Use the method and composition of Suramine, pentosan polysulfate ester, telomerase antisense body and telomerase inhibitorCross-reference with related application
The application requires to obtain provisional application sequence number 60/444,061 right of priority, this application was submitted on January 31st, 2003, title is " Suramine, pentosan polysulfate ester and a human telomerase antisense body to the active restraining effect of Telomerase (inhibition of telomerase activity by suramin, pentosanpolysulfate or antisense to human telomerase ".With also having of the application's cross reference: the U.S. Patent application 10/646 that on June 18th, 2003 submitted to, 018, title for " by telomere destroy the active method and composition of regulating medicine (Methods and compositions for modulating drug activitythrough telomere damage "; And the U.S. Patent application 09/587 of submission on June 5th, 2000,559, title is " method and composition (Methods and compositionsfor modulation cell proliferation and cell death) of regulating cell proliferation and necrocytosis ", and its full text content is incorporated in herein as reference.
The research of government-funded
(license number is R01 CA77091, R01CA97067, R37CA49816 by U.S. Department of Health and Human Service (the United StatesDepartment of Health and Human Services) subsidy on working portion ground described in the application; R01CA78577; R01CA74179; R21CA91547; And U01CA76576).
Background of invention
Technical field
The invention describes a kind of target human telomerase and suppress the antisense molecule (hTR-antisense body (anti-sense)) of the telomerase activation in the human cancer cell.The present invention has also described the purposes of Suramine, pentosan polysulfate ester (PPS) or hTR-antisense body inhibition telomerase activation.The present invention instructed further another kind of telomerase inhibitor 3 ' zidovudine (3 '-azido-deoxythymidine) (AZT) (it produces the Plasma Concentration of nmole level when non-toxic dosage) be in the purposes of treatment in the tumour.The present invention has also instructed when reducing (tumor size reduction) (cell subtracts go out (cytoreduction)) with other operation methods or non-operation gross tumor volume or afterwards, has treated the method for tumour with telomerase inhibitor.The present invention had also instructed before non-operation gross tumor volume reduces treatment, used telomerase inhibitor, can improve the susceptibility of tumour cell to this treatment effect.The present invention has also instructed how to use telomerase inhibitor, comes the development of prophylaxis of tumours as Suramine, PPS or hTR antisense body.
The explanation of prior art
Telomere and Telomerase. telomere is the cap-like structure of end of chromosome, and is very important for keeping chromosomal integrity and replication.Since archaeal dna polymerase can not duplicated chromosome 3 ' end, each cell fission meeting makes telomere shorten 50 to 200bp.Telomere loss reaches and is lower than a certain critical minimum length and will causes necrocytosis (Lingner, etal., Science, 269:1533-1534,1995), or cause cell senesce (senescence) (as the forfeiture multiplication capacity) (Goldstein, Science, 249:1129-1133,1990; Martin, et al., Lab.Invest., 23:86-92,1979).The Telomerase this kind of enzyme comprises RNA and protein ingredient, and it can recover the length of telomere, almost exists at large in tumour cell, and does not generally exist in normal somatic cell.(Hiyama，etal.，J?Natl?Cancer?Inst，88：116-122，1996).
Use the method for telomerase inhibitor. the importance of the function of Telomerase, with and optionally be present in and drawn initial hypothesis in the tumour cell, be that Telomerase is the ideal tumour-specific target, telomerase inhibitor is a kind of antitumor drug that treatment effectiveness is arranged.(as: in Huminiecki, L., ActaBiochimica Polonica, 43:531-538,1996). propose this usage and be based on following hypothesis: the activatory Telomerase is the startup of tumour and keeps necessary.Still there is not the clinical validity that report confirms telomerase inhibitor.In fact, after this have several important discoveries to be described, they all point out the therapeutic value of telomerase inhibitor limited.At first, generally acknowledged at present other telomeres that Telomerase does not rely on Telomerase in addition prolong mechanism (as, Gan, etal., FEBS Lett., 527:10-14,2002).Secondly, even Telomerase is suppressed fully, the telomere that is pre-stored in the tumour cell also often has enough length, is enough to keep the cell proliferation in a plurality of cycles.Therefore, telomerase inhibitor will could produce cytotoxicity through after one period quite long retardation time.For example, telomerase inhibitor just produces cytotoxicity after through 23 to 26 cell multiplications in the HeLa cell.(Feng，et?al.，Science，269：1236-1241，1995)。Because uncontrolledly growing, tumour will cause fatal tumor load; as; the death of tumour mediation can take place in a patient usually after tumor doubling is less than 10 times; so; suppressing to cause that necrocytosis or old and feeble characteristic make it can not be practical thereby long lag-phase of this needs of telomerase inhibitor one could produce Telomerase, is not a kind of useful medicine.Though before nearly 10 years, just proposed hypothesis first,, be that the medicine of telomerase inhibitor detects in human patients as antitumor drug without any a kind of clearly being accredited as up to today with telomerase inhibitor treatment tumour.(U.S. Patent application the 5th, 489, No. 508).
In No. the 10/464th, 018, United States Patent (USP) please in, the applicant has described following discovery: with taxol (taxol) treatment cancer, thereby can cause the telomere damage to lower telomerase activation, cause that this tumour produces tolerance to the treatment of taxol; Can reduce this tolerance and give a kind of telomerase inhibitor (as AZT, hTR antisense body) simultaneously, strengthen the antitumor action of taxol.
The application has expanded the application that the applicant invents before this, has instructed to make Telomerase suppress to become a kind of method of effective antitumour treatment.On the one hand, the present invention has instructed before giving telomerase inhibitor or has used cell to subtract the therapy of going out simultaneously, reduce tumor load, like this length that telomerase inhibitor can corrode telomere make its be reduced to be lower than the critical level that produces apoptosis and cell aging before, tumor load can not reach lethal level, thereby makes Telomerase suppress to become a kind of effective therapy.Therefore, the present invention has instructed and has used telomerase inhibitor to resist the method for tumour, comprise: give patient's cell and subtract the therapy of going out, (promptly reduce the treatment of gross tumor volume, as, surgical operation, radiotherapy, chemotherapy etc.), give a kind of telomerase inhibitor then, wherein the plasma concentration of this telomerase inhibitor should maintain the level that Telomerase suppresses, and continues one period long period, for example, several weeks or several months.This telomerase inhibitor also can give when cell subtracts the treatment of going out.Here the content of being instructed is based on the following discovery in the application: cell subtract in treatment (as the chemotherapy) process of going out and/or end after, making in the blood plasma Suramine maintain Telomerase suppresses and atoxic concentration level, can promote tumour to dwindle, delay tumour progression, prolong human cancer patient's survival time.
Purposes after another aspect of the present invention has instructed telomerase inhibitor to be used for cytotoxic treatments to stop, for example, because of dose limitation (dose-limiting) toxicity of cytotoxic treatments stops.Cytotoxic treatments, often to the tumour patient toxigenicity, therefore can not be used (promptly using during this period of time in the phase of keeping as supportive care) indefinitely such as chemotherapy.With clinical viewpoint, this is an important problem, because most of chemotherapeutical tumour all will make progress, and can not eradicate usually.For example, under the situation that chemotherapy can be eradicated, the nonsmall-cell lung cancer patient less than 1% can reach fully and reply (Schiller, et al., New Eng J Med, 346:92-98,2002) in tumour.Therefore, residual tumour cell continued growth again after cytotoxic treatments stops.On the contrary, because the Telomerase selectivity is expressed in tumour cell, telomerase inhibitor is not easy toxigenicity, therefore can indefinitely or long-time the application.Like this, the ability of telomerase inhibitor inhibition cell proliferation or inducing cell aging just can stop the regrowth of tumour, shows survival advantage.
For example, a kind of commonly used and the effective chemical treatment is taxol (225mg/m in the human patients of nonsmall-cell lung cancer2) unite with carboplatin (AUC=6), per three weeks are administered once.The neurotoxicity of taxol can be accumulated, and reaches dose limitation usually after 4 to 6 treatments, forces treatment to stop.Tumour begins continued growth under the common situation in several months, so mean survival time (MST) is about 8 months (Schiller, etal., 2002).
About the prior art in this field, the peptide nucleic acid(PNA) antisense body molecule (peptide nucleic acid antisense molecules) of having instructed Telomerase among WO 97 38013 A (PNA) with the use of uniting of antitumor drug.Kondo has described the inhibition Telomerase can improve the susceptibility (Kondo, etal., Oncogene, 16:3323-3330,1998) of tumour to the medicine of damage dna.Therefore, these early stage publications have been instructed this general admitted notion: adopt the combination therapy of two kinds of effective therapeutic modalities to use these two kinds of therapeutic modalities more effective more respectively to cancer.But these publications do not have to disclose and will use cell to subtract to go out treatment to reach the purpose that reduces gross tumor volume to be enough to make the treatment of telomerase inhibitor effective.These publications do not have to disclose yet and can use telomerase inhibitor to be used as a kind of mode of resisting cancer after cytotoxic treatments stops.Therefore, the theory that the application found is new, and promptly to subtract the treatment of going out be very necessary for making Telomerase suppress to become a kind of effective antitumour therapy to cell, because there is not this usage before this, also do not report this type of result.The applicant finds, when subtracting the treatment of going out, share in cell the Suramine of low dosage, Suramine concentration is maintained be enough to suppress Telomerase, but be not enough to produce the level of anti-tumor activity, still can effective antitumor in human patients, this discovery also is surprising, because prior art shows that the Suramine of low dosage does not have anti-tumor activity the mankind like this.(Dreicer，et?al.，Invest.New?Drugs，17：183-189，1999；Falcone，etal.，Tumori，84：666-668，1998；Falcone，et?al.，Cancer，86：470-476，1999；Hussain，etal.，J?of?Clin.Oncol.，18：1043-1049，2000；Miglietta，etal.，J?Cancer?Res.Clin.Oncol.，123：407-410，1997；Mirza，et?a/.，Acta?Oncologica，36：171-174，1997；Motzer，etal.，Cancer，72：3313-3317，1993；Rapoport，etal.，Ann.Oncol.，4：567-573，1993；Rosen，et?al.，J.Clin.Oncol.，14：1626-1636，1996)。
The present invention has also instructed with a kind of telomerase inhibitor and has done pre-treatment (pretreatment), adds the antitumous effect of intense prior chemotherapy.This is based on the application's discovery, and promptly the Suramine that for example reaches 50 micromolar plasma concentrations with low dosage is done pre-treatment, and such concentration is enough to suppress Telomerase, but is not enough to produce cytotoxicity, can increase chemotherapeutic activity.The Suramine of low dosage was used before or after tumour is established (tumor establishment), or do not use during threat to life as yet, all observed the chemotherapy sensitizing effect of Suramine at tumor load.This discovery is novel, because do not report this usage and this result before this.In fact, this discovery is surprising, because prior art shows that the Suramine of low dosage is in inhuman animal like this, still all do not have after tumour is established before tumour is established anti-tumor activity (as, Pesenti, etal., Br.J.Cancer, 66:367-372,1992).
