growth cell, all clinical chemotherapeutic treatments of accepting use big drug dose, and it also causes the heavy damage of the host cell of normal propagation.Therefore, for treatment for cancer, need more optionally targeting.Another difficult problem relevant with chemotherapy is the intrinsic or acquired resistance that exists in the tumor of many types antineoplastic agent.In a word, present conventional chemotherapy provides the long-term benefit seldom to most of malignant tumor, and follows the adverse side effect that reduces life length and reduce quality of life usually.Therefore, needs can provide the long-term control of tumor to allow the completely new approach of good quality of life simultaneously.

Certainly, efficacy of drugs, to send with side effect be to need the difficult problem that solves in the new chemotherapeutic of exploitation.In solid tumor, when medicine is difficult for being penetrated into different cellular layers, may be very difficult to sending of hypoxic zone.In order to eliminate these uncertainties, design has metabolism in the sub-micro molar range at least, and to suppress the anticarcinogen of constant seemingly rational.May advocate between cancerous cell line be heredity with phenotype on different.Yet all tumor cell lines all depend on the glycolysis and the oxidative phosphorylation of ATP supply.Concentrating on the Warburg effect allows design based on the physics between tumor and the normal cell-and the medicine of biochemical capacity volume variance, to promote to send the design with therapeutic strategy, it optionally influences only tumor metabolism and growth, and can unhealthful host tissue and organ dysfunction.

People's such as Bingham US patent 6,331,559 and 6,951,887 and people's such as Bingham U.S. Provisional Application No.60/912,598 (all being incorporated herein by reference) disclose new class lipoic acid derivatives therapeutic agent, and it is the diseased cells of targeting and kill tumor cell and some other types optionally.These patents further disclose pharmaceutical composition and its using method of the such lipoic acid derivatives that comprises effective dose and pharmaceutically acceptable carrier.Yet,, in arbitrary piece of patent, all do not indicate these derivants to be used to regulate the structure and/or the expression of PDH complex and/or regulate the PDH complex activity though these patents have been described the general service of the structure of these lipoic acid derivatives.

Because the structure and/or activity of verified PDH complex are the decisive determiners of tumor promotion, then useful structure that provides this PDH complex and/or activity or even the acceptable regulator of pharmacy of expression, and using method.

Purpose of the present invention and commercial Application

Therefore, an object of the present invention is to provide that a kind of to be used for the treatment of or to diagnose with the cell hyperproliferation be disease, disease or the syndrome of the feature pharmaceutical composition such as cancer, it demonstrates selective active in tumor cell.

A further purpose of the present invention provides and a kind ofly is used for the treatment of or diagnoses such aforementioned diseases, disease or syndromic pharmaceutical composition, the side effect minimum that it causes when using.

Of the present invention one further purpose provide and a kind ofly be used for the treatment of or diagnose such aforementioned diseases, disease or syndromic pharmaceutical composition, it is easily with minimum possible cost preparation, and can preserve the longest possible period.

Of the present invention one further purpose provide and a kind ofly be used for the treatment of or diagnose such aforementioned diseases, disease or syndromic pharmaceutical composition, it regulates mitochondrial energy metabolism via structure, activity and/or the expression of PDH complex in the tumor cell mitochondrion especially.

Summary of the invention

In order to reach aforementioned purpose, the present invention provides a kind of being used for the treatment of widely, diagnosis or prevention comprise in the human Homoiotherm with at least a enzyme and/or multienzyme complex, or its subunit is such as the structure of PDH complex, express and/or active adjustment or the interferential disease of changing into feature, the pharmaceutical composition of disease or syndrome or its symptom, described disease, disease or syndrome comprise with the cell hyperproliferation being those of feature, such as cancer, wherein said pharmaceutical composition comprises at least a lipoic acid derivatives and at least a pharmaceutically acceptable carrier of effective dose, described lipoic acid derivatives is included in United States Patent (USP) 6,331,559 and 6,951,887 and U.S. Provisional Application No.60/912, those that describe in 598 all are incorporated herein by reference all these applications.

By suppressing mitochondrial energy metabolism, lipoic acid derivatives of the present invention causes mitochondrial membrane potential forfeiture and other the mitochondrial consequence in the diseased cells, causes irreversibly trigger cell death.Lipoic acid derivatives of the present invention also can and/or suppress PDP or suppress acetone acid via the E1 subunit activity that suppresses the PDH complex to change into more that hypotoxicity molecule acetoin suppresses mitochondrial energy metabolism by activation PDK.The synthetic inhibition of acetoin will make other process undesired, comprise redox equilibrium, and can cause that also toxic by-products comprises the generation of acetaldehyde, superoxides, hydrogen peroxide and hydroxyl radical free radical, the result, these by-products self cause the mitochondrial irreversible destruction of diseased cells.

Pharmaceutical composition of the present invention can be regulated the effect of PDK1, PDK2, PDK3, PDK4 and mutant separately or isoform.Described medical compounds is the effect of scalable PDP1, PDP2 and isoform separately thereof also.

Pharmaceutical composition of the present invention is the expression of the enzyme component of phosphorylase, kinases and the dehydrogenase found in the PDH complex of scalable also.This adjusting can take place transcribing, translate or translate in the after-stage, and the described stage comprises the outer genetic silencing of suitable gene.

As fundamentally relevant with tricarboxylic acid cycle molecule with by expansion glycolysis derived compounds, pharmaceutical composition of the present invention shows selectivity and absorbs in the tumor cell.And such selectivity tumor cell absorbs to have minimized uses the side effect of this pharmaceutical composition to the non-transformed cell and the tissue of health.

In one embodiment of the invention, described lipoic acid derivatives has general formula (I) or its salt:

R wherein ₁And R ₂Be independently selected from the group of forming by following: hydrogen, alkyl C _nH _2n+1, alkene C _nH _2n, thiazolinyl C _nH _2n-1, alkynes C _nH _2n-2, alkynyl C _nH _2n-3, alkyl sulfide CH ₃(CH ₂) _n-S-, alkyl disulfide CH ₃CH _t-S-S-, thiocarbamate (thiocarbamic ester) (CH ₂) _nC=NH-and hemimercaptol CH ₃CH (OH)-S-, wherein n is that 1-10 and t are 0-9; Aromatic group; Be defined as R ₃C (O)-acyl group; Heteroaryl; Be defined as R ₄C (=NH)-imines acyl (imidoyl); The organic metal aryl; Alkyl organic metal aryl; With hemiacetal R ₅CH (OH)-S-;

R as defined above wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃Be selected from the group of forming by following: hydrogen, thiazolinyl, alkynyl, alkylaryl, heteroaryl, miscellaneous alkyl aryl and organic metal aryl, wherein any can be that replace or unsubstituted;

R wherein ₄Be selected from the group of forming by following: hydrogen, thiazolinyl, alkynyl, aryl, alkylaryl, heteroaryl and miscellaneous alkyl aryl, wherein any can be that replace or unsubstituted;

R wherein ₅Be CCl ₃, CF ₃Or COOH;

Wherein x is 0-16.

In second embodiment of the present invention, described lipoic acid derivatives is defined as second general formula (II) or its salt:

Wherein M be covalent bond ,-[C (R ₁) (R ₂)] _z-or metallo-chelate or other metal complex, wherein said metal is not a palladium;

R wherein ₁And R ₂Be independently selected from the group of forming by following: hydrogen, acyl group R ₃C (O)-, alkyl C _nH _2n+1, be defined as C _mH _2m-1Thiazolinyl, be defined as C _mH _2m-3Alkynyl, aryl, heteroaryl, alkyl sulfide CH ₃(CH ₂) _n-S-, be defined as R ₃C (=NH)-the imines acyl and be defined as R ₄The hemiacetal of CH (OH)-S-;

R as defined above wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃And R ₄Be independently selected from the group of forming by following: hydrogen, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclic radical, wherein any can be that replace or unsubstituted;

R wherein ₅Be selected from the group of forming by following :-CCl ₃,-CF ₃Or-COOH;

Wherein x is 0-16, and z is 0-5, and n is that 0-10 and m are 2-10.

In the 3rd embodiment of the present invention, described lipoic acid derivatives has the 3rd general formula (III) or its salt:

R wherein ₁And R ₂Be independently selected from the group of forming by following: hydrogen, alkyl C _nH _2n+1, alkene C _nH _2n, thiazolinyl C _nH _2n-1, alkynes C _nH _2n-2, alkynyl C _nH _2n-3, alkyl sulfide CH ₃(CH ₂) _n-S-, alkyl disulfide CH ₃CH _t-S-S-, thiocarbamate (CH ₂) _nC=NH-and hemimercaptol CH ₃CH (OH)-S-, wherein n is that 1-10 and t are 0-9, aromatic group, is defined as R ₄C (O)-acyl group, heteroaryl, be defined as R ₅C (=NH)-imines acyl, organic metal aryl, alkyl organic metal aryl, hemiacetal R ₆CH (OH)-S-, aminoacid, saccharide, nucleic acid, lipid and polymer and combination;

R as defined above wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃Be selected from the group of forming by following: aminoacid, saccharide, nucleic acid, lipid and polymer thereof;

R wherein ₄Be selected from the group of forming by following: hydrogen, thiazolinyl, alkynyl, alkylaryl, heteroaryl, miscellaneous alkyl aryl and organic metal aryl, wherein any can be that replace or unsubstituted;

R wherein ₅Be selected from the group of forming by following: hydrogen, thiazolinyl, alkynyl, aryl, alkylaryl, heteroaryl and miscellaneous alkyl aryl, wherein any can be that replace or unsubstituted;

R wherein ₆Be CCl ₃, CF ₃Or COOH;

Wherein x is 0-16.

In the 4th embodiment of the present invention, described lipoic acid derivatives is defined as the 4th general formula (IV) or its salt:

Wherein M be covalent bond ,-[C (R ₁) (R ₂)] _z-, or metallo-chelate or other metal complex, wherein said metal is not a palladium;

R wherein ₁And R ₂Be independently selected from the group of forming by following: hydrogen, acyl group R ₄C (O)-, alkyl C _nH _2n+1, be defined as C _mH _2m-1Thiazolinyl, be defined as C _mH _2m-3Alkynyl, aryl, heteroaryl, alkyl sulfide CH ₃(CH ₂) _n-S-, be defined as R ₄C (=NH)-the imines acyl, be defined as R ₆Hemiacetal, aminoacid, saccharide, nucleic acid, lipid and polymer thereof and the combination of CH (OH)-S-;

R as defined above wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃Be selected from the group of forming by following: aminoacid, saccharide, nucleic acid, lipid and polymer thereof;

R wherein ₄And R ₅Be independently selected from the group of forming by following: hydrogen, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclic radical, wherein any can be that replace or unsubstituted;

R wherein ₅Be selected from the group of forming by following: CCl ₃, CF ₃Or COOH;

Wherein x is 0-16, and z is 0-5, and n is that 0-10 and m are 2-10.

And, because any or all these universal architecture can be at cell or mitochondrion intracellular metabolite, expect clearly the above-mentioned structure of mentioning metabolite all within the scope of the present invention.

In each general formula, (the R)-isomer of each specific lipoic acid derivatives all has the physiologically active bigger than (S)-isomer.Therefore, described lipoic acid derivatives should be only exists with its (R)-isomeric forms or with (R)-and (S)-mixture of isomers form.

Of the present invention one further aspect, the method of a kind of diagnosis, treatment or disease, disease, syndrome or its symptom of prevention in comprising human Homoiotherm is provided, described disease, disease, syndrome or its symptom comprise adjustment or the interferential change such as structure, expression and/or the function of PDH complex of at least a enzyme and/or multienzyme complex or its subunit, comprise with the cell hyperproliferation being those of feature, such as cancer, wherein said method comprises pharmaceutical composition disclosed herein from effective dose to such animal that use.

Of the present invention one further aspect, provide a kind of diagnosis and prediction to present the method for being benefited among the patient of disease, disease or syndromic symptom, described disease, disease or syndrome comprise structure, expression and/or active adjustment or the interferential change such as the PDH complex of at least a enzyme and/or multienzyme complex or its subunit, comprise with the cell hyperproliferation being those of feature, such as cancer, described method comprises from the patient and obtains cell sample, use the pharmaceutical composition of the present invention of effective dose and obtain the result thus to described cell external.

The accompanying drawing summary

Following accompanying drawing has been set forth embodiment of the present invention, and does not mean that the application's that restriction as whole description and claim are included scope.

Fig. 1 has described targeting and has entered mitochondrion and general signal transducers and effect thereof from wherein coming out.

Fig. 2 display line plastochondria kinases and phosphatase and localized catalogue within mitochondrion thereof.

Fig. 3 A illustrates in the mitochondrial outer membrane PKA to the phosphorylation of BAD, and goes up reversible phosphorylation.

Fig. 3 B has presented under the effect of PDH complex that acetone acid changes into S-acetyl-coenzyme-A in the mitochondrial matrix, and goes up reversible phosphorylation.

Fig. 3 C shows COX in the effect of hydrogen reduction Cheng Shui and pump being got in the proton cross-line plastochondria inner membrance, and goes up reversible phosphorylation.

The substrate during the glycolysis of Fig. 4 diagram acetone acid generates and the structure of product have also shown the generation of ATP and NADH and relevant enzyme.

Fig. 5 shows the adjusting of HIF-1 to glucose metabolism.

The difference of energy metabolism between intravital normal structure of Fig. 6 A diagram and the cancerous tissue.

Fig. 6 B describes the biological form of thioctic acid in the PDH complex and forms difference between the lipoic acid derivatives of pharmaceutical composition part of the present invention.

Fig. 6 C shows that sulfur decoyl residue is to the adjusting to the PDH complex of the effect of PDK.

Fig. 7 shows the effect of pharmaceutical composition of the present invention to the xenotransplantation tumor growth.

Fig. 8 shows the therapeutic effect of pharmaceutical composition of the present invention to three kinds of tumor cells and a kind of no transformed cells.

Fig. 9 A shows with the ATP level in the lung carcinoma cell behind the medicine composite for curing of the present invention of lethal threshold or above amount.

Fig. 9 B relatively comprises in the medium of glucose in the medium contrast that comprises acetone acid, and pharmaceutical composition of the present invention is to the synthetic inhibition of ATP.

Relatively in breast cancer cell and normal breast cell, pharmaceutical composition of the present invention is to the synthetic inhibition of ATP for Fig. 9 C.

Fig. 9 D has compared pharmaceutical composition of the present invention in the lung carcinoma cell, thioctic acid and inactive form of the present invention to the synthetic inhibition of ATP.

Figure 10 diagram pharmaceutical composition of the present invention is to the influence of the tumor cell mitochondrion level of PDH complex and ketoglurate dehydrogenase (α KDH) enzymatic activity.

Figure 11 A show to use by oneself extract two dimension gel Western of lung carcinoma cell medicine composite for curing of the present invention or the simulation treatment analyzes.

Figure 11 B shows with amplification medicine composite for curing of the present invention and the paired two-dimentional gel sample simulation treatment.

Regulating action when Figure 12 A has described endogenous thioctic acid that PDK is covalently bonded in PDH complex E2 subunit and regulates.

Figure 12 B describes the possibility mechanism of pharmaceutical composition of the present invention to the differential inactivation of tumor cell PDH complex.

Figure 13 presents the influence of pharmaceutical composition of the present invention to the mitochondrial membrane potential in the H460 lung carcinoma cell.

Figure 14 shows that pharmaceutical composition wherein of the present invention causes the Western engram analysis result of the cell death approach of kinds of tumor cells type.

Detailed Description Of The Invention

The present invention relates generally to and be used for the treatment of, diagnose or prevent disease, illness or syndrome in the homeothermal animal or the pharmaceutical composition of its symptom, described disease, illness or syndrome or its symptom comprise that at least a enzyme and/or multienzyme complex or its subunit are such as structure, expression and/or the adjustment of activity or the change of interference of PDH compound, comprise take cell hyperproliferation as feature those, such as cancer or its symptom. Such animal comprises those in the mammal species, such as the mankind, horse, ox, comprise the domestic animal of dog and cat etc., suffers from the disease take cell hyperproliferation as feature and other pathology illness and the syndrome that comprise cancer. Pharmaceutical composition of the present invention comprises at least a lipoic acid derivatives of effective dose, is included in United States Patent (USP) 6,331,559 and 6, those (being also referred to as thioctan) and pharmaceutically acceptable carrier or the excipient described in 951,887 and U.S. Provisional Application No.60/912,598. As not only being the common derivative of within mitochondria, finding, and help to increase the molecule of the glycolysis activity of tumour cell, as as shown in the Warburg effect, lipoic acid derivatives of the present invention is suitable for selectively being delivered to and effectively concentrating within the mitochondria of cell take hyper-proliferative as feature and tissue especially, described cell and tissue be such as tumour cell and tissue, thereby avoid normal cell and tissue to be subjected to the impact of described composition.

Pharmaceutical composition of the present invention can regulate PDK1, PDK2, PDK3, PDK4 and separately isoform via the effect of reversible phosphorylation. Described pharmaceutical composition also can regulate PDP1, PDP2 and its separately isoform and/or mutant also by the effect of reversible phosphorylation. Such adjusting can be by promoting or suppressing kinases or phosphatase activity takes place.

By suppressing mitochondrial energetic supersession, lipoic acid derivatives of the present invention causes mitochondrial membrane potential forfeiture and other the mitochondrial consequence in the diseased cells simultaneously, causes irreversibly trigger cell death. Lipoic acid derivatives of the present invention also can and/or suppress PDP or suppress pyruvic acid via the E1 subunit activity that suppresses the PDH compound by activation PDK to change into more that hypotoxicity molecule 3-hydroxy-2-butanone suppresses mitochondrial energetic supersession. The synthetic inhibition of 3-hydroxy-2-butanone will make other process undesired, comprise redox equilibrium, and can cause that also toxic by-products comprises the generation of acetaldehyde, superoxides, hydrogen peroxide and hydroxyl radical free radical, the result, these accessory substances self cause the mitochondrial irreversible destruction of diseased cells.