Telomerase inhibitor. the application has instructed Suramine, PPS, or the usage of hTR antisense body in suppressing Telomerase.This is based on the application's discovery, i.e. Suramine, and PPS, or hTR antisense body is effective telomerase inhibitor, can make the contraction in length of planting telomere in the tumour of animal.Prior art has been instructed the several compounds with Telomerase rejection characteristic.(as, U.S. Patent number Nos.5,656,638,5,760,062,5,767,278,5,770,613, and described in 5,863,936 each file).Prior art has also instructed cis-platinum can suppress the Telomerase restraining effect, may be because crosslinked (Burger, et al., EurJ Cancer, 33:638-644,1997) of telomere repeat sequence.Prior art has further been instructed use peptide nucleic acid(PNA) (PNA) antisense molecule and thiophosphoric acid (phosphorothioate) oligonucleotide, by the RNA composition of target Telomerase, suppresses the purposes (Norton of telomerase activation, etal., Nat Biotechnol, 14:615-619,1996)..But these reports are not all instructed Suramine, PPS, or hTR antisense body is as the purposes of telomerase inhibitor.
Telomerase inhibitor is as (cancer) chemoprophylaxis. and suggestion is done chemoprophylaxis with telomerase inhibitor.For example, Akama has pointed out and has given the possibility (Akama, etal., U.S.Patent No.6,452,014,2002) of thiazole (thiazolidinone) compounds class telomerase inhibitor as prophylactic.But Akama does not instruct the use Suramine, PPS, or hTR justice body suppresses the usage of Telomerase.
Use the method Suramine of Suramine, a kind of how Sulfonated naphthyl urea (polysulfonatednaphthylurea) has multiple pharmacological activity (Ahmann, et al., Proc.Am.Soc.Clin.Oncol., 10:178,1991; Armand, Bonnay, Gandia, Cvitkovic, De Braud, Bertheault, Droz, Carde, Schlumberger, and Fourcault, 1991; Dreicer, et al., 1999; Falcone, et al., 1998; Falcone, et al., 1999; Garrett, et al., Proc.Natl.Acad.Sci.USA, 81:7466-7470,1984; Grazioli, et al., Int J Immunopharmacol, 14:637-642,1992; Hawking, Adv.Pharmacol.Chemother., 5:289-322,1978; Hensey, et al., FASEB, 258:156-158,1989; Hosang, J.Cell.Biochem., 29:265-273,1985; Hussain, et a/., 2000; Manetti, etal., Bioorganic ﹠amp; Med.Chem., 6:947-958,1998; Mills, et al., Cancer Res., 50:3036-3042,1990; Myers, et al., Proc.Am.Soc.Clin.Oncol., 9:113,1990; Ono, eta/., Eur.J.Biochem., 172:349-353,1998; Pollak, et al., Proc.Am.Soc.Clin.Oncol., 9:54,1990; Pollak, et al., J.Natl.Cancer Inst., 82:1349-1352,1990; Stein, Cancer Res., 54:2239-2248,1993; Takano, et al., CancerRes., 54:2654-2660,1994; Wade, et a/., J.Surg.Res., 53:195-198,1992; Waltenberger, etal., J.Mol.Cell Cardiol., 28:1523-1529,1996). its anti-tumor activity it is believed that it is owing to suppressed archaeal dna polymerase α and reversed transcriptive enzyme, suppress somatomedin (promptly, platelet-derived somatomedin, fiber mother cell growth factor or FGF, conversion growth factor β, Urogastron, vascular endothelial growth factor and insulin-like growth factor-i) combination and IL-2 and Transferrins,iron complexes and receptors bind separately, suppress phosphorylation activity and the glycosaminoglycan metabolism of PKC.Suramine can also suppress the Na/K-ATP enzyme, tumour necrosis factor and topoisomerase II.The Suramine of any concentration all is unknown to the inhibition activity of Telomerase, is the application's new discovery.
Suramine has shown certain activity (DeClercq, Cancer Letters, 8:9-22,1979 in prostate cancer; Eisenberger, et al., Cancer Treat Rev., 20:259-273,1994; Fotes, etal., Biochim Biophys Acta., 38:262-272,1973; Huang, et al., Oncogene, 9:491-499,1994), in multiple solid tumor, carry out overtesting, both as independent medicament use also can with other chemotherapy couplings.Suramine therapeutic plasma concentration is at 100 to 200 μ M (140-280 μ g/ml) (Funayama, et al., Anticancer Res., 13:1981-1988,1993), and the wide region of indication be 70 to 210 μ M (100-300 μ g/ml) (Klohs, etc., US 5,597,830,1997).When these concentration, Suramine shows significant toxicity in the patient, and activity only reaches moderate.A plurality of study group represent that the Suramine single therapy does not have appreciable antitumous effect in human patients, and to human patients, and using the uniting of the Suramine of high dosage and cytotoxic drug compares with single medicine does not produce useful result yet.Based on these discoveries, these investigators recommend not use Suramine, no matter its to be single medicine use or unite use (Dreicer, eta/., 1999 with other cytotoxic drugs; Falcone, eta/., 1998; Falcone, et a/., 1999; Hussain, et a/., 2000; Miglietta, et al., 1997; Mirza, et al., 1997; Motzer, etal., 1993; Rapoport, etal., 1993; Rosen, etal., 1996).Therefore, there is not the discovery of institute of the present invention basis, just do not use the motivation of Suramine, no matter be the single medicine or the Suramine of any dosage and other cytotoxic drug couplings, or with Asia treatment (subtherapeutic) dosage and be the Suramine and the cytotoxic drug coupling of non-toxic dosage.Unique exception just is to use the Suramine of inferior therapeutic dose, so that strengthen the effect of other form of therapy.This usage is found by the applicant, at United States Patent (USP) 6,599, has a detailed description in No. 912 files, and this usage need be before being carried out other forms of treatment to the patient, in the therapeutic process or given Suramine afterwards in a short time, as chemotherapy or radiotherapy.The present invention supports the exception usage or the different usage of Suramine, and points out emphatically long total treatment phase of needs, for example several weeks or several months, several years, or one uncertain period are so that suppress Telomerase, make telomere shorten to enough degree, suppress cell proliferation or cause necrocytosis.
The present invention has instructed the needs of lasting inhibition Telomerase.Therefore, need a kind ofly in body, remove slower compound (compound).Suramine meets this demand.It is feature that the pharmacokinetics of Suramine in human patients reduces (triphasic plasma concentration decline) with three grades of plasma concentrations, and the transformation period is 5.5 hours, 4.1 days and 78 days.Total human body clearance rate (total body clearance) is 0.0095 liter/hour/m2 (Jodrell, etal., J Clin.Oncol., 12:166-175,1994).Suramine is also very slow in the intravital distribution of dog, and the final transformation period (terminal half-life) is about 13 days (seeing example 8)
Using the method .PPS of PPS is a kind of semisynthetic heparitin (heparinoid), has anticoagulation (Wellstein, et al., Breast Cancer Res.Treat., 38:109-119,1996).In the chicken chorioallantoid membrane chemical examination (chicken chorioannantoic membrane assay), in patient's blood plasma, do not have anticoagulation and be lower than under 1000 times the concentration conditions of its toxic action, PPS suppresses FGF and its receptors bind, and inhibition vasculogenesis (Parker, etal., J.Natl.Cancer Inst., 85:1068-1073,1993; Wellstein, et al., 1996).Only when surpassing 1 μ g/ml (Parker, etal., 1993), concentration just finds anticoagulation.In vivo under the condition, if treatment just begins when tumour is also untouchable, PPS can suppress rat MAT-LyLu growth of tumor speed, but for almost not effect of the tumour that has formed, transfer (McLeskey, et al., the Br.J.Cancer that can not suppress mouse MCF7 tumour, 73:1053-1062,1996; Nguyen, etal., AnticancerRes., 13:2143-2148,1993; Wellstein, et al., J.Natl.Cancer Inst., 83:716-720,1991).The anti-tumor activity of PPS in clinical pre-neoplastic model makes it obtain clinical assessment.The result shows, when the PPS dose titration when avoiding its anticoagulation, tolerance is good, but do not show anti-tumor activity in the patient (Lush, etal., Ann Oncol., 7:939-944,1996; Swain, et al., Invest.New Drugs, 13:55-62,1995).As a result, no longer PPS is assessed as potential effective antitumour or anti-angiogenic medicaments.Retrieval all ongoing cancer clinical trials on January 27th, 2000 in PDQ clinical testing data storehouse (http://www.nci.nih.gov) show that there is 1,790 test in the whole world, but do not have the test about PPS.Therefore, do not have this discovery, just do not use the motivation of PPS treatment cancer.
The PPS dosage that is used for the anti-tumor activity assessment is about every square metre of 400mg every day (being equivalent to 10mg/kg every day), oral administration.Every day successive administration, plasma concentration versus time increases, at the 200-460ng/ml that reached in the 15th day of treatment.At this dosage level, in the time of 1-3 month haematics toxicity appears, usually as diarrhoea, gastrointestinal hemorrhage or rectitis (proctitis).Rectitis is dose-limiting toxicity (Marshallet al., Clin.Cancer Res., 3:2347-2354,1997) in this test.The concentration (0.5-0.6 μ g/ml sees embodiment 3) that telomerase activation will be suppressed 50% required PPS is higher than far away and causes the toxic concentration of rectitis.This consideration and following discovery: can in target tissue, reach Telomerase inhibition concentration (as 5 to 100 μ g/g) to target organ topical administration Suramine, and the concentration of Suramine is quite low (as 0.1 μ g/ml in the blood plasma, see embodiment 13), cause the PPS part is used for this invention of organ that needs suppress Telomerase.This new usage has alleviated the potential problems of systemic host toxicity.
Use the people such as method .Melana of AZT to sum up the account of the history (Melana, et al., 1998) of AZT recently.AZT is used for the treatment of the patient that human immunodeficiency virus infects.AZT develops as antitumor drug at first.But it no longer is regarded as the potential antitumor drug, because in drinking-water during administration, its cytotoxicity in animal is higher relatively.Found AZT afterwards when the mode administration of bolus injection, toxicity is very low.AZT is carried out I phase and II clinical trial phase, existing single medicine uses also to have with other drug unites use, is used for the treatment of gastrointestinal cancer.According to obtainable data computation, the plasma concentration that the AZT dosage that these early stage researchs are used (as 7 to 10g/m2/ days) produces surpasses 10 micromoles, is listed below.Common oral dosage is per 4 hours 2.5mg/kg or after 15mg/kg/ days, the average steady state concentration of AZT is 1.06 μ g/ml (Physician ' s Desk Reference, 2003) in patient's blood plasma.Carry out the cytotoxicity treatment with AZT, the minimum dose that uses is 3g/m2/ day (United States Patent (USP) the 5th, 116,823) are about 85mg/kg/ days after the conversion.The Css of 1.06 μ g/ml during with 15mg/kg/ days carries out linear extrapolation, obtains the expection plasma concentration of 6 μ g/ml, is 22 μ M for 85mg/kg/ days situations.The prior art instruction, AZT is a kind of telomerase inhibitor, produces 50% concentration range that suppresses and fluctuates in the micromole to the mmole order of magnitude (Strahl, et al., Nucleic Acid Res, 22:893-900,1994; Strahl, etal., Mol.CellBiol., 16:53-65,1996).