In first embodiment of the present invention, described lipoic acid derivatives is defined as first general formula (I) or its salt:

Wherein x is 0-16. R₁And R₂Can be independently:

(1) the acyl group R that connects by thioester bond₃C (O)-, R wherein₃Be alkyl, aryl or organic metal aryl, include but not limited to acetyl group and bytyry (butaryl), instantiation is the diacetyl lipoic acid;

(2) aromatic group that connects by thioester bond includes but not limited to benzoyl or benzoyl derivative, and instantiation is the dibenzoyl lipoic acid;

(3) the alkyl C that connects by thioester bond_nH _2n+1, be 1-10 at this n, this alkyl with other parts (as, for instance ,-OH ,-Cl or-NH₂) replace, include but not limited to methyl, ethyl, butyl, decyl and 6,8-, two carbamyl methyl lipoic acids;

(4) the thiazolinyl C that connects by thioester bond_nH _2n-1, be 2-10 at this n, include but not limited to propylene, 2,3 dimethyl-2-butylene and heptene;

(5) the alkynyl C that connects by thioester bond_nH _2n-2, be 2-10 at this n, include but not limited to acetylene, propine and octyne;

(6) alkyl, thiazolinyl and alkynyl, it can be open chain or alicyclic ring, alicyclic group contains the interpolation of either carbon or replaces to consist of heterocycle, includes but not limited to cyclopropane, cyclopentene and 6,8 methyl-succinimide base lipoic acid;

(7) alkyl, thiazolinyl and alkynyl, it can have interpolation at their either carbon, includes but not limited to hydroxyl and amine;

(8) aromatic group or the aryl that connect by thioester bond, it is the derivative of benzene or benzene, includes but not limited to toluene and aniline;

(9) the alkyl sulfide group CH that connects by disulfide bond₃(CH ₂) _n-S-, wherein n can be but be not limited to 0-9;

(10) the imines acyl CHR that connects by thioester bond₄C (=NH)-, wherein n can be but be not limited to 1-10; And

(11) hemiacetal group R₅CH (OH)-S-, wherein R₅Be limited to the compound that strong electron-withdrawing substituent is arranged, include but not limited to trichloroacetaldehyde and pyruvic acid.

The R that more than defines₁And R₂Do not replace or replace, also can contain the thioesters of the oxidized generation sulfoxide of energy or sulfone, respectively such as C-S (O)-R and C-S (O)₂-R。

R ₁And R₂Can further contain the disulphide that can be oxidized to sulfo-sulfinic acid or thiosulfonic acid, respectively as: C-S (O)-S-R and C-S (O)₂-S-R。

R ₃Be selected from the group that is formed by hydrogen, thiazolinyl, alkynyl, alkylaryl, heteroaryl, miscellaneous alkyl aryl and organic metal aryl, its any replacement or unsubstituted. Similarly, R₄Be selected from the group that forms by by hydrogen, thiazolinyl, alkynyl, aryl, alkylaryl, heteroaryl and miscellaneous alkyl aryl, its any replacement or unsubstituted.

R ₅Be-CCl₃、-CF ₃Or-COOH.

In second embodiment of the present invention, described lipoic acid derivatives is defined by second general formula (II) or its salt:

Wherein, M be covalent bond ,-[C (R₁)(R2)] _z-or metal chelate or other metal complexs, wherein said metal is not palladium;

Wherein, R₁And R₂Be independently selected from the group that is formed by following: hydrogen, acyl group R₃C (O)-, alkyl C_nH _2n+1, with C_nH _2n-1The definition thiazolinyl, with C_nH _2n-3Alkynyl, aryl, heteroaryl, the alkyl sulfide CH of definition₃(CH ₂) _n-S-, with R₃C (=NH)-definition the imines acyl and with R₄The hemiacetal of CH (OH)-S-definition;

Wherein, the R that more than defines₁And R₂Do not replace or replace;

Wherein, R₃And R₄Be independently selected from the group that is formed by hydrogen, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclic radical, its any replacement or unsubstituted;

Wherein, R₅Be selected from by-CCl₃、-CF ₃Or-group that COOH forms;

And wherein x is that 0-16, z are 0-5, and n is 0-10.

In the third embodiment of the present invention, described lipoic acid derivatives has the third general formula (III) or its salt:

Wherein, R₁And R₂Be independently selected from the group that is formed by following: hydrogen, alkyl C_nH _2n+1, alkene C_nH _2n, thiazolinyl C_nH _2n-1, alkynes C_nH _2n-2, alkynyl C_nH _2n-3, alkyl sulfide CH₃(CH ₂) _n-S-, alkyl disulfide CH₃CH _t-S-S-, thiocarbamate (CH₂) _nC=NH-and hemimercaptol CH₃CH (OH)-S-, wherein n be 1-10 and t be 0-9, aromatic series, with R₄The acyl group of C (O)-definition, heteroaryl, with R₅C (=NH)-definition imines acyl, organic metal aryl, alkyl-organic metal aryl, hemiacetal R₆CH (OH)-S-, amino acid, carbohydrate, nucleic acid, lipid and their polymer and combination;

Wherein, the R that more than defines₁And R₂Do not replace or replace;

Wherein, R₃Be selected from the group that is formed by amino acid, carbohydrate, nucleic acid, lipid and its polymer;

Wherein, R₄Be selected from the group that is formed by hydrogen, thiazolinyl, alkynyl, alkylaryl, heteroaryl, miscellaneous alkyl aryl and organic metal aryl; Its any replacement or unsubstituted;

Wherein, R₅Be selected from the group that is formed by hydrogen, thiazolinyl, alkynyl, aryl, alkylaryl, heteroaryl and miscellaneous alkyl aryl, its any replacement or unsubstituted;

Wherein, R₆CCl₃、CF ₃, or COOH;

And wherein, x is 0-16.

In the 4th kind of embodiment of the present invention, described lipoic acid derivatives is defined by the 4th kind of general formula (IV) or its salt:

Wherein, M be covalent bond ,-[C (R₁)(R ₂)] _z-or metal chelate or other metal complexs, wherein said metal is not palladium;

Wherein, R₁And R₂Be independently selected from the group that is formed by following: hydrogen, acyl group R₄C (O)-, alkyl C_nH _2n+1, with C_mH _2m-1The definition thiazolinyl, with C_mH _2m-3Alkynyl, aryl, heteroaryl, the alkyl sulfide CH of definition₃(CH ₂) n-S-, with R₄C (=NH)-definition the imines acyl, with R₆Hemiacetal, amino acid, carbohydrate, nucleic acid, lipid and their polymer and the combination of CH (OH)-S-definition;

Wherein, the R that more than defines₁And R₂Do not replace or replace;

Wherein, R₃Be selected from the group that is formed by amino acid, carbohydrate, nucleic acid, lipid and its polymer;

Wherein, R₄And R₅Be independently selected from the group that is formed by hydrogen, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclic radical; Its any replacement or unsubstituted;

Wherein, R₅Be selected from by CCl₃、CF ₃Or the group of COOH composition;

And wherein, x is 0-16, and z is 0-5, and n is that 0-10 and m are 2-10.

And, because any or all these universal architecture can be at cell or mitochondria intracellular metabolite, expect clearly the above-mentioned structure of mentioning metabolite all within the scope of the present invention.

Observe: in first kind and second kind of general formula, (R)-ratios of the isomers (the S)-isomer of the lipoic acid derivatives that each is specific has stronger physiologically active.Therefore, though consider to use two kinds of isomers in the present invention, in particularly preferred embodiments, especially consider preferential use (R)-isomer or in (S)-mixture of isomers, have (R)-isomer.

Pharmaceutical composition of the present invention is the expression of phosphatase, kinases and the dehydrogenase enzyme component found in the PDH complex of scalable also.This adjusting can occur in and transcribe, translates or translate after-stage, comprises the back silence of giving birth to that is fit to gene.

The present composition can further comprise pharmaceutically acceptable carrier or excipient.The example of pharmaceutical acceptable carrier is well-known in the art and comprises in those pharmaceutical compositions of being everlasting and using, as, but be not limited to absorption enhancer, antimicrobial and and their combination of antioxidant, buffer agent, intercalating agent, flavorant, coloring agent, antiseptic, raising bioavailability.The amount of these additives depends on required characteristic, and this can be determined by those skilled in the art easily.

Pharmaceutical composition of the present invention can contain salt, buffer agent, antiseptic and compatibility carrier usually, randomly makes up the other treatment composition.When using in medicine, salt should be that pharmacy is acceptable, but the acceptable salt of non-pharmacy also can suitably use preparing the acceptable salt of its pharmacy, and is not excluded from scope of the present invention.Those that the acceptable salt of this pharmacology and pharmacy includes but not limited to prepare from following acid: hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, palicylic, p-methyl benzenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid.And the acceptable salt of pharmacy can be prepared as alkali metal or alkali salt, as sodium salt, potassium salt or the calcium salt of hydroxy-acid group.

The present invention also provides the method for the treatment of or diagnose the patient by at least a therapeutic agent from effective dose to cell that send or diagnostic agent with therapeutic agent or diagnostic agent, described therapeutic agent or diagnostic agent are used for implementing prevention, diagnosis or treatment disease, disease or syndrome or its symptom, described disease, disease or syndrome or its symptom comprise structure, expression and/or active adjustment or the interferential change of at least a enzyme and/or multienzyme complex or its subunit, comprise with the cell hyperproliferation being those of feature.Regulate the PDH complex and especially come into one's own, comprise after the treatment of the primary tumo(u)r by control tumor cell proliferation, vascularization, transitivity growth, apoptosis and the excision or the treatment of small transfer progress simultaneously as the improved treatment of cancer; And the radiotherapy of primary tumo(u)r or other chemotherapies.Pharmaceutical composition of the present invention is to useful such as following cancer types: former or metastasis melanin tumor, lymphoma, sarcoma, pulmonary carcinoma, hepatocarcinoma, Hodgkin lymphoma and non-Hodgkin lymphoma, leukemia, uterus carcinoma, cervical cancer, bladder cancer, renal carcinoma, colon cancer and adenocarcinoma such as breast carcinoma, carcinoma of prostate, ovarian cancer and cancer of pancreas.

For treatment and diagnostic application, described pharmaceutical composition can directly be applied to the patient after in conjunction with pharmaceutically acceptable carrier.This method can be by using separately or implement with the another kind treatment of effective dose or the treatment or the diagnostic agent of diagnostic agent combination, described another kind of treatment or diagnostic agent can be but be not limited to glycolytic inhibitor, microtubule interaction agent (microtubule-interacting agent), press down the cell growth stimulator, folic acid inhibitor, alkylating agent, topoisomerase enzyme inhibitor, cheese ammonia kinase inhibitor, podophyllinic acid lactone or derivatives thereof, antitumor antibiotics, chemotherapeutics, inducer of apoptosis, anti-angiogenic agent, chlormethine, nucleic acid intercalating agent, and their combination.These therapeutic agents can further comprise other metabolism and suppress reagent.Many this therapeutic agents are known in the art.This combination therapy provides simultaneously, is used for the treatment of in succession or separately the disease that needs amplify or the assurance patient replys Therapeutic Method.

The inventive method can use the acceptable method of application of any medical science to implement, and can produce the effect level of reactive compound and do not cause clinical unacceptable side reaction.Although preferably be particularly suitable for the preparation that parenteral is used, but the present composition also can aerosol, the form of spray, powder, gel, lotion, Emulsion, suppository, ointment and analog, is arranged in suction, oral, local, percutaneous, intranasal, ophthalmic, pulmonary, rectum, mucosa, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, the pleura, in the intrauterine, tumor or infusion methods or use.If need these preparations, also can comprise other additives well-known in the art to give the required denseness of preparation and other characteristics.

The ad hoc fashion that one skilled in the art will appreciate that administering therapeutic or diagnostic agent depends on: specific dose of selection; Use in order to treat, to diagnose or prevent disease, disease, syndrome or its symptom; The order of severity of the medical conditions of treatment or diagnosis; And required dosage is renderd a service in treatment.For instance, the optimal way of the leukemic anticarcinogen of administering therapeutic comprises that intravenous uses, and the method for optimizing of treatment skin carcinoma comprises part or intradermal administration.

As used herein, " effective dose " refers to treat or dosage or the multiple dosage of diagnostic agent when obtaining desired therapeutic or diagnosis effect.Substantially, the effective dose of treatment or diagnostic agent can change according to following: specific dose activity of use, the metabolic stability of this agent and effect duration; Experimenter's species, age, body weight, general health, diet situation (dietary status), sex and diet (diet); Mode of using and time; Discharge rate; Drug regimen (if having); And the performance degree of the particular disorder that will treat and/or the order of severity and difference.Accurately but the many tests of dosage no mistake in treatment are determined by those of ordinary skills, reach expected result to use once a day or several times, and dosage also can be adjusted by independent doctor and reaches the expectation therapeutic effect or adjust when any complication takes place.Importantly, when being used for the treatment of cancer, the dosage of the therapeutic agent of use should be enough to suppress or kill tumor cell and normal cell preserves from basically.

But treatment that comprises in pharmaceutical composition of the present invention or diagnostic agent can be prepared with the maximum amount of any requirement to safely use in the patient.The weight range of diagnostic agent or therapeutic agent can be from being less than 0.01mg/mL to being higher than 1000mg/mL, preferably about 50mg/mL.

Generally speaking, pharmaceutical composition of the present invention will be sent effectively regulating the structure of PDH complex and/or the mode that is applied to the patient of active amount being enough to.Therefore, dosage range can be from about 0.3mg/m ²To 2000mg/m ², preferably about 60mg/m ²Dosage can potion or is used with each form of separating agent, as once a day to four times or more times.Replying under the insufficient situation of experimenter under a certain dosage, can use the high dose more effectively more high dose of different, more concentrated route of delivery (or by), to the degree of patient's tolerance.For reaching treatment or suitable system or the targeting level of diagnostic agent, can consider multi-agent every day.

In another one embodiment of the present invention, lipoic acid derivatives of the present invention also can be used as external diagnosis and prediction agent.As previously mentioned, according to the specific tumors cell or the cell type of touching upon, different lipoic acid derivatives may have more or less effectiveness by regulating the PDH complex to suppressing different tumor kinds.Therefore, for instance, in diagnosis or select under the suitable chemotherapy strategy situation of difficult, provide a kind of tumor type and optional method of effectively treating identified to the mensuration of tumor cell in vitro culture with the lipoic acid derivatives of known targeting specific tumors cell type.

Turn to accompanying drawing, energy metabolism many one of may differences in normal structure and the tumor cell in vivo in Fig. 6 A diagram.Compare with the normal cell of corresponding state, the ATP of tumor cell produces and often depends on cytoplasmic glycolysis more but not the mitochondrion energy metabolism.The PDH complex is expressed and the change of adjusting obviously is the part that this tomour specific adapts to.The reduction of PDH catalyst component level and/or the increase of level that produces the inhibition PDK of these effects can make the tumor cell ratio normal cell to the medicament of attacking PDH complex susceptible more.

The structure of the thioctic acid when Fig. 6 B describes the normal reaction that catalysis relates in the acetone acid synthesis of acetyl coenzyme A from the PDH complex.In vivo, thioctic acid is connected in the ε amino of lysine on the E2 sulfur decoyl domain avtive spot by the c-terminus on its non-peptide amide bond.Notice that simultaneously oxidation/reduction that PDH E2-fault sulphur is sad/acetylation state is that active kinases of controlled PDH and phosphatase are monitored by the phosphorylation inactivation of control PDH E1 α subunit.This figure has also described the structure of three kinds of representational lipoic acid derivatives that may use in the present invention.CPI-613 and CPI-045 have high anticancer tiring, and CPI-157 pair cell culture seldom or non-activity and be used as contrast in several tests.

Fig. 6 C shows the relation between the PDH complex component, and PDH complex component comprises E2, the E1 of its bonded thioctic acid, the PDK of adjusting.The high level of acetyl-thioctic acid or dihydrolipoic acid (not shown) activates PDK, and it is conversely by inactivation E1 α, and promptly the subunit of the first step in the catalysis PDH complex catalytic reaction suppresses the more multithread amount by the PDH complex.As Fig. 3 A finding, this process is served as the actuator of the carbon/energy stream by the PDH complex, and this adjustment process is changed significantly to support the energy metabolism of tumor cell variation.

Fig. 7 shows the effect of pharmaceutical composition of the present invention to the xenotransplantation tumor growth.As described in embodiment 2, be implanted into the mice drosal part under the cell skin.Then, injectable drug (or vehicle only in mouse peritoneum; " simulation "), initial natural law is as shown in FIG..Left side diagram the present invention presses 1mg/kg or vehicle contrasts the pancreas tumor model of 3 injections weekly.This test is typical case's representative made from twice of AsPC-1 cell that twice usefulness BxPC-3 cell done.Right side three width of cloth figure show press shown in concentration weekly (circle), time (inverted triangle) or time (the vehicle treatment is a triangle on every Fridays on every Wendesdays; Drug therapy be the square) injection H406 lung tumor model.This test is four typical case's representatives of making of the H460 cell.

Fig. 8 shows that pharmaceutical composition of the present invention is by 200-300 μ M (" treatment ") or simulation treatment (" the simulation treatment ") treatment effect to three kinds of tumor cell types and a kind of no transformed cells type (MDCK).Cell was treated 48 hours in containing the suitable tissue culture medium (TCM) of 10% serum.Observe by embodiment 2 described methods by apoptosis or a large amount of cell deaths of class apoptosis pathway (also seeing Figure 11) in three kinds of cancer cell systems.On the contrary, unconverted mdck cell is not obviously influenced by the Drug therapy of this dosage.

Fig. 9 A shows the ATP level with H460 lung carcinoma cell behind lethal threshold (200 μ M are in 10% serum) or the above medicine composite for curing of the present invention.The treatment of concentration shown in the dotted line representative is pressed.The treatment of time shown in the representative of the solid line of corresponding thickness continues is removed medicine and recovery 60 minutes in no pharmaceutical culture medium subsequently.Arrow is represented the interval that ATP recovers.