The present invention has instructed the use of low dosage AZT.Applicant's discovery produces the AZT of the non-toxic dosage of nanomole plasma concentration, and the tumour that causes fully having determined in the animal is dwindled.In animal, add the anti-tumor activity that also can strengthen taxol with the AZT of this non-toxic dosage.Although AZT is considered to antitumor drug and telomerase inhibitor, prior art is not described usage and the effectiveness of low dosage AZT like this as yet.In fact, the application's discovery, the anti-tumor activity that promptly produces the low dosage AZT of nanomolar concentration is surprising, because prior art shows that it is at higher dosage that AZT produces anti-tumor activity, the plasma concentration that described dosage produces is at micromole's level (Clark, et al., J Cancer Res.Clin.Oncol., 122:554-558,1996).It also is surprising that the AZT of nmole strengthens this discovery of chemotherapeutic activity, because prior art shows that cell toxicant or the Telomerase inhibition concentration of AZT are (Strahl, et al., 1994 in the scope of micromole or mmole; Strahl, et al., 1996; (Melana, et al., Clin.Cancer Res., 4:693-696,1998; Table 1 in the example 1).
Telomerase antisense body antisense body construct is to the existing report of the active restraining effect of Telomerase, and the possibility that they are applied to oncotherapy also was suggested (Kelland, LancetOncol, 2:95-102,2001).But Bao Dao hTR antisense body was not described as yet here.And, unite long-term Telomerase suppression therapy and cell with telomere antisense body construct and subtract the method for the treatment of coupling of going out and be not suggested or described by utilizing.
Summary of the invention
The application is based on several relevant discoveries of shortening about Telomerase inhibition and telomere.At first, the applicant finds Suramine, and PPS and hTR antisense body are effective telomerase inhibitors, can shorten the telomere length of planting in the tumour of animal.Second point discovery is, to the abundant tumour of moulding in the animal of load tumour, carries out pre-treatment with Suramine and can make the chemotherapeutic activity enhancing.Thirdly find it is that telomerase inhibitor such as Suramine and PPS etc., can strengthen the anti-tumor activity of the chemotherapy and radiation of cancer.The 4th point discovery is, during cell subtracts the treatment of going out and make the blood plasma Suramine maintain Telomerase inhibition concentration after finishing can to promote tumour to dwindle the survival time that delays tumor growth and prolong human tumour patient.The 5th point discovery is, the Suramine part is used for the treatment of the target tissue that is intended to, making local organization concentration reach is enough to make the degree that Telomerase is suppressed, telomere is shortened in this tissue, and while concentration of Suramine in blood plasma is extremely low, is not enough to suppress Telomerase.The 6th point discovery is that use produces the AZT of the non-toxic dosage of the extremely low plasma concentration of measuring, and causes the fully definite oncolysis of animal, and makes the anti-tumor activity enhancing of taxol in animal.
Therefore, the applicant provides and can be applied in the kinds of tumors treatment, and the method that treatment result is made moderate progress than standard care, compound and composition.
First aspect, the present invention has instructed the use Suramine, and PPS or hTR antisense body by with Telomerase or contain the cells contacting of Telomerase, suppress the method for Telomerase.
Second and related fields, instructed the method that suppresses the telomerase activation in patient's preferred mammal, this patient suffers from the disease of Telomerase mediation, and described method comprises Suramine or other telomerase inhibitors that gives this patient treatment significant quantity.
The third aspect has been instructed the method for improving the treatment result of tumour patient preferred mammal, comprises giving Suramine or other telomerase inhibitors that the patient uses telomere inhibitor amount.Giving the time that the mode of Telomerase suppression therapy should make tumour be exposed to telomerase inhibitor keeps several proliferating cycles at least, under the preferable case, keeps 10 or 20 proliferating cycles at least.
Fourth aspect has been instructed the method for the treatment that strengthens cancer patients's preferred mammal, is included in cell and subtracts during the treatment of going out or after finishing, give Suramine or other telomerase inhibitors of patient's use side granzyme amount of suppression.It can be the surgical excision or the non-operative treatment of tumour that this cell subtracts the treatment of going out, as radiotherapy, and chemotherapy, photodynamic therapy.In order to obtain better result, can use one or more telomerase inhibitors.
The 5th aspect, the present invention has instructed method for cancer among the treatment patient, described method may suffer from cancer or suffer from the little non-detectable cancer that arrives by at first confirming the patient, gives this patient end granzyme and suppresses medicine, thereby reach the purpose for the treatment of cancer or suppressing cancer development.It will be effectively that this class patient use side granzyme is suppressed pharmacological agent, because initial gross tumor volume is less, threatens at tumor load before patient's the health, also allows many tumor proliferation cycles.
The 6th aspect, the present invention is a kind of method for the treatment of the catabasis patient, the described catabasis is after his or her cancer therapy success, and described patient also has the potentially dangerous of New Development cancer or cancer return.Especially suitable to the little or avirulent medicine of patient's toxicity, because it has good risk-benefit ratio.Suramine, PPS or AZT etc., very little or nontoxic to patient's toxicity when suppressing the required low dosage of telomerase activation, and this good risk-benefit ratio is provided.
The method that suppresses Telomerase with hTR antisense body transfectional cell has been instructed in the 7th aspect.
Eight aspect, this aspect have been instructed and have been used the AZT of nontoxic dose to resist method for cancer, and the plasma concentration that described nontoxic dose produces is lower than micro-molar range, for example in the nmole scope.
The 9th aspect, telomerase inhibitor can local applications, are used near the position the known cancer, or are used for suspicious present or tumorigenic in the future organ.The telomerase inhibitor of topical application can be near application site tumour cell or tissue in the effective Telomerase inhibition concentration of generation.Local application, for example injection or embedment method can adopt the form of storage vault (depot), such as the device that slowly discharges.The topical of telomerase inhibitor will produce Telomerase inhibition concentration in the tissue of treatment institute target, and need not produce Telomerase inhibition concentration in the blood plasma of non-treatment target or other organs.
In addition, the patent application of cross reference (U.S. Patent application the 09/587th, No. 559) in, the applicant has described such discovery, promptly acid and basic fibroblast growth factor (FGF) can cause the broad-spectrum tumor tolerance to chemotherapy, and the FGF inhibitor can strengthen cancer chemotherapeutic and radiocurable anti-tumor activity.Suramine and PPS are the FGF inhibitor.The present invention has instructed the inhibition of telomerase of these two kinds of preparations.Suramine and PPS also can produce the Telomerase restraining effect in the concentration that produces the FGF inhibition.Therefore, Suramine or PPS can unite with the cell toxicant anticancer therapy and give, and to strengthen the effect of cell toxicity medicament, also suppress Telomerase simultaneously, telomere length is shortened, thereby obtain additional useful antitumous effect.After the cytotoxicity therapy finishes, can continue to give Suramine or PPS, to obtain the effect of long term inhibition telomerase activation.These (two) plant medicine reach be target FGF again the purposes of the mechanism of target Telomerase provide convenience for the doctor of patient and treatment, also improved the result (outcome) of cytotoxic treatments.
As explained above, carry out effective anticancer therapy with telomerase inhibitor and need in a plurality of proliferating cycles of tumour cell, (over multiple proliferation cycles of the tumor cells) suppress telomerase activation.This lasting being suppressed under use has long t1/2 (for example surpassing a week) in patient's body the situation of medicine realized most effectively, so that continue to keep the restraining effect of telomerase activation in not frequent treatment.The removing transformation period of Suramine surpasses a week in human (Jordell etal., 1994) and non-human animal's (seeing example 8), satisfy this requirement.
Compound of the present invention has multiple valuable application as harmless telomerase activation inhibitor, for example is used for the treatment for cancer of Mammals such as philtrum.Pharmaceutical composition of the present invention can be applied to the treatment plan of kill cancer cell in vivo or be used at the treatment plan of kill cancer cell that exsomatizes.Therefore, the invention provides treatment treatment for cancer compound and composition, and the method for the disease of cancer in the treatment mankind and other Mammalss (for example, dog, cat, ox, horse and the related animal of other veterinary science) and the mediation of other Telomerases.
Other characteristics of the present invention and advantage can obviously embody by the following detailed description and claims.
Detailed Description Of The Invention
For simplicity, with specification sheets, some term that uses in embodiment and claims is gathered in this, so that guarantee clear for the understanding of the present invention unanimity.
Definition
This paper term " misgrowth (aberrant growth) " refers to a kind of cell phenotype, and its normal phenotype with cell is different, specifically refers to and direct or indirect those relevant cell phenotypes such as diseases such as cancer.
This paper term " administration (adminstering) " refers to medicament is introduced cell, for example external; Or the intravital cell of animal, promptly in vivo; Or more described cell is put back to after the introducing cell in the animal body and (promptly exsomatized).
This paper term " medicament ", " medicine ", " compound ", " anticarcinogen ", " chemotherapeutics (therapeutic) ", " antitumor agent (antineoplastic) " reach " antitumour drug " and can exchange use, refer to have the medicament (unless being further limited) of inhibition or the abnormal cell growth that slows down (as tumour) characteristic.Aforementioned term also is intended to comprise cytotoxicity, kills cellularity or cell growth-inhibiting medicament.Term " medicament " comprises small molecules, macromole (as, peptide, albumen, antibody or antibody fragment), and nucleic acid (as, gene therapy construct, recombinant virus, nucleic acid fragment (comprising, for example the nucleic acid fragment).
This paper term " apoptosis " refers to any non-necrosis, the form of cell death of cell regulate and control, and the standard of clearly having formulated as this area defines.
Art-recognized implication is pressed in the definition of this paper term " benign ", " before the cancer " and " virulent ".
This paper term " cancer ", " tumour cell ", " tumour ", " leukemia ", or " leukemia cell " can exchange use, refer to any tumour (" true tumor "), such as cancer (deriving from epithelial cell), gland cancer (deriving from gland tissue), sarcoma (deriving from reticular tissue), lymphoma (deriving from Lymphoid tissue) or hematological system tumor (for example, leukemia or erythroleukemia (erythroleukemia)).Term " cancer " and " tumour cell " also are intended to comprise cancerous tissue or tumour agglomerate, are interpreted as the set (compilation) of cancer cells or tumour cell.In addition, term " cancer " and " tumour cell " comprise optimum, property and virulent cancer or cell before the cancer.Generally cancer or tumour cell can show various characteristics well known in the art (hallmark), such as the somatomedin dependent/non-dependent, lack cell/cells contacting growth-inhibiting and/or abnormal karyotype.In contrast, generally normal cell in substratum, inoculate can only carry out limited number of times go down to posterity and/or show various art-recognized belong to Normocellular characteristics (as, growth factor dependency, contact inhibition, and/or normal karyotype).