Fig. 9 B compared with acetone acid (with the form of methyl-acetone acid) as the culture medium neutralization of main carbon source (dotted line) with glucose as the culture medium of main carbon source (solid line) in the synthetic inhibitory action of ATP.Notice that pharmaceutical composition of the present invention finally produces cell death with identical threshold concentration in two kinds of culture medium; Yet, contain total cell ATP level in the culture medium of acetone acid and consume height in early days and contain in the culture medium of glucose and do not have early stage consumption.And, 300 μ M cell death initial faster than the time at 200 μ M drug level.

Fig. 9 C has compared the synthetic inhibitory action of ATP of pharmaceutical composition of the present invention in SK-Br-3 breast cancer cell and HMEC normal breast cell.Be different from the result of the test that is depicted in Fig. 6 A and Fig. 6 B, these tests are done in serum-free medium (MEBM).As a result, the lethal threshold of medicine is lower, about 50 μ M.Note a small amount of decline of ATP level in 22-hour sample of normal cells and drug dose is irrelevant and the normal test of reflection difference.

Fig. 9 D has compared pharmaceutical composition of the present invention (left figure), thioctic acid (middle figure) and inactive form of the present invention (right figure) to the synthetic inhibition of ATP in the H460 lung carcinoma cell.In Fig. 6 C, these tests are done in serum-free medium, so the lethal threshold of medicine is about 50 μ M.

Figure 10 shows the effect of pharmaceutical composition of the present invention (with 400 μ M in the DMEM that contains 10% serum) to the tumor cell mitochondrion level of PDH (PDC) and (α KDH) enzymatic activity.Notice that PDH is subjected to press down strongly, α KDH is not then.As described in embodiment 2, enzyme activity level is to use the "diazoresorcinol" reduction of the carbon source that responds to adding to measure in the mitochondrial extract of purification.The background line is corresponding to the "diazoresorcinol" reduction that lacks the carbon source of adding.

Then, in Figure 11 A, the extract that the H460 lung carcinoma cell of (-) is treated in (+) or the simulation of the pharmaceutical composition of the present invention of using by oneself (continuing 120 minutes by 400 μ M in containing the RPMI culture medium of 10% serum) treatment carries out the Western analysis of two-dimensional electrophoresis gel.Western shifts thing and is surveyed by the mixture at the monoclonal antibody of PDH complex E1 α and E2 subunit.Western shifts thing and aligns at the E2 subunit.The hyperphosphorylation form level of noting E1 in the Drug therapy sample obviously raises and low phosphorylation form level obviously reduces.Left side vertically white line shows one of standard to homogenous gel, the migration of E2 subunit.The vertical white line in right side passes the alpha form than low phosphorylation E1, the enzyme active component of supposing.

Figure 11 B shows the amplification of the pairing two-dimensional electrophoresis gel sample of medicine composite for curing of the present invention and simulation treatment.Part A is the amplification of the part of Fig. 8 A.Part B is the SK-Br-3 breast cancer cell that continues simulation treatment (-) in 180 minutes and treatment (+) with 80 μ M compositionss in MEBM serum-free galactophore epithelial cell culture medium.Portion C is the SK-Br-3 breast cancer cell that continues simulation treatment (-) in 240 minutes and treatment (+) with 80 μ M compositionss in MEBM serum-free galactophore epithelial cell culture medium.Part D is the HEMC normal breast cell that continues simulation treatment (-) in 240 minutes and treatment (+) with 80 μ M compositionss in MEBM serum-free galactophore epithelial cell culture medium.Vertically white line passes the alpha form than low phosphorylation E1, the enzymatic activity composition of supposing.

Figure 12 A and 12B have described pharmaceutical composition of the present invention effect hypothesis powerful in vivo, the selectivity anticarcinogenic effect.Regulating action when having shown that such as, Figure 12 A endogenous thioctic acid that PDK is covalently bonded in PDC E2 subunit is regulated.Respond to the high level of reductive and/or acetylation thioctic acid under the PDK normal condition and make the PDC inactivation, this process is obviously changed in tumor cell.

Correspondingly, Figure 12 B has shown the huge quantitative differences of the ratio of PDK and its substrate PDC-E1 among the PDC, is considered to the normal and tumor cell of difference in vivo.Low-level being considered to of PDK replaces " migration " (by subunit of two dimerization) around the PDH complex in the normal cell, makes the E1 phosphorylation gradually.The dephosphorylation of this phosphorylation and PDP is in (not shown) in the homeostasis.In this illustrated effect hypothesis, thioctan, activates one or more PDK isotypes thus artificially and makes E1 α inactivation in conjunction with the same loci stimulation PDK of acetyl group-thioctic acid and/or two dehydrogenation thioctic acid by normally.In the cancerous cell, obviously higher PDK level may make this stimulation more be effective in by thioctan and close PDC enzymatic activity and mitochondrion energy metabolism.

Provide following limiting examples to promote to understand pharmaceutical composition of the present invention.

Embodiment 1

The chemosynthesis of thioctan

Lipoic acid derivatives (being thioctan) CPI-613 and CPI-157 are with 6, and the 8-dimercapto octanoic acid passes through to use the U.S. 6,331 that improves as starting material, the 559B1 and the U.S. 6,951, and the described method of 887B2 is synthetic.Thioctan CPI-045 is according to US 6,331, and 559B1 is described synthetic.

The structural analysis of these three kinds of thioctan is as follows.A plurality of independently the synthesizing on its anticancer property of CPI-613 and CPI-157 is undistinguishable.Measure the purity of the CPI-613 that uses in (Fig. 9) above 99% in heteroplastic transplantation (Fig. 7) and ATP.Every other prepared product is greater than 98% purity.

CPI-613:6,8-dibenzyl sulfide alkyl is sad: white crystalline solid, fusing point 65-66 ℃ (document ¹67.5-69 °); ¹H-NMR (250MHz, CDCl ₃): δ 7.15-7.4 (m, 10H), 3.66 (s, 2H), 3.64 (s, 2H), 2.52-2.62 (m, 1H), 2.50 (t, J=7.6Hz, 2H), 2.29 (t, J=7.6Hz, 2H), 1.2-1.8 (m, 8H); ¹³(62.9MHz, CDCl3): δ 179.6,138.6,138.5,128.9,128.8,128.5,128.4,126.9,44.1,36.4,35.1,34.4,33.8,28.7,26.0,24.4. for C-NMR

CPI-157:6,8-diethyl sulfide alkyl is sad: colorless oil; TLC (EtAc: hexane: HAc, 200: 200: 1v/v): R _f=0.60; ¹H-NMR (300MHz, CDCl ₃): δ 2.64-2.76 (m, IH), 2.65 (t, J=7.5Hz, 2H), 2.52 (q, J=7.5Hz, 2H), 2.49 (q, J=7.2Hz, 2H), 2.36 (t, J=7.4Hz, 2H), 1.40-1.85 (m, 8H), 1.25 (t, J=7.2Hz, 3H), 1.22 (t, J=7.5Hz, 3H); ¹³C-NMR (75MHz, CDCl ₃): δ 180.0,44.3, and 34.6,33.9,28.9,26.2,25.9,24.5,24.2,14.9,14.7; IR (film): 2963,1708,1449,1423,1283,1263cm ^-1

CPI-045:6,8-two-benzoyl sulfane base is sad: colourless viscosity grease; TLC (hexane: EtAc: HAc, 100: 50: 1v/v): R _f=0.30; ¹H-NMR (250MHz, CDCl ₃): δ 7.9-8.1 (m, 4H), 7.38-7.60 (m, 6H), 3.8-4.0 (m, IH), 3.0-3.3 (m, 2H), 2.34 (t, J=7.1Hz, 2H), 1.4-2.2 (m, 8H); ¹³C-NMR (62.9MHz, CDCl3): δ 191.7,191.5, and 179.7,137.0,136.9,133.3,128.5,127.3,127.1,43.6,35.0,34.6,33.8,26.4,26.2,24.3; IR (film): 2973,1710,1704,1667,1665,1662,1448,1207,1175,911,773,757,733,688,648cm ^-1

Embodiment 2

Be used for determining the method for THIOCTAN anticancer function

Cell: human tumor cell line is obtained from ATCC and breeds according to the ATCC suggestion.People's galactophore epithelial cell (HMEC), stingy tract epithelial cell (SAEC) and normal person's epidermal keratinocytes (NHEK) primary cell is obtained from LONZA Walkersville, and Inc (Walkersville, MD).Every kind of cell line all supplier's exploitation, keep and breed according to supplier's explanation in available from supplier's appropriate culture medium.The normal cell in 3 to 6 generations is used in the test of report herein.

Tumor growth suppresses research: people BxPC-3 or AsPC-1 pancreatic tumor cell or H460NSCLC are transplanted the female Mus in CD1-Nu/Nu by subcutaneous injection (SC).After about 8-12 days, by dosage shown in the legend and timetable intraperitoneal (IP) injection mice.Medicine or vehicle are by the about 2ml injection of every 25gm body weight.Drug level is 1.25mg/ml (approximately 3.1mM) or still less.Vehicle/solvent is that 25mM or triethanolamine aqueous solution still less constitute by concentration.The vehicle of simulation treatment animal injection always with this time test in injection the highest drug dose solvent phase with.Monitor health and the mortality rate of mice every day.Measure body weight and gross tumor volume every day before the treatment, measures weekly about three times in the treatment and after the treatment.Mice is maintained at bright/dark circulation in 12 hours, arbitrarily obtains food and stable breeding in Stony Brook university Animal House according to mechanism's guideline.