This paper term " cell " comprises any eukaryotic cell, mammalian cell or clone such as somatocyte or germ cell line, as, HeLa cell (mankind), NIH3T3 cell (mouse), embryonic stem cell and various cell type such as hemopoietic stem cell, sarcoplast (myoblast), liver cell, lymphocyte and epithelial cell and various clones for example described herein.
This paper term " the affirmation patient suffers from maybe will suffer from (identifying a patient having or about tohave) " is meant confessed diagnosis in various this areas or the prognosis technology used, comprise for example prostate specific antigen (PSA) test, BRCA1 and/or BRCA2 gene type (genotyping), the heredity technology such as (genetic profiling) of drawing, the patient has been made a definite diagnosis to be suffered from or sees to suffer from cancer from the statistics angle.This term also is intended to comprise only to be known or accepts that any prompting patient is just suffering from or with cancered information.
This paper term " suppress or slow down cell growth ", for example, cancer cells is to instigate its growth and/or transfer to be slowed down, disturb or stagnate, and may but be not to need, for example, the misgrowth of cell is eliminated fully.This term also is intended to comprise by necrocytosis (apoptosis) or necrosis and suppresses or the cell growth of slowing down.
This paper term " local ground (locally) " reaches " ground, zone (regionally) " can exchange use, finger is used for treatment in the tumour agglomerate, or have in the organ of tumour, or overall tumour scope maybe can be planted the zone of metastasis, or in the precancerosis kitchen range, for example special organ to certain tumor type, as prostate-specific in prostate cancer.
This paper term " tumor load " is the term of extensively generally acknowledging in this area, refers to part or all of quality, volume or the size of patient's in-vivo tumour tissue.Tumor size will be determined by the clinical means of standard, generally includes palpation or iconography method (for example, x line, cat scan, PET scanning, ultrasonic scanning figure (ultrasound sonogram)).Tumor load can be estimated by the summation of tumor size.Tumor load is a kind of index of clinical process of generally acknowledged reflection disease, the big indication prognosis mala of tumor load, and tumor load is little then to be prognosis bona's sign.It has been generally acknowledged that tumor load is relevant to patient's mortality with tumour.Although human patients often can bear the tumor load of about 1kg, it can not be considered as universal law, because tumorigenic position is the fatefulue important determinative relevant with the tumour agglomerate with the destruction that tumour may cause.For example, the metastatic tumour of growing in brain can be just fatal when size only has several centimetres, and occur in liver or internal organ (viscera) even tumour much biggerly also can bear.Therefore, for the patient that metastases has taken place in the brain, fatefulue tumor load amount can be little more a lot of than the patient who does not have to shift in the brain.
This paper term " systematicness " refers to the treatment approach that adopts for the systematicness that makes medicine be distributed widely in the experimenter, such as oral or through the intravenously medication." system " concentration refers to whole intravital concentration equally, as detected concentration in the circulating plasma.
This paper term " pharmaceutically useful carrier " is to generally acknowledge in the field, comprises a kind of pharmaceutically useful material, composition or vehicle (vehicle), is suitable for being used for medicine of the present invention is used for Mammals.
This paper term " pharmaceutical composition " comprises the preparation that is applicable to Mammals (as the mankind).When compound of the present invention is used for Mammals as medicament, during as the mankind, with this medicament prototype or with the form medication of pharmaceutical composition, described pharmaceutical composition for example contains 0.1 to 99.5% activeconstituents (as, treatment significant quantity) and the pharmaceutically useful carrier of (comparatively preferably 0.5 to 90%).
This paper term " experimenter " mean comprise human and the non-human animal (as, mouse, rat, rabbit, cat, dog, domestic animal and primates).Preferred human animal comprises the human patients of suffering from certain illness, and the characteristics of this illness are unusual cell growths, as cancer.
This paper term " telomere " refers to the chromosomal end of eukaryotic cell, its in cancer cells through the prolongation of regular meeting's abnormality.
This paper term " Telomerase " phalangeal cell enzyme or enzymic activity, it is at the Nucleotide polymerization or safeguard structure end of chromosome, that be known as telomere.
This paper term " Telomerase inhibition medicine " and " telomerase inhibitor " refer to suppress the medicine of (completely or partially) telomerase activation.
This paper term " Telomerase restraining effect " refers to the restraining effect to Telomerase that can directly measure, for example, confirms by the TRAP assay method that adopts improvement, wherein Gai Liang TRAP assay method is described (Gan eta/. by people such as Gan, Pharm.Res., 18:488-493,2001); Or according to the shortening that confirms average telomere length in all cells with the TALA assay method, wherein TALA assay method card is described (Ganeta/., Pharm.Res., 18:1655-1659,2001) by people such as Gan; Or confirm the loss (erosion) of the single telomere in each cell with the FISH assay method, wherein the FISH assay method is described (Gan et al., Pharm.Res., 18:1655-1659,2001) by people such as Gan.
This paper term " chemical sensitizer " refers to a kind of medicament, and it can strengthen second kind of medicament, as the antitumous effect of anticancer chemotherapy medicament.Term " chemical sensitization effect " refers to by chemical sensitizer, and Comparatively speaking the anti-tumor activity of cancer chemotherapy medicament anti-tumor activity of this cancer chemotherapy medicament when not using this chemical sensitizer increases.
The generation of with medicament prophylaxis of tumours, cancer instigated in this paper term " chemoprophylaxis ".The medicament that term " chemoprophylaxis medicament " refers to can prophylaxis of tumours, cancer takes place.
Herein, " the therapeutics significant quantity " that Telomerase suppresses medicament and/or chemotherapeutic refers to that this medicament separately or unite amount when using, described amount can effectively suppress activity (suppressing medicament for Telomerase) or the inhibition or for example cancer (for chemotherapeutic) of abnormal cell growth that slows down of Telomerase after giving experimenter's (as human patients) with single agent or multi-agent.
This paper term " pentosan polysulfate ester (pentosan polysulfate) " or " PPS " refer to (sulfated) polyanion of semisynthetic sulphating, it is made of β-D-xylopyranose residue, characteristic and heparin are similar, and molecular weight is in the 1500-5000 scope.For instance, Merck index, the 10th edition, the 1025th page, Merck ﹠amp; Co, Inc has the description to this compound in 1983.Other titles of representing this compound also have: xylan hydrosulphuric acid ester (xylan hydrogen sulfate), xylan poly sulfuric ester (xylansulfate), CB 806l; Fibrase; Hemoclar.
This paper term " biological marker " is identical with recognized meanings in the field, refers to molecule or compound, and as albumen or gene, its existence or its level can be pointed out the existence of disease or the possibility raising of disease, described disorders such as cancers take place.For example, the patient that raises of prostate specific antigen level suffers from probably or prostate cancer takes place.The patient that BRCA1 or BRCA2 gene have a sudden change suffers from probably or mammary cancer and ovarian cancer takes place.
The test that this paper term " exsomatize (ex vivo) " instigates the viable cell that is used in the tissue culture to carry out.
Telomerase inhibitor
As mentioned above, the infinite multiplication of cell often relates to activation of telomerase etc.More particularly, analyze the activity of Telomerase verified the relation between the ability of telomerase activation and many tumor cell lines maintenance infinite multiplication, described tumor cell line comprises skin, reticular tissue, fat, mammary gland, lung, stomach, pancreas, ovary, uterine neck, uterus, kidney, bladder, colon, prostate gland, central nervous system, retina and (the Kim et a/. of blood tumor cell system, 1994, Science, 266:2011-2014).This information and prior art replenish aging (replicative senescence) (WO 93/23572) that the shortening all point out telomere length can produce apoptotic signal or replicability, show that suppressing telomerase activation in the sufficiently long time can be used as a kind of effective anticancer therapy.
In a relevant embodiment, the present invention is the method that suppresses cell proliferation or duplicate.In this method, one or more preparations that can suppress telomerase activation described in the invention provide when cellular replication.As explained above, Telomerase fully duplicates the end of linear chromosomal dna and 5 ' the terminal end last base that can not lose per share chain plays an important role.The cell of infinite multiplication and rapid proliferating cells are utilized Telomerase to add telomeric dna at end of chromosome and are repeated.The Telomerase inhibition can cause proliferating cells can not add telomere, and these cells will stop division gradually.The method of this inhibition ability of cell proliferation can be effective to treat with cell proliferation rate and improve relevant disease, such as in cancer (telomerase activation in the malignant cell), reach for example hemoposieis (hematopoiesis) (telomerase activation in the hemopoietic stem cell), this also is conspicuous to those skilled in the art.
Therefore, on the one hand, the present invention has instructed Suramine, PPS and hTR-antisense body to suppress the purposes of Telomerase, is used for prevention or treats polytype malignant tumour.Specifically be, compound of the present invention can provide the method for height wide range of therapeutic malignant tumour, as the human tumor cell line of expressing Telomerase and tumour high per-cent proved.The more important thing is, the compound of describing among the present invention can effectively provide selectivity to attack the therapy of malignant cell, therefore avoided the multiple deleterious side effect relevant with the cytotoxicity chemotherapeutic agents, described medicament is the cell that does not kill and wound in the division with making any distinction between.
Compound of the present invention can expand to the analogue of Suramine and PPS, and these analogues also suppress Telomerase.
On the other hand, the invention provides the compound that is used to suppress Telomerase, pharmaceutical composition and the method relevant, or their pharmaceutically useful salt with these compounds, comprising Telomerase is contacted with compound or its pharmaceutically useful salt, wherein this compound is Suramine or PPS.
In an embodiment preferred, the Telomerase that will suppress is mammiferous Telomerase, as people's Telomerase.
The anti-tumor activity of telomerase inhibitor
A second aspect of the present invention and related fields thereof are, have found that Suramine maintains can effectively suppress the level of telomerase activation the time in the intravital amount of experimenter, can shorten the length of tumour telomere.Therefore, of the present invention this instructed the method that suppresses patient's telomerase activation on the one hand, and this class patient preferably Mammals suffers from the disease that Telomerase mediates, and described method comprises Suramine or other telomerase inhibitors of administration patient treatment effective dose.
Compound described in the invention can suppress the Telomerase in cell extract, cultured cells and the intact animal cell.The activity of preparation also can be showed with technology described herein among the present invention.
One of method that is used for identifying compound of the present invention be make cell, tissue, even more preferably cell extract or other contain the prepared product of Telomerase, contact in the buffer reagent compatible with the test-compound of several concentration known with telomerase activation.Detect the telomerase activation of the test-compound of each concentration, and the IC of compound50Value (can reach 50% test-compound concentration of initial or control value) to the restraining effect that enzymic activity produces with and IC90Use measured by standard techniques.Can adopt to be used for determining the other method of the present invention's preparation to the inhibition concentration of Telomerase, its information disclosed according to the present invention is conspicuous for those skilled in the art.
According to method as described above, detect and find the IC of Suramine and PPS50Value is lower than 10 μ M.
About using compounds for treating malignant disease described herein, expect that compound described in the invention can induce delitescence (crisis) in Telomerase dependent cells system.To the clone that Telomerase relies on, as human throat FaDu cell, expection is treated the shortening that also can cause the telomere length in the cell for the treatment of with compound of the present invention.
The compound of describing among expection the present invention can cause that also the telomere in human tumour cell line's fission process shortens described human tumour cell line such as FaDu cell and human prostate PC3 cell.But importantly, in normal human subject cell in contrast, as the BJ cell in fibroblast source, observed telomere shortens and only uses vehicle, and the cell of handling as physiological saline does not have difference.Compound described in the invention does not have significant cellulotoxic effect yet in normal cell when the concentration that the Telomerase of recommending suppresses.
In addition, the compound of the present invention's description can pass through they activity (IC to Telomerase the specificity of Telomerase50) with them the specific activity of other enzymes is estimated.As confessed in this area, enzyme is the molecule that promotes biological respinse.For instance, in the in vitro tests, have and combine with the similar similar nucleic acid of Telomerase or the enzyme of modification activities comprises dna polymerase i, the HeLa rna plymerase ii, the T3RNA polysaccharase, MMLV reversed transcriptive enzyme I, topoisomerase I, topoisomerase II, terminal enzyme (DNA) and single-stranded DNA binding protein (SSB).Comprise that also other do not belong to the enzyme of this group.If compound is for the IC of Telomerase50Value with respect to it for by the IC of other enzymes of examination50Be worth lowlyer, think that this compound has specificity to Telomerase.Perhaps, if compound for the IC of Telomerase90Value with respect to it for by the IC of other enzymes of examination90Be worth lowly, just think that this compound has specificity to Telomerase.
Also can carry out in vivo test with the mouse xenograft models, described mouse xenograft models is as implanting nude mice with the FaDu tumour cell, the mouse for the treatment of with compound of the present invention is had the tumour agglomerate by expection, in general, may reduce, continue constant or even after the initial dose medication, can increase in one period, but along with the treatment that continues, oncotherapy (mass) will be dwindled.In contrast, expection will continue to increase with the tumour agglomerate of the control animals generation of physiological saline processing.
According to aforementioned information, one of ordinary skill in the art would recognize that the present invention also provides the method that the treatment plan that relates to compound administration of the present invention is selected.For this purpose, carry out end limit fragment (terminal restriction fragment, TRF) it is helpful analyzing, and in this analysis, it is not the restriction enzyme enzymolysis of telomeric sequence (T2AG3) n that the DNA of tumour cell is used specific sequence.An example of this analytical procedure is a patent application (No. the 10/464th, 018, U.S. Patent application) in the past.
After the DNA enzymolysis, carry out gel electrophoresis restriction inscribe fragment is separated by size.The fragment of separating detects with telomeric sequence specific nucleic acid primer, measures the length that contains the terminal fragment of the telomeric dna of cell in the sample.By measuring the length of telomeric dna, people can estimate to answer the administration of telomerase inhibitor how long should continue, and whether also should adopt other methods of treatment (as, surgical operation, chemotherapy and/or radiotherapy).In addition, in treatment, people can detect cell and judge whether telomere length shortening has taken place through (progressive) cell fission of carrying out property, so that the effect of proof treatment.
Telomerase before chemotherapy suppresses pre-treatment
The 3rd aspect of the present invention is to have found to treat the validity that can strengthen chemotherapy of tumors with Suramine before cancer chemotherapy begins, exist minimum remaining pathology (minimal residual disease) this moment, and the amount of the Suramine that gives can effectively suppress telomerase activation.Therefore, of the present invention this instructed the method for the treatment result of improving cancer patients's preferred mammal on the one hand, comprises Suramine or other telomerase inhibitors to patient's use side granzyme amount of suppression.Preferably, give the time that the mode of Telomerase suppression therapy should make tumour be exposed to telomerase inhibitor to keep several proliferating cycles at least, preferred situation is kept 10 or 20 proliferating cycles at least.Preferable methods is to give telomerase inhibitor before giving the cytotoxicity scheme.Interchangeable preferred method be before giving the cytotoxicity scheme and during give Telomerase.Embodiment preferred is, described telomerase inhibitor is a Suramine, and the amount that gives Suramine can effectively suppress telomerase activation, and is not enough to produce anti-tumor activity.
Cell continues the Telomerase restraining effect after subtracting the treatment of going out
A fourth aspect of the present invention is to have found during cell subtracts the treatment of going out or the concentration of keeping Suramine in the blood plasma after finishing can promote tumour to dwindle in Telomerase inhibition level, delays tumor growth, and prolongs the survival time of tumour patient.Therefore, of the present invention this provides the method for strengthening cancer patients's treatment on the one hand, and described cancer patients is Mammals preferably, is included in Suramine or other telomerase inhibitors that cell subtracts during the treatment of going out or uses the treatment significant quantity after finishing to the patient.It can be surgical excision tumour or non-operative treatment that this cell subtracts the treatment of going out, as, radiotherapy, chemotherapy, optical dynamic therapy (photodynamic).Preferably, giving the time length that the mode of Telomerase suppression therapy will make tumour be exposed to telomerase inhibitor is equivalent to several proliferating cycles at least, more preferably should reach at least 10 to 20 proliferating cycles, the plasma concentration of telomerase inhibitor should be known can produce the concentration that Telomerase suppresses to tumour.Preferable methods is an administration Telomerase during giving the cytotoxicity treatment plan and afterwards, the cytotoxicity treatment plan can reduce patient's tumor load like this, thereby can provide sufficiently long leading time (lead time) for telomerase inhibitor, make telomere shorten to be lower than to cause necrocytosis and aged critical length.In another embodiment, realize dwindling of tumor size by the surgery mode.In another embodiment, after the cytotoxicity treatment plan stops back (as owing to the dose limitation toxic action in the cancer patients stops), give telomerase inhibitor as the mode of resisting cancer.Preferably, after the cytotoxicity treatment plan stops, give telomerase inhibitor, and the medication of telomerase inhibitor should continue to minority week, several months, several years, or more preferably, the use in indefinitely.
The applicant finds that Suramine and PPS make the treatment of tumour cell pair cell toxicity increase (U.S. Patent Application Serial Number 09/587,559 as the susceptibility of cancer chemotherapeutic and radiotherapy; Embodiment 10).Therefore, the uniting use and can be used for making tumour cell pair cell toxicity anticancer therapy susceptibility to increase of any or two kinds of compounds in these two kinds of compounds, and the additional benefits that suppresses telomerase activation also is provided simultaneously, thus promote the shortening of telomere.After cytotoxicity oncotherapy scheme stops, can continue with the treatment of the inhibitor of Suramine and PPS or other telomerase activations.This double treatment of Suramine and PPS benefits to have represented the unexpected advantage of using these compounds for treating.
Utilize the inhibiting chemoprophylaxis of Telomerase
The 5th aspect, the present invention has instructed a kind of method of treatment patient's cancer, it is undertaken by following process: confirm that the patient will suffer from cancer, or intravital (harbor) cancer is little during to the non-detectable degree of ordinary method, give the patient end granzyme and suppress medicine, thereby reach the purpose for the treatment of cancer or suppressing cancer development.For example, some ordinary method that detects tumour is physical method (as, palpation), the pathology method (as, just urine or in blood) or the iconography method (as, X line, cat scan, PET scanning, ultrasonic scanning figure).To may suffer from cancer or body in have the patient of undetectable cancer to confirm also can finish by monitoring bio mark or genetic flaw.For example, it be 4ng/ml that the level of patient's blood plasma prostate specific antigen (PSA) may surpass normal dividing value, but digital rectal examination may not have the tumour that can touch, can't see tumour by ultra sonic imaging yet.The PSA level that raises is pointed out the formation of carcinoma of prostate or is existed possibility big, and it is quite little that physical detection does not find then to point out tumour, or be in forerunner (precursor) state.Other lifts an example, and female patient does not have the tumour that can measure at present, but has the sudden change of BRCA1 or two kinds of genes of BRCA2, shows that its easy trouble quality of suffering from mammary gland or ovarian cancer is very strong.Other of generally acknowledging in the field are used to estimate in being also included within the tumor load method of tumour possibility occurrence.This class patient use side granzyme is suppressed pharmacological agent may be effectively because initial gross tumor volume is less, tumor load become to coerce the patient health and peaceful and comfortable before, also leave many tumor proliferation cycles.
The 6th aspect, the present invention is a kind of method for the treatment of the catabasis patient, this class patient also has big (substantial) danger of New Development cancer or cancer return after his or her cancer therapy success.For example, a patient uses following treatment plan scheme through intravenous administration because of filling the air the large B cell lymphoid tumor of sending out: endoxan (cyclophosphamide) 750mg/m2, Zorubicin (doxorubicin) 50mg/m2, vincristine(VCR) (vincristine) 1.4mg/m2, and oral prednisone (prednisone) 100mg/m2, the result shows that the patient is slow to this therapeutic response, reaches alleviation gradually, the possibility higher (Armitage, et a/., J Clin.Oncol., 4:160-164,1986) on the statistics that patient's cancer takes place to recur.This patient will benefit with the telomerase inhibitor treatment.If patient's tumour occurs once more, telomerase inhibitor can effectively be resisted this patient's cancer.Especially suitable to the little or avirulent medicine of patient's toxicity, because it has good risk-benefit ratio.Suramine, PPS or AZT etc., very little or nontoxic to patient's toxicity when suppressing the required low dosage of telomerase activation, also just have this good risk-benefit ratio.
Carrying out Telomerase with hTR antisense body suppresses
The 7th aspect of the present invention is based on applicant's discovery, promptly uses the hTR antisense body transfectional cell can be in the activity of external effective inhibition Telomerase.Among the embodiment 4, obviously demonstrated the nucleotide sequence of definition hRT antisense body.Therefore, the present invention has instructed the method that suppresses Telomerase with hRT antisense body cells transfected.
Low dosage AZT
Eight aspect, this aspect have been instructed and have been used the AZT of nontoxic dose to resist method for cancer, and the plasma concentration of its generation is lower than micro-molar range, for example the nmole scope.In the embodiment, give tumour patient, do not follow other treatment AZT.Another embodiment subtracts the treatment back of going out at the operation cell and uses AZT to the cancer patients.Another embodiment, before non-operation cell subtracts the treatment of going out, during or use AZT thereafter.
Telomerase inhibitor is in the local application of target position or target organ
The 9th aspect, telomerase inhibitor can be near known cancer the local or regional medication in position, or be used for suspicious present tumorigenic organ or estimate tumorigenic in the future organ.The telomerase inhibitor of topical application can be near medicine-feeding part tumour cell or tissue in generation live end granzyme inhibition concentration.The locality medication, for example injection or embedment method can adopt the form of storage vault, such as the device that slowly discharges.According to the difference of therapentic part, implantation may need surgical operation.For example, the patient of a high-level epidermis bladder cancer may not have known tumors remaining after successfully treating, but the risk height of tumor recurrence on the statistics.In such patient, can insert art (transurethralinsertion) is directly inserted chronic release telomerase inhibitor at intravesical device by per urethra, the time that continues to discharge can be in several weeks, several months or several years.The telomerase inhibitor that discharges can produce effective inhibition concentration at the mucous layer or the muscle layer of bladder, thereby suppresses the formation of tumour, and finally suppresses any recurrent tumor.Other lifts an example, raise and the patient of suspection prostate cancer in prostate specific antigen (PSA) concentration, can adopt at prostate gland inner or near it locality give or implant the compound of slow release, send one or more telomerase inhibitors, reduce the probability that Symptomatic tumour takes place.