Cell death is measured: when not suppressed to disturb by the synthetic thioctan of early stage ATP when the time long enough, most of cell survival is measured and is used CellTiter-Glo reagent (Promega).(Fig. 9) in the model experiment, cell is plated on black, clear bottom 96-orifice plate by 5,000 cells in every hole.After 18-25 hour, replace culture medium with the fresh culture that contains the CPI-613 thioctan in medicine solvent (in the triethanolamine aqueous solution blood serum medium by in 2.8mM and the serum-free medium by 0.7mM) or the same solvent.According to drug dose, add the back according to producer's explanation at medicine and measured in 24 or 48 hours.

In some cases, cell is plated on the 48-orifice plate by 10,000 cells in every hole, and after 18-25 hour, replaces culture medium with the fresh culture that contains the CPI-045 thioctan of variable concentrations in medicine solvent (about 1% ethanol final concentration) or the same solvent.Cell is maintained at solvent or the pastille culture medium is tested with continuation.Observed flat board in 24,48,72 hours behind the adding medicine, and the estimation cell number is fusion percentage ratio.Under these conditions, under the dosage near threshold value, the inductive cell death of thioctan is the height apoptosis, and the cell number of estimation is the very reliable indicant of death.(Figure 10) the cell integrity that kept at 72 hours if having, is measured by trypanblue exclusion method.

Table 2 provides the external effect data to tumor cell about thioctan.Listed is people's tumor and the people's primary cell that we have investigated the sensitivity that CPI-613 and/or CPI-045 are killed and wounded.The cell death of apoptosis or paranecrosis appears in "+" expression cell under the dosage at serum-free medium at about 200-300 μ M (containing 10% serum) dosage and about 50 μ M.(Fig. 8 and 9) "--" represents that these cells need about 5 times of high doses to come inducing cell death in corresponding culture medium." nt " represents untested combination.As the MDCK normal cell among Fig. 8, all tumor cells tie up in the appropriate culture medium that contains 10% serum and analyze.In addition, HMEC, SAEC, NHKC people's primary cell and SK-BR-3, A549 and H460 tumor cell line are also analyzed in suitable serum-free medium.Primary cell be touched suppress and cell transformed density suitable.

ATP measures: cell is plated on 96-orifice plate at the bottom of the black transparent by 5,000 cells in every hole.Behind the 18-25h, by shown in interval and drug level replace culture medium with containing medicine solvent (triethanolamine) or thioctan (CPI-613 or CPI-157) or the fresh culture of thioctic acid.Cell survival and integrity are measured with trypanblue exclusion method by removing to reclaim behind the medicine.Use CellTiter-Glo luminescence assays (Promega) to measure ATP according to shop instruction.All measurements all repeat to carry out, and the apparent altitude concordance.The standard error scope of average is the 0.1-2% of measured value.Therefore, error bar is omitted among Fig. 9.Methyl-prop keto acid culture medium among Fig. 9 is made of the RPMI (Invitrogen) of no glucose, be added with 10% hyclone, 5mM HEPES (pH7.4) and 10mM methyl-prop keto acid (Sigma-Aldrich), and corresponding dextrose culture-medium is conventional RPMI (Invitrogen) through dialysis.

PDH and α KDH enzymatic determination: tumor cell grows to 80% fusion on the 15cm flat board, and treats with CPI-613 shown in pressing.According to Moreadith and Fiskum ¹The method separate mitochondria.Mitochondrion cracking in 0.4% lauryl maltoside.50 μ l mitochondrion lysates are added to the 96-orifice plate.(50mM Tris, pH 7.5,2mM β-NAD+, 225uMv TPP, 2mM acetone acid or α-Tong Wuersuan, 150 μ M coenzyme As, 2.6mM cysteine, 1mM MgCl to add 50 μ l reactant mixtures to the mitochondrion lysate ₂), and mixture hatched 45 minutes at 37 ℃.At this moment, add 15 μ M "diazoresorcinol"s and 0.5U/ml diaphorase to mixture and continued to hatch 5 minutes.Use 530nm excitation wavelength and 590nm emission wavelength to measure the generation that fluorescence is monitored NADH by going up in microplate reader (Fluorostar).All mensuration all repeat to carry out, and show the height concordance.The standard error scope of average is the 0.3-4% of measured value.Therefore, error bar is omitted among Fig. 7.

E1 α phosphorylation:

The 2-D gel of cell lysate: cell grows to 95% and merges on the 60mm plate, and with shown in medicine or solvent treat.With 450 μ l lysis buffer A[455 μ l Zoom 2D albumen cosolvents, 1 (Invitrogen), 2.5 μ l IM Tris alkali, 5 μ l 100X protease inhibitor cocktails (Complete min, no EDTA, Roche); 5 μ l 2M DTT] the original position cell lysis.Cell lysate is transferred to 1.5ml microcentrifugal tube and ultrasonic 15 samsaras of 50% power on ice.Incubated at room adds 2.5 μ l DMAAs after 10 minutes (DMA Sigma-Aldrich), and continues to hatch 10 minutes lysates.Add 5 μ l 2M DTT with the excessive DMA that neutralizes.With lysate centrifugal 15 minutes at 16,000 * g.

The 2-D gel: we use Zoom Benchtop protein groups system (Invitrogen) to specifications.In brief, with the ampholyte of 30-50 μ l lysate and 0.8 μ l pH 3-10,0.75 μ l 2M DTT mixes, and is settled to 150 μ l with Zoom 2D albumen cosolvent 1.150 μ l sample pipetting volumes to the IPG electrophresis apparatus, and are added pH 3-10NL IPG adhesive tape.With soaked overnight under the IPG adhesive tape room temperature.Step-by-step program is used for isoelectrofocusing (250V, 20 minutes; 450V, 15 minutes; 750V, 15 minutes 2000V, 30 minutes).Adhesive tape was handled 15 minutes with 1 * sample-loading buffer, and reuse adds 1 * sample-loading buffer of 160mM iodoacetic acid to be handled 15 minutes.Adhesive tape is gone up electrophoresis at NuPAGE 4-12%Bis Tris ZOOM glue (Invitrogen).

Table 2:thioctan is to the vitro effect of tumor and primary cell

Western: Western blot is in PVDF 4.5 μ m films.Use monoclonal antibody (Invitrogen) to detect PDHE1 α and E2.

Caspase-3 and PARP cracking: according to Roy and Nicholson ⁴³Detect cracked Caspase-3 at the Western trace.In brief, after the treatment of medicine or solvent, scrape cell and 6,000 * g is centrifugal with medium/cells/apoptosis body mixture.With lysis buffer C (4M carbamide, 10% glycerol, 2%SDS, 0.003%BPB; The 5%2-mercaptoethanol) cracking precipitation.With sample on the total lysis albumen of every hole 30 μ g to 12%Bis-Tris glue.Western blot is in PVDF 4.5 μ m films.Monoclonal antibody (Mus monoclonal [31A1067] with anti-Caspase-3; Abcam) detect former-Caspase-3 and activity-Caspase-3.Use anti--poly-(ADP-ribose) polymerase monoclonal antibody, clone C-2-10 (Sigma-Aldrich) detects the PARP cracking.

Mitochondrion Ca ^{+ 2}Detect: cell is by 3 * 10 ⁵Be inoculated in 35mm glass film plates (BD Biosciences), grow overnight, and with shown in the treatment of medicine or solvent.Then, (4 μ M Invitrogen) add like cell, and hatch 10 minutes at 37 ℃ to use calcium dyestuff Fluo-4, X-Rhod-1 or Rhod-2 in no phenol red medium.PBS washes cell once, and uses the FITC filter plate, uses Axiovert 200M (Zeiss) microscope that deconvolutes to catch picture in fixed open-assembly time.Use the software that provides by producer to carry out fluorescent quantitation.The result of X-Rhod-1 and Rhod-2 similar (Figure 10) illustrates these dyestuff slotted line plastochondrias Ca ^{+ 2}Signal. ^3-4

List of references:

1.Moreadith RW and Fiskum G.Isolation of Mitochondria from Ascites Tumour-Cells Permeabilized with Digitonin (the mitochondrial separation from the ascites tumour cell of lanatoside dialysis) .Analytical Biochemistry 137,360-367 (1984).

2.Roy S and Nicholson DW.Criteria for identifying authentic caspase substrates during apoptosis (differentiating the standard of Caspase substrate real in the apoptosis) .Apoptosis 322,110-125 (2000).

3.Gerencser AA and Adam-Vizi V.Selective, high-resolution fluorescence imaging of mitochondrial Ca ²⁺Concentration (mitochondrion Ca ^{+ 2}The selectivity high-resolution fluorescence imaging of concentration) .Cell Calcium 30,311-321 (2001).

4.Gyorgy H, Gyorgy C, Das S, Garcia-Perez C, Saotome M, Roy SS and Yi MQ.Mitochondrial calcium signalling and cell death:Approaches for assessing the role of mitochondrial Ca ²⁺Uptake in apoptosis (conduction of mitochondrial calcium signal and cell death: mitochondrion Ca in the assessment apoptosis ^{+ 2}The method of absorption effect) .Cell Calcium 40,553-560 (2006).