The topical of telomerase inhibitor will produce Telomerase inhibition concentration in the tissue of treatment institute target, and not at blood plasma or be not to produce Telomerase inhibition concentration in other organs of target of treatment.Of the present invention this is based on following discovery on the one hand, and the Suramine part is used for target organ, and the result produces Telomerase inhibition concentration (as, 5 to 100 μ g/g) in target tissue, and the Suramine concentration very low (as 0.1 μ g/ml or lower) in the blood plasma.
Locality administration telomerase inhibitor is compared with systemic route of administration has some advantage.An advantage is the needs that it has promptly avoided frequent drug administration, and it is uninterrupted to guarantee that again the Telomerase restraining effect continues.Another advantage is that local application has reduced telomerase inhibitor may to the toxigenicity effect of organizing of other sick non-treatment targets.Even do not have overt toxicity behind general administration low dosage telomerase inhibitor such as the Suramine yet, wherein need in the cancer patients, provide the Telomerase inhibition concentration (as, see embodiment 7), reduce non-tumour and carry the probability that the exposure of organ can further reduce allergy or other rare incidents.Consider the application that may show toxic other telomerase inhibitors after the systemic medication, this advantage is just more outstanding.
Another uses in the embodiment of PPS (or Suramine), and the present invention can treat matter inflammation between bladder as injection or implantation by adopting the bladder topical, and described medicine can be the form in storage vault such as chronic releasing device.The treatment result that this method can be used for making treatment suffer from the patient of matter inflammation between bladder strengthens, and realize: Suramine, the pharmaceutically useful salt of the pharmaceutically useful salt of Suramine, pentosan polysulfate ester (PPS) and PPS by mode with one or more material topical patients with significant quantity.
Suppress or reduce the method for cell growth
On the one hand, the invention describes the method that suppresses or reduce the cell growth, the for example misgrowth of growing of described cell as hyperplasia or the growth of hypertrophy (hypertrophic) cell, contacts with at least a Telomerase inhibition medicine and realizes by cell and at least a cell being subtracted the medicine that goes out.In general, these methods comprise a step, and the cell (as cancer cells) that is about to the pathologic hyper-proliferative suppresses medicine with at least a Telomerase and contacts, and its dosage can effectively reduce or suppress the propagation of cell or cause cell killing.
Present method can be used for cultured cells, as, under external or isolated condition, also can be used for the intravital cell of experimenter, as, as the part of interior therapeutic test.Treatment plan can be implemented on the mankind or other animal subjects.The treatment validity of combination therapy of the present invention strengthens, and has represented a kind of scheme likely, and it can substitute traditional high toxicity antitumor drug scheme.
Also can use separately though Telomerase suppresses medicine, this medicine preferably with the cytotoxic drug coupling, to obtain to be better than the result of treatment of the expected effect that arbitrary medicine list uses.Further, these medicines can also with other cancer therapy drug couplings, as anti-microtubule medicine, topoisomerase I inhibitor, topoisomerase II inhibitor, antimetabolite, mitotic inhibitor, alkylating agent, intercalator, can influence the medicament of conversion of signals approach (as inhibitors of protein kinase C, as hormone antagonist, antibody as growth factor receptors), promote apoptosis and/or downright bad medicament, biological response modifier is (as Interferon, rabbit, as interleukin, as tumour necrosis factor), operation or radiotherapy.
Use above-mentioned strategy, cell toxicity medicament and Telomerase are enhanced when suppressing drug combination and preferably collaborative effect, improved the validity of cancer therapy drug, thereby allow to use than these medicines of low dosage one or more (even, for example, can use the inferior therapeutic dose of medicine, as long as its only tested or use when independent use rather than coupling); Reduce thus inductive side reaction among experimenter such as the human cancer patient (as, generally acknowledge in any field with the relevant side reaction of chemotherapeutics that gives not change dosage, reduce (neutropenia), intestinal epithelial cells come off (intestinal epithelialcell sloughing) etc. as, alopecia, neutrophil leucocyte).
The treatment method for cancer
Method among the present invention can be used for the treatment of the malignant disease of multiple tract, as influence lung, mammary gland, lymphoid tissue (lymphoid), gi tract (as, colon, rectum), urogenital tract (as, prostate gland, bladder, testis), the pharynx malignant disease, and gland cancer, it comprises following malignant disease, such as colorectal carcinoma, the rectum cancer, renal cell carcinoma, prostate cancer and/or tumor of testis, lung non-small cell carcinoma, carcinoma of small intestine and esophagus cancer.
Can comprise for example fibrosarcoma (fivrosarcoma) with the common noumenal tumour of method treatment of the present invention, myosarcoma (myosarcoma), liposarcoma (liposarcoma), chondrosarcoma (chondrosarcoma), osteogenic sarcoma (ostergeinc sarcoma), chordoma (chordoma), angiosarcoma (angiosarcoma), endotheliosarcoma (endotheliosarcoma), lymphangiosarcoma (lymphangiosarcoma), lymphangioendothelial sarcoma (lymphangioendotheliosarcoma), synovioma (synovioma), mesothelioma (mesothelioma), ewing's sarcoma (Ewing ' s tumor), leiomyosarcoma (leiomyosarcoma), rhabdosarcoma (rhabdomyosarcoma), cancer of the stomach (gastric cancer), the esophageal carcinoma (esophageal cancer), colorectal carcinoma (colon carcinoma), the rectum cancer (rectal cancer), carcinoma of the pancreas, mammary cancer, ovarian cancer, prostate cancer, uterus carcinoma, the cancer (cancer of the head and neck) of head and neck, skin carcinoma, the cancer of the brain, squamous cell carcinoma, sebaceous carcinoma (sebaceous gland carcinoma), papillary carcinoma (papillary carcinoma), papillary carcinoma (papillary adenocarcinoma), cystadenocarcinoma (cystadenocarcinoma), medullary carcinoma (medullary carcinoma), segmental bronchus source property cancer (bronchogenic carcinoma), renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma (choriocarcinoma), spermocytoma (seminoma), embryonal carcinoma (embryonal carcinoma), the WilmShi knurl (Wilm ' s tumor), cervical cancer, carcinoma of testis, lung cancer, small cell lung cancer, nonsmall-cell lung cancer, bladder cancer, cell carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic tumor, oligodendroglioma (oligodentroglioma), meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma or Ka Boqi sarcoma (Kaposi ' s sarcoma).
Method of the present invention also can be used to suppress or reduce the growth of the cell of hematopoietic cell origin, as derives from medullary system, lymphatic system or red system or its any precursor cell.For example, the present invention relates to the treatment of diseases of various derived from bone marrow, described disease comprise acute promyelocytic leukemia (acute promyeloidleukemia) (APML), acute myeloblastic leukemia (acute myelogenous leukemia) (AML) and chronic myelocytic leukemia (chronic myelogenous leukemia) (CML), but be not limited thereto.(summarizing in Vaickus L. (1991) Crit.Rev.Oncol./Hemotol.11:267-97).Can comprise with the lymphocytic malignant disease of this method treatment, acute lymphoblastic leukemia (acutelymphoblastic leukemia) (ALL, comprise that B is that ALL and T are ALL), lymphocytic leukemia (chronic lymphocytic leukemia) (CLL), prolymphocytic leukemia (prolymphocytic leukemia) (PLL), hairy cell leukemia (the cell leukemia of ha) (HLL) and WaldenstromShi macroglobulinemia (WM) (Waldenstrom ' s macroglobulinemia), but be not limited thereto.
Expection can use the malignant lymphoma of methods of treatment other types of the present invention also to comprise, non Hodgkin lymphoma (non-Hodgkin ' s lymphoma) and variant thereof, as lymphoma peripheral T cell (peripheralT-cell lymphomas), adult T cell leukemia/lymphoma (adult T-cellleukemia/lymphoma) (ATL), cutaneous T cell lymphoma (cutaneous T-cell lymphoma) (CTCL) and large granular lymphocyte leukemia (large granular lymphocytic leukemia) (LGF), but be not limited thereto.
Other can comprise the malignant disease that erythroleukemia, lymphoma, Hokdkin disease and source are not clear with the malignant disease of method treatment of the present invention, for example, the malignant diseases of malignant diseases that are difficult to classify and performance various kinds of cell type are such as some embryonal carcinoma or teratoma.
Illustrate, the experimenter can be the patient who suffers from nonsmall-cell lung cancer, use taxol, carboplatin and long-term Suramine combination therapy, after its reaction terminating being needed stop using in taxol/carboplatin combination therapy toxicity relevant because of medicine or because of the patient, Suramine is treated and can successfully be continued to carry out.Perhaps, the patient who suffers from nonsmall-cell lung cancer can use gemcitabine (gemcitabine), cis-platinum and long-term Suramine combination therapy.
Other lifts an example, the experimenter can be the patient of hormone intractable (refractory) prostate cancer, uses Estracyte (estromustine phosphate), taxotere (taxotere) and long-term Suramine combination therapy or Zorubicin, KETOKONAZOL (ketoconazole) and long-term Suramine combination therapy.
Illustrate, the experimenter can be the patient who suffers from metastatic breast cancer again, and it is used endoxan, Zorubicin, 5 Fluracils and long-term Suramine combination therapy, or Zorubicin, taxotere and long-term Suramine combination therapy.In the relevant example, the experimenter be suffer from overexpression with HER2/neu oncogene late period (advanced) mammary cancer the patient, use Trastuzumab (Herceptin) and long-term Suramine to treat coupling or not coupling taxol or cis-platinum to it.
Illustrate, the experimenter can be the patient who suffers from the colorectal cancer of progressivity or transfer again, uses according to sharp for health (irinotecan) and long-term Suramine combination therapy.In the relevant example, the experimenter is the patient who suffers from the progressivity colorectal carcinoma, uses 5 Fluracils, formyl tetrahydrofolic acid (leucoorin) and long-term Suramine combination therapy.
Medication
In the preferred embodiments of the invention, telomerase inhibitor is that systematicness is used.For example, selected compound can through the gi tract external administration (as, through subcutaneous, through intravenously, through intramuscular, through intraperitoneal, through intracutaneous, in sheath etc. mode), oral, intranasal, inhaling type feeding drug into pulmones, per rectum administration and/or transdermal administration.
In another embodiment, telomerase inhibitor is local or regional medication.For example, selected compound can through intravesical (intravesically) administration (as, be administered to intravesical), administration in prostate gland, administration or locality (topically) administration in tumour.
In another embodiment, present method comprises the identical or different medicament that uses repeated doses further, and some details will further be discussed hereinafter.
Dosage regimen
General medicine doctor of this area or animal doctor can determine at an easy rate and give significant quantity described medicament (as, with the form of pharmaceutical composition).For example, generally, physician or animal doctor can bring into use compound of the present invention from the level that is lower than required dosage, so that start required result of treatment, and improve dosage gradually, until reaching required effect.
Generally speaking, the dosage that compound of the present invention is suitable is to produce the subliminal dose that result of treatment promptly can be treated experimenter's disorders such as cancers.This effective dose is usually by above-mentioned factor decision.Usually, with the dosage of compound of the present invention through vein and subcutaneous administration patient, to about 100mg, preferred scope is about 0.01 to about 10mg per kilogram of body weight by per kilogram of body weight about 0.0001, and more preferably about 0.01 arrives about 4mg per kilogram of body weight.If desired, can with effective per daily dose of active agents in whole day with appropriate intervals be divided into two, three, four, five, six or more a plurality of sub-doses come administration, be chosen as with unit dosage form.
In the embodiment preferred, described telomerase inhibitor is a Suramine, and the concentration of Suramine is enough to suppress telomerase activation, but is not enough to produce one or more situation of following effects: (i) on cell proliferation has remarkable inhibition; (ii) in the mankind or animal tumor cell, cause significant necrocytosis; The antitumous effect that can measure and/or (iv) cell cycle arrest are (iii) arranged in experimenter such as human experimenter.Estimating its effect in cultured cells can use the system of describing among the embodiment 14 to determine.
In the embodiment preferred, telomerase inhibitor is a Suramine, and the level of its medication makes the Suramine plasma concentration that exists following one or more situation can not occur: (i) significant cell cycle arrest; (ii) significant necrocytosis; (iii) significant cell growth-inhibiting, for example, plasma concentration is such level, if the Suramine of same concentration is used for cultured cells, had at least 50% by in the culturing cell for the treatment of, more preferably then be at least 80%, most preferred is 99% can continue following one or more situation with after the Suramine treatment: (cycling) cell in the mitotic cycle continues to advance in the cell cycle; Cell maintains vigour or cell still can be bred.
Preferably, the plasma concentration that the Suramine volume production that gives is given birth to is about 0.001 to 100 μ g/ml, even more preferably about 0.1 to about 70 μ g/ml, and being more preferably is about 0.5 to 30 μ g/ml.It is feature that the pharmacokinetics of Suramine is removed with three grades of concentration, and the transformation period was respectively 5.5 hours, 4.1 days and 78 days.Total human body clearance rate is 0.0095 liter/hour/m2(Jodrell, etal., J Clin.Oncol., 12:166-175,1994).Based on the pharmacokinetics principle, the professional in this area can calculate, and the predose that on average should give the patient is about 240mg/m2Thereby the plasma concentration that reaches will be in 168 hours be reduced to by about 90 μ g/ml (63pM) and is about 14 μ g/ml (10 μ M).Selected 168 hours or the interval in 1 week for example, be because locate further consultation by this timed interval to the physician who treats usually.Interim between required other treatment, also can similarly calculate the predose of determining to give Suramine, so that reach preferred Suramine plasma concentration.In treatment cycle thereafter, also can calculate equally for regulating the required maintenance dose of plasma concentration.
In preferred embodiments, total Suramine exposed amount should be less than 7840 μ M-days in 112 days in the preferred blood plasma, in 112 days less than 7100 μ M-days, in 84 days less than 5,880 μ M-days, in 84 days less than 5,300 μ M-days, in 20 days less than 2,000 μ M-days, preferably should be in 96 hours less than 800 μ M-days, preferably should be in 96 hours less than 600 μ M-days, preferably should be in 96 hours less than 500 μ M-days, preferably should be in 96 hours less than 400 μ M-days, preferably should be in 96 hours less than 300 μ M-days, preferably should be in 96 hours less than 252 μ M-days, preferably should be in 96 hours less than 200 μ M-days, preferably should be in 96 hours less than 150 μ M-days, preferably should be in 96 hours less than 100 μ M-days, most preferably should be in 96 hours less than 52 μ M-days.Total Suramine exposed amount represented with μ M-days, be with the drug plasma concentration that μ M-days was unit representation (as the average micro-molar concentration in 24 hours) with the sky be both product during the treatment of unit representation.For example, with the Suramine of 13 μ M the experimenter was treated 4 days, causing total drug exposure was 52 μ M-days in 96 hours.
Preferably, the plasma concentration that amount produced that gives Suramine should be less than 100 μ M/ml, preferably should be less than 90 μ M/ml, preferably should be less than 80 μ M/ml, preferably should be less than 60 μ M/ml, preferably should be less than 40 μ M/ml, more preferably should be less than 15 μ M/ml, most preferably should be less than 10 μ M/ml.
In preferred embodiments, described telomerase inhibitor is a Suramine, give Suramine or Suramine concentration in the blood plasma maintained to be enough to suppress telomerase activation level during surpass one month, or be more preferably and surpass 1 year, or be more preferably indefinitely.
In preferred embodiments, described telomerase inhibitor is a Suramine, give Suramine or Suramine concentration in the blood plasma maintained to be enough to suppress telomerase activation level during above 60 days, preferably above 100 days, preferably surpass 150 days, preferably surpass 1 year, be more preferably above 2 years, most preferred is indefinitely, surpass give cell subtract the treatment of going out during.
In preferred embodiments, described telomerase inhibitor is a Suramine, the mode that gives Suramine is the topical target organ, Suramine treatment institute maintained at the Suramine concentration in organizing the level that is enough to suppress telomerase activation during above 1 month, or preferably surpass 1 year, or be more preferably indefinitely.For example, when using Suramine to carry out cancer prevention, treatment may need to use the several years, extends to patient's the whole remaining years.
In an embodiment preferred, described telomerase inhibitor is a Suramine, the mode that gives Suramine is the topical target organ, Suramine treatment institute maintained at the Suramine concentration in the tissue level that is enough to suppress telomerase activation during surpass 60 days, preferably above 100 days, preferably above 150 days, preferably above 1 year, be more preferably and surpass 2 years, most preferred be indefinitely, surpass give cell subtract go out treat during.
In preferred embodiments, described telomerase inhibitor is a Suramine, the administration Suramine or with the concentration of Suramine in the blood plasma maintain be enough to suppress telomerase activation or strengthen cell subtract level that the treatment of going out renders a service during be more than 30 days, preferably above 60 days, preferably surpass 100 days, preferably surpass 150 days, preferably above 1 year, most preferred for surpassing 2 years, described during for give cell before subtracting the treatment first day of going out during.
Method described herein uses Suramine to strengthen the antitumous effect that cell subtracts the treatment of going out, select Suramine dosage to make it be lower than 100 μ g/ml subtracting the plasma concentration that is produced in the Mammals of the therapy for treating that goes out with cell, preferably be lower than 80 μ g/ml, preferably be lower than 60 μ g/ml, more preferably less than 40 μ g/ml, most preferably be lower than 15 μ g/ml.The administration of Suramine can be before using at least a anticarcinogen or other cells to subtract to go out treatment, simultaneously or carry out afterwards.Animal experiment shown in this paper shows, with two weekly (two weekly) 10mg/kg intravenously bullet formula Suramines the treatment of mouse is strengthened the curative effect of the antitumour drug of using thereafter (as taxol), and can not produce additional losing weight.Calculating this dosage of employing can be so that the blood plasma Suramine concentration that produces after 72 hours at dosed administration (dose administration) be about 10 μ M (～14 μ g/ml).The method of prior art is used the Suramine of high dosage, it is used separately or with the cell toxicity medicament coupling, for human patients, produce measurable antitumor action and the concentration of blood plasma Suramine need be maintained 150 to 300 μ g/ml (Eisenberger et a/., (1995) JClinOncol 13:2174-2186; Klohs, United States Patent (USP) the 5th, 597, the 830 and the 5th, 767, No. 110).Purpose is to keep the Suramine plasma concentration and consists of at the common Suramine dosage timetable of 150 to 300 μ g/ml: initial dosage is 2100mg/m in first week2, and per 28 days repeat administration subsequent dose, described administration continues 6 months or more of a specified duration; Bayes' theorem (Bayesian) pharmacokinetics method (Dawson et al., Clin Cancer Res 4:37-44,1998 are used in the adjustment of subsequent dose; Falcone eta/., Cancer86:470-476,1999).With this dosage and long-term treatment, Suramine can cause following toxic action to human patients: adrenal insufficiency, blood coagulation disease (coagulopathy), peripheral neurophaty and muscle weakness proximal (Dorr and Von Hoff, Cancer Chemotherapy Handbook, 1994, pp 859-866).The incidence that knows these toxic actions is relevant with dose accumulation with seriousness, and method described herein can reduce to it minimum.
In the embodiment preferred, described telomerase inhibitor is PPS, and preferably, the telomere that the concentration of administration Suramine is enough to suppress telomerase activation and cause tumour cell shortens, but is not enough to produce following one or more influence: (i) significant anticoagulating active; (ii) in the mankind or animal tumor cell, cause significant necrocytosis; The antitumous effect that can measure and/or (iv) cell cycle arrest are (iii) arranged in experimenter such as human experimenter.
Though medicament of the present invention can give separately also can unite with other drug to give, preferred described medicament is as the form administration of pharmaceutical composition.
In an embodiment preferred, described telomerase inhibitor is a Suramine.
Composition and prescription
On the other hand, the present invention relates to pharmaceutical composition, it comprises at least a telomerase inhibitor and pharmaceutically useful carrier.Preferably, the amount of the medicament of existence can effectively suppress the activity of Telomerase in the human tumor, and strengthens killing and wounding the cell of hyper-proliferative.
In an embodiment preferred, have operation instruction in the packing of described pharmaceutical composition or multiple pharmaceutical composition, as described herein.
The present invention also comprises (timed-release) prescription that regularly discharges, for example, and the prescription of the slow release of telomerase inhibitor and pharmaceutically useful carrier.
In another embodiment, pharmaceutical composition is applicable to intravenous injection.This composition also is suitable for locality, regionality or systemic medication.
In another embodiment, pharmaceutical composition can comprise one or more pharmaceutically useful carriers.And in another embodiment, invention has a nano particle of relating to, and it comprises and intersects gel and the healing potion that links to each other, as telomerase inhibitor, and for example Suramine or PPS etc.In another embodiment, the present invention relates to comprise the mixture of nano particle and pharmaceutically useful carrier in addition.This carrier for example, goes for systemic administration, regional administration or topical.In one embodiment, the nano particle diameter is about about 500 to about 1 μ m, or is about 600nm to about 800nm.