Embodiment 3

Thioctan interfering line mitochondrial membrane potential and Ca ^{+ 2}Take in

Thioctan shows that to the synthetic substrate effect (Fig. 9) that suppresses of ATP the TCA in this medicine interfering line mitochondrial matrix circulates.If so, we expect mitochondrial membrane potential really ¹May reduce at lethal threshold dosage and when above.Use the dyestuff TMRE of current potential-sensitivity, we observe the expectation effect.(Figure 13) along with the beginning of Drug therapy, mitochondrial membrane potential descends fast.The kinetics that transmembrane potential descends extremely is similar to the synthetic loss of ATP when having the mitochondrion substrate.(Fig. 9)

The minimizing of ATP excites the Ca that comprises that absorption discharges from Cytoplasm deposit (comprising endoplasmic reticulum) in the known mitochondrion ^{+ 2}The balance homeostatic reaction of picked-up.And, think this Ca ^{+ 2}Input to mitochondrial matrix needs mitochondrial membrane potential.Therefore, we expect that thioctan can produce lasting Cytoplasm Ca at lethal threshold or above treatment based on the transmembrane potential that continues decline ^{+ 2}Release follows this instantaneous ionic mitochondrion to take in.Use X-Rhod-1 and Rhod-2 to measure mitochondrion Ca ^{+ 2}And use Fluo-4 to measure Cytoplasm Ca ^{+ 2}, we observe these intended effects.(Figure 13).

To with lethal threshold and high slightly CPI dosage (comparison diagram 9 and 13) about 2 hours, along with mitochondrial membrane potential decline, this initial mitochondrion Ca ^{+ 2}Transition descends.The second largest mitochondrion Ca that is considered to relevant with the unlatching of calcium dependent cell death approach appearred at 4-6 hour subsequently ^{+ 2}The peak. ³

List of references:

1.Garrett R and Grisham CM.Biochemistry.Thomson Brooks/Cole, Southbank, Vic, Australia; Belmont, CA. (2007).

2.Graier WF, Frieden M and Malli R.Mitochondria and Ca ²⁺Signaling:old guests, new functions (mitochondrion and Ca ²⁺Signal conduction: old thing, new function) .Pflugers Archiv-European Journal of Physiology 455,375-396 (2007).

3.Gyorgy H, Gyorgy C, Das S, Garcia-Perez C, Saotome M, Roy SS and Yi MQ.Mitochondrial calcium signalling and cell death:Approaches for assessing the role of mitochondrial Ca ²⁺Uptake in apoptosis (conduction of mitochondrial calcium signal and cell death: mitochondrion Ca in the assessment apoptosis ²⁺The method of absorption effect) .Cell Calcium 40,553-560 (2006).

Embodiment 4

Thioctan brings out the dead program of various kinds of cell

Although detailed mechanism is not clear fully, the reduction of known mitochondrion in some cases energy metabolism is relevant with the decision that enters the cell death approach. ^1-3Be higher than lethal threshold but in 2 times of this minimal lethal doses the time, the cancerous cell type experience morphology of all tests mainly is similar to apoptotic cells death (Fig. 8) at thioctan dosage.Apoptosis formula death under these situations confirms (result does not show) by the annexin immunostaining and the TUNEL DNA end-marker determination of routine.

Higher drug dose (it is about more than 2 times to surpass lethal threshold), active thioctan inducing cell death (as with bed board (replating) survival mensuration and trypan blue or third pyridine eliminating are evaluated again), not have morphology relevant with apoptosis, hints downright bad sample approach (result does not provide).

These data acknowledgements thioctan CPI-613 suppresses the mitochondrion energy metabolism and really induces relevant with cell death.

Known or supposition contains different and various cell death approach ⁴Various tumor cell of inactivation sudden change all be killed (Fig. 8 and table 2) with extremely similar thioctan dosage, this discovery is startling.This finds these drug-induced a kind of main signals that can participate in the far-end cell death execution path of a plurality of, potential surplus of hint. ⁵

Consistent with this probability, we find that Z-VAD-FMK class Caspase inhibitor changes the cell death form in the thioctan treatment cell knifeedge, but the lethal threshold dosage of medicine is not had discernible effect.

Can carry out the probability that mechanism are carried out by a plurality of terminals for further studying the inductive cell death of thioctan, we have checked the diagnosis of Caspase-3 with PARP-1 cracking-different cell death approach. ⁵We find that thioctan CPI-613 and CPI-045 induce the level of the alterable height of these two kinds of cracking incidents in different cells.(Figure 14)

In a word, these results show: thioctan can induce the strategic assurance to death, and according to drug dose and cell type, it is unpredictable that this strategic assurance is carried out for the terminal tactics of this decision.

List of references:

1.Watabe M and N akaki T.ATP depletion does not account for apoptosis induced by inhibition of mitochondrial electron transport chain in human dopaminergic cells (the ATP minimizing can not be explained by mitochondrion electron transport chain in people's dopaminergic cell and be subjected to press down inductive apoptosis) .Neuropharmacology 52,536-541 (2007).

2.Yuneva M, Zamboni N, Oefner P, Sachidanandam R, with Lazebnik Y.Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells (glutamine but not glucose defective are induced the apoptosis that MYC-relies in people's cell) .Journal of Cell Biology 178,93-105 (2007).

3.Skulachev VP.Bioenergetic aspects of apoptosis, necrosis and mitoptosis (the bio-energy aspect of apoptosis, necrosis and mitochondrion apoptosis) .Apoptosis 11,473-485 (2006).

4.Johnstone RW, Ruefli AA, and Lowe SW.Apoptosis:A link between cancer genetics and chemotherapy (apoptosis: the .Cell108 tie between cancer heredity and the chemotherapy), 153-164 (2002).

5.Cregan SP, Dawson VL and Slack RS.Role of AIF in caspase-dependent and caspase-independent cell death (effect in the cell death that relies on non-Caspase that AIF relies at Caspase) .Oncogene 23,2785-2796 (2004).

Embodiment 5

The structure of lipoic acid derivatives analog

A plurality of limiting examples of the following lipoic acid derivatives analog that provides have been produced and are open at this paper.

Aforementioned discussion only disclosure and description exemplary embodiment of the subject disclosure.According to this discussion and claims, those skilled in the art will recognize the spirit and scope of the present invention that wherein can make various changes, improvement and adjustment and not deviate from following claim and limited easily.And though explained exemplary embodiment at this, other embodiments that implement this area also can be learned other designs of the present invention or use.Therefore, though the present invention is described together with its exemplary, the many improvement that should understand in design and use are obvious to those skilled in the art, and expectation the application comprises its any adjustment or change.Therefore expect that clearly the present invention is limited by claim and equivalent thereof only.

Claims (53)

1. comprise at least a enzyme in the mitochondrion of human Homoiotherm diseased cells and/or multienzyme complex or its subunit structure, expression and/or active adjustment or the acceptable regulator of interferential pharmacy such as pyruvic dehydrogenase (PDH) complex of modifying.

2. regulator as claimed in claim 1, wherein said adjustment or interference comprise reversible phosphorylation or dephosphorylation.

3. regulator as claimed in claim 2, wherein said reversible phosphorylation or dephosphorylation occur on kinases, phosphatase and/or the dehydrogenase of enzyme or multienzyme complex or its subunit.

4. regulator as claimed in claim 3, wherein said regulator promote or the inhibition kinase activity.

5. regulator as claimed in claim 4, wherein said kinases are selected from the group that comprises pyruvic dehydrogenase kinase (PDK) 1, PDK2, PDK3, PDK4 and isoform separately thereof.

6. regulator as claimed in claim 3, wherein said regulator promote or the inhibition phosphatase activity.

7. regulator as claimed in claim 6, wherein said phosphatase are selected from the group that comprises pyruvic dehydrogenase phosphatase (PDP) 1, PDP2 and isoform separately thereof.

8. regulator as claimed in claim 3, wherein said regulator promote or the inhibition dehydrogenase activity.

9. regulator as claimed in claim 2, wherein said reversible phosphorylation or dephosphorylation occur on the described PDH complex.

10. regulator as claimed in claim 9, wherein said adjusting occur on the E1 α subunit of described PDH complex.

11. regulator as claimed in claim 10, wherein said adjusting is by inactivation PDP and isoform and mutant forms generation.

12. regulator as claimed in claim 11, wherein said inactivation PDP expresses generation by suppressing PDP.

13. regulator as claimed in claim 10, wherein said adjusting is by activation PDK and isoform and mutant forms generation.

14. it is responsive or insensitive that regulator as claimed in claim 1, wherein said diseased cells demonstrate the treatment with the described regulator of claim 1.

15. regulator as claimed in claim 14, wherein treatment-insensitive diseased cells can be by the enzyme of at least a modification of abduction delivering or multienzyme complex or its subunit, so that they are to treating sensitivity.

16. regulator as claimed in claim 15, it is inductive by genetic manipulation wherein expressing.

17. regulator as claimed in claim 16, wherein said inducing by transcribing operation realized.

18. regulator as claimed in claim 16, wherein said inducing by translating operation realized.

19. regulator as claimed in claim 16, wherein said inducing by translating the back operation realized.