The present invention relates in addition and contains medicine such as telomerase inhibitor, as the nano particle of Suramine or PPS etc.In one embodiment, described nano particle diameter arrives about 100 μ m for about 500nm, and about 500nm is to about 50 μ m, about 500nm is to about 25 μ m, and about 500nm is to about 20 μ m, and about 500nm is to about 15 μ m, about 500nm is to about 10 μ m, about 750nm is to about 10 μ m, and about 1 μ m is to about 10 μ m, and about 750nm is to about 7.5 μ m, about 1 μ m is to about 7.5 μ m, about 2 μ m are to about 7.5 μ m, and 3 μ m are to about 7 μ m, or about 5 μ m.In another embodiment, invention has the compound of relating to, and it comprises nano particle and pharmaceutically useful carrier.This pharmaceutically useful carrier for example, goes for systemic administration, regional administration or topical.The invention still further relates to treatment patient's method, comprise patient's administration nano particle of the present invention and pharmaceutically useful carrier.
In another embodiment, the present invention relates to be applicable to oral administration or systemic administration patient's nano particle, it contains taxol, and wherein said nano particle diameter is about 5 μ m.In another embodiment, the present invention relates to be applicable to oral administration or systemic administration patient's nano particle, it contains Suramine or PPS, and wherein said nano particle diameter is about 5 μ m.
The invention still further relates to test kit, i.e. goods, it is used for treatment for cancer.This test kit comprises the telomerase inhibitor in the pharmaceutically useful carrier, container, and use described telomerase inhibitor to suppress or reduce the specification sheets of cell growth, described cell growth for example with the misgrowth of tumour or related to cancer.For example, may comprise in the test kit of the present invention be used in advance, the telomerase inhibitor of follow-up or administration simultaneously.Test kit also can provide the telomerase inhibitor that is mixed with formulation and be applicable to the carrier of part, zone or systemic medication.Further, test kit also can provide prognosis, diagnosis and/or cancer by stages, for example is used for, and estimates susceptibility and resistibility to cancer.
The pharmaceutical composition that contains mixture of the present invention can contain wetting agent, emulsifying agent and lubricant, as Sodium Lauryl Sulphate BP/USP (sodium lauryl sulfate) and Magnesium Stearate, and tinting material, releasing agent, coating agent, sweeting agent, seasonings and sweetening agent, and sanitas.
Prescription of the present invention comprises the prescription that is suitable for various administering modes, that described administering mode comprises is oral, intranasal, part, suction, transdermal, through cheek, through the hypogloeeis, per rectum, transvaginal and/or through the gi tract external administration.Adopt suitable form by various routes of administration.For example, can be with tablet or capsular form administration, by injection, suction, eye drop, ointment, suppository etc., by injection, infusion or inhalation; With lotion or ointment topical; With the administration of suppository per rectum.This prescription is easy to provide with the form of unitary dose, can prepare with well-known method in the pharmaceutical technology.Normally can produce the amount of result of treatment in single agent form with the amount of carrier substance bonded activeconstituents.Generally, precentagewise example meter, the amount of activeconstituents can be about 1% to about 99%, preferably about 5% to about 70%, most preferably about 10% to about 30%.
The form that the present invention is suitable for the prescription of oral administration comprises that capsule, flat capsule, pill, tablet, lozenge (adopt sapid matrix, normally sucrose and Sudan Gum-arabic or tragakanta), medicinal powder, granule, or solution in water or the on-aqueous liquid or suspension, or make oil-in-water or water in oil liquid emulsion, or make elixir or syrup, or make pastille (adopting inert base, as gelatinum or glycerine, or sucrose and Sudan Gum-arabic) and/or collutory etc.
Transdermal patch has extra advantage, can be so that medicine of the present invention controlledly is released in the body.This medicine type can be by with described compound dissolution or be dispensed in the suitable medium and make.Also can improve the percutaneous flow velocity of compound with absorption enhancer.This flow velocity can be controlled by one of following dual mode: rate-controlling membrane is provided or active compound is distributed in polymeric matrix or the gel.
Ophthalmology prescription, eye drop, medicinal powder, solution etc. also can be thought within the scope of the present invention.
The present invention is suitable for pharmaceutical composition through the gi tract external administration and contains in the compound of the present invention one or more, itself and one or more following material associating: Zhang Hanshui such as pharmaceutically useful sterilization or non-aqueous solution, dispersion agent, suspensoid, or emulsion, or sterile powder, it will be dissolved in aseptic the injectable solvent or dispersion agent before use, wherein can contain the solute that buffer reagent, fungistat, feasible prescription and purpose receptor blood etc. are opened, or suspension agent or thickening material.
In some situation, for the effect of prolong drug, need be by through subcutaneous or through the slow down absorption of medicine of the mode of intramuscularly.This can realize by the amorphous substance that uses crystalline suspension or poorly water-soluble.The uptake rate of medicine depends on its dissolution rate, and dissolution rate is then by crystalline size and crystalline form decision.Perhaps can be by with medicine dissolution or be suspended in the oily carrier to make medicament forms through the gi tract external administration to absorb to slow down.
The preparation of injectable file layout is by for example in many lactic acid polyglycolide (polylactide-polyglycolide), forming the microencapsulated matrix of test-compound at Biodegradable polymeric.According to the character of medicine and polymeric ratio and used special polymers, the release rate of medicine can be controlled.The degradable polymeric example of other biological comprises, for example poly-(ortho ester (orthoester)) and poly-(dehydrate (anhydride)).Injectable file layout also can prepare in another way, is about to medicine embedding (entrapping) in the liposome or microemulsion compatible with body tissue.
No matter select which kind of route of administration, compound of the present invention and/or pharmaceutical composition of the present invention all are the pharmaceutically useful formulations that is mixed with the well-known ordinary method of this area professional, and wherein this compound can be used with suitable hydrated form.
The actual dose level of activeconstituents may have nothing in common with each other in the pharmaceutical composition of the present invention, so that obtain a certain amount of activeconstituents, and the described a certain amount of required reaction that can effectively realize to concrete patient, composition and administering mode, and the patient is not had toxicity.
Selected dosage level is determined by multiple factor, the activity that comprises particular compound of the present invention or its ester, salt or acid amides, the excretion rate of route of administration, administration time, the particular compound that adopted, the time that treatment continues, with other drug, preparation and/or material, the patient's age of treat, sex, body weight, state, general health situation and the medical history of used medication combined use, and the interior generally acknowledged similar factor of medical field.
Embodiment
Brief description the present invention now provides following examples to help further understanding.Provide these embodiment to be intended to illustrate practical application area more of the present invention, and unrestricted application of the present invention.
In described all embodiment, unless stated otherwise, the technology that the present invention used is routine techniquess such as chemistry, molecular biology, microbiology, recombinant DNA technology, cell cultures, animal rearing, all belongs to the technology in the prior art personnel ability scope and detailed description is arranged in the literature.As referring to Sambrook, Fritsch and Maniatis, Molecular Cloning:Cold Spring HarborLaboratory Press (1989); DNA Cloning, the 1st and 2 volumes, (D.N.Glover, Ed.1985); Harlow and Lane, Antibodies:a Laboratory Manual, (1988) Cold Spring Harbor; Oligonucleotide Synthesis (M.J.Gait, Ed.1984); Nucleic Acid Hybridization (B.D.Hames and S.J.Higgins, Eds.1984); (Academic Press Inc.), particularly the 154th and the 155th rolls up (Wu and Grossman, Eds to the series Methods In Enzymology; AndCurrent Protocols in Molecular Biology, eds.Ausubel et a/., John Wiley ﹠amp; Sons (1992)).
Materials and methods:
Basic methodologyMaterial requested (for example, medicine, chemical and reactant, people's mammary gland MCF7 cell, because of the FaDu cell, prostate gland PC3 cell), intravital FaDu tumor xenogeneic graft of immune deficiency mouse and three-dimensional tumor tissues are cultivated all and are used the patent sequence number according to the U.S.: 09/587,662, andGanet al., FEBS Letters, 527:10-14,2002 described acquisitions, preparation and use.The measuring method of the effect of drugs of tissue culture cells such as the U.S. use the patent sequence number: 09/587,662 is described.
The active inhibition of Telomerase in cell lysates and the intact cellThe telomeric repeat amplification scheme (Modified Telomeric Repeat Amplification Protocol) of improvement (TRAP) is analyzed (Gan, etal., Pharm.Res., 18:488-493,2001) be used for measuring the telomerase activation of cell lysates.The active mensuration of Telomerase adopts TRAP analysis in the cell in the intact cell, and is as described below: with PBS washed cell (1 * 105) twice, and centrifugation.Cell precipitation is resuspended among the 100 microlitre serum-free RPMI1640 of streptolysin (Streptolysin) O, 2 little TS primers that rub and the 50 little dNTP of rubbing that contain the 5u/ milliliter room temperature insulation 5 minutes.The enzyme streptolysin O increases the permeability of cytolemma to the TS primer.After entering cell, the TS primer is prolonged by Telomerase original position in the cell.Then, the TS primer after prolonging is separated from cell, as the masterplate of PCR amplification.In cell, add 200 microlitres and contain the RPMI1640 substratum of 10%PBS with termination reaction.Mixture 30 ℃ of insulations 30 minutes, is extended TS primer in the cell to utilize Telomerase.Afterwards, with cytolysis, use the TRAP method directly the cytolysis thing that contains the TS primer that has been extended to be analyzed.
The mensuration of the Telomerase length in the cultured cellsTwo kinds of methods can be used for measuring the length of Telomerase.First method is based on the solution hybridization method (TALA) (Gan, etal., Pharm.Res., 18:1655-1659,2001) of Telomerase Content and length measurment, measures the mean length of terminal limitations fragment (TRF).Second method is that original position fluorescent hybridization method (FISH) is measured the Telomerase signal, and estimates about length of each telomere of end of chromosome.FISH method such as the U.S. use patent sequence number .09/587, and 662 and Gan etc., 2001 is described.
The mensuration of senile cellCan use (Dimri etc., 1995) described 4-tilactase staining to measure senile cell.
Embodiment 1 Suramine and AZT are effective telomerase inhibitors-in cell extract and cultured cells
The inhibition of TelomeraseSuramine is the effect of slight inhibition reverse transcriptase activity to be arranged but the uncertain medicament that whether can suppress Telomerase, in multiple human tumour cell line, it is studied, described clone comprises human pharynx FaDu, human benign prostatic PC3 and human mammary MCF7 cell.Its activity is compared with the AZT activity.
Treatment processUse the cells contacting growth interface of processing in allowing culturing bottle of Suramine or AZT to begin afterwards.Test the same day, substratum is shifted out, with the substratum displacement that contains inhibitor.Used drug level is respectively 0,0.1,1,5,10,50 little Suramines that rub, or 0,0.1,1,10,100 little AZT that rub.Growth 7-15 measures telomerase activation and telomere length in cytolysis thing and the intact cell after week in the substratum that contains 0 to 50 little Suramine that rubs or AZT.
Suramine and AZT can be suppressed by Suramine and AZT in human cancer cell Telomerase Activity the active influence of Telomerase, and concentration dependent is arranged.Produce 50% concentration that suppresses and see Table 1.The required concentration of generation 90% inhibition is less than 50 millis and rubs.
The inhibition of table 1 telomerase activation
| Inhibitor | IC50, little rubbing (mean value ± SD) |
| MCF7 | PC