20. regulator as claimed in claim 15, it is inductive by outer genetic manipulation wherein expressing.

21. regulator as claimed in claim 15, it is inductive by the phenotype operation wherein expressing.

22. regulator as claimed in claim 14, wherein when with the described modulators for treatment of claim 1, described diseased cells is expressed the enzyme or the multienzyme complex of at least a modification.

23. regulator as claimed in claim 1, wherein said regulator influences the expression of PDK and isoform and mutant forms.

24. regulator as claimed in claim 1, wherein said regulator influences the expression of PDP and isoform and mutant forms.

25. as claim 23 or 24 described regulators, wherein said expression changes on the level after transcribing, translate or translating.

26. regulator as claimed in claim 25, wherein said change is epigenetic.

27. regulator as claimed in claim 9, wherein said regulator suppresses the generation of toxic metabolite.

28. regulator as claimed in claim 9, wherein said regulator promotes the detoxifcation of toxic metabolite.

29. as claim 27 or 28 described regulators, wherein said metabolite is selected from the group of being made up of acetaldehyde, superoxides, hydrogen peroxide and hydroxyl radical free radical.

30. regulator as claimed in claim 28, wherein regulating action is to observe by the reduction that acetoin generates.

31. regulator as claimed in claim 9, wherein said reversible phosphorylation or dephosphorylation become irreversible.

32. regulator as claimed in claim 31, wherein said phosphorylation or dephosphorylation effect cause cell death.

33. regulator as claimed in claim 32, wherein said effect is an apoptosis.

34. regulator as claimed in claim 32, wherein said effect are downright bad.

35. regulator as claimed in claim 1 comprises at least a lipoic acid derivatives and at least a its pharmaceutically acceptable carrier.

36. regulator as claimed in claim 35, wherein said lipoic acid derivatives have following formula, its metabolite or its salt:

R wherein ₁And R ₂Be independently selected from the group of forming by following: hydrogen, alkyl C _nH _2n+1, alkene C _nH _2n, thiazolinyl C _nH _2n-1, alkynes C _nH _2n-2, alkynyl C _nH _2n-3, alkyl sulfide CH ₃(CH ₂) _n-S-, alkyl disulfide CH ₃CH _t-S-S-, thiocarbamate (CH ₂) _nC=NH-and hemimercaptol CH ₃CH (OH)-S-, wherein n is that 1-10 and t are 0-9; Aromatic group; Be defined as R ₃C (O)-acyl group; Heteroaryl; Be defined as R ₄C (=NH)-the imines acyl; The organic metal aryl; Alkyl organic metal aryl; With hemiacetal R ₅CH (OH)-S-;

R as defined above wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃Be selected from the group of forming by following: hydrogen, thiazolinyl, alkynyl, alkylaryl, heteroaryl, miscellaneous alkyl aryl and organic metal aryl, wherein any can be that replace or unsubstituted;

R wherein ₄Be selected from by the following group of forming: hydrogen, thiazolinyl, alkynyl, aryl, alkylaryl, heteroaryl and miscellaneous alkyl aryl, wherein any can be that replace or unsubstituted;

R wherein ₅Be CCl ₃, CF ₃Or COOH;

Wherein x is 0-16.

37. regulator as claimed in claim 35, wherein said lipoic acid derivatives have following formula, its metabolite or its salt:

Wherein M be covalent bond ,-[C (R ₁) (R ₂)] _z-or metallo-chelate or other metal complex, wherein said metal is not a palladium;

R wherein ₁And R ₂Be independently selected from by the following group of forming: hydrogen, acyl group R ₃C (O)-, alkyl C _nH _2n+1, be defined as C _nH _2n-1Thiazolinyl, be defined as C _nH _2n-3Alkynyl, aryl, heteroaryl, alkyl sulfide CH ₃(CH ₂) _n-S-, be defined as R ₃C (=NH)-the imines acyl and be defined as R ₄The hemiacetal of CH (OH)-S-;

R as defined above wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃And R ₄Be independently selected from the group of forming by following: hydrogen, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclic radical, wherein any can be that replace or unsubstituted;

R wherein ₅Be selected from the group of forming by following :-CCl ₃,-CF ₃Or-COOH;

Wherein x is 0-16, and z is that 0-5 and n are 0-10.

38. regulator as claimed in claim 35, wherein said lipoic acid derivatives have following formula, its metabolite or its salt:

R wherein ₁And R ₂Be independently selected from the group of forming by following: hydrogen, alkyl C _nH _2n+1, alkene C _nH _2n, thiazolinyl C _nH _2n-1, alkynes C _nH _2n-2, alkynyl C _nH _2n-3, alkyl sulfide CH ₃(CH ₂) _n-S-, alkyl disulfide CH ₃CH _t-S-S-, thiocarbamate (CH ₂) _nC=NH-and hemimercaptol CH ₃CH (OH)-S-, wherein n is that 1-10 and t are 0-9, aromatic group, is defined as R ₄C (O)-acyl group, heteroaryl, be defined as R ₅C (=NH)-imines acyl, organic metal aryl, alkyl organic metal aryl, hemiacetal R ₆CH (OH)-S-, aminoacid, saccharide, nucleic acid, lipid and polymer and combination;

R wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃Be selected from the group of forming by following: aminoacid, saccharide, nucleic acid, lipid and polymer thereof;

R wherein ₄Be selected from the group of forming by following: hydrogen, thiazolinyl, alkynyl, alkylaryl, heteroaryl, miscellaneous alkyl aryl and organic metal aryl, wherein any can be that replace or unsubstituted;

R wherein ₅Be selected from the group of forming by following: hydrogen, thiazolinyl, alkynyl, aryl, alkylaryl, heteroaryl and miscellaneous alkyl aryl, wherein any can be that replace or unsubstituted;

R wherein ₆Be CCl ₃, CF ₃Or COOH;

Wherein x is 0-16.

39. regulator as claimed in claim 35, wherein said lipoic acid derivatives have following formula, its metabolite or its salt:

Wherein M be covalent bond ,-[C (R ₁) (R ₂)] _z-, or metallo-chelate or other metal complex, wherein said metal is not a palladium;

R wherein ₁And R ₂Be independently selected from the group of forming by following: hydrogen, acyl group R ₄C (O)-, alkyl C _nH _2n+1, be defined as C _mH _2m-1Thiazolinyl, be defined as C _mH _2m-3Alkynyl, aryl, heteroaryl, alkyl sulfide CH ₃(CH ₂) _n-S-, be defined as R ₄C (=NH)-the imines acyl, be defined as R ₆Hemiacetal, aminoacid, saccharide, nucleic acid, lipid and polymer thereof and the combination of CH (OH)-S-;

R wherein ₁And R ₂It can be unsubstituted or replacement;

R wherein ₃Be selected from the group of forming by following: aminoacid, saccharide, nucleic acid, lipid and polymer thereof;

R wherein ₄And R ₅Be independently selected from the group of forming by following: hydrogen, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclic radical, wherein any can be that replace or unsubstituted;

R wherein ₅Be selected from the group of forming by following: CCl ₃, CF ₃Or COOH;

Wherein x is 0-16, and z is 0-5, and n is that 0-10 and m are 2-10.

40. as claim 36,37,38 or 39 described regulators, wherein said lipoic acid derivatives only exists with its (R)-isomer.

41. as claim 36,37,38 or 39 described regulators, wherein said lipoic acid derivatives exists with its (R)-isomer and (S)-mixture of isomers.

42. regulator as claimed in claim 1, wherein said regulator be used for the treatment of and diagnose the illness, disease or syndrome or its symptom, described disease, disease or syndrome or its symptom comprise structure, expression and/or the active change of at least a enzyme and/or multienzyme complex or its subunit.

43. regulator as claimed in claim 42, wherein said at least a multienzyme complex is the PDH complex.

44. regulator as claimed in claim 42, wherein said disease, disease or syndromic further feature are cell hyperproliferations.

45. regulator as claimed in claim 44, wherein said disease, disease or syndrome are cancers.

46. an adjusting presents at least a enzyme among disease, disease or the syndromic patient and/or the method for multienzyme complex or its subunit, described disease, disease or syndrome comprise structure, expression and/or the active change of described at least a enzyme and/or multienzyme complex or its subunit, and described method comprises the described regulator of the claim 1 of using effective dose.

47. method as claimed in claim 46, wherein at least a multienzyme complex is the PDH complex.

48. method as claimed in claim 46, wherein said disease, disease or syndromic further feature are cell hyperproliferations.

50. method as claimed in claim 48, wherein said disease, disease or syndrome are cancers.

51. diagnose and predict the method for being benefited among the patient who presents disease, disease or syndromic symptom for one kind, described disease, disease or syndrome comprise structure, expression and/or the active change of at least a enzyme and/or multienzyme complex or its subunit, described method comprises from the patient and obtains cell sample, use the described regulator of claim 1 of effective dose and obtain the result thus to described cell external.

52. method as claimed in claim 51, wherein at least a multienzyme complex is the PDH complex.

53. method as claimed in claim 51, wherein said disease, disease or the syndromic cell hyperproliferation that further is characterized as.

54. method as claimed in claim 53, wherein said disease, disease or syndrome are cancers.

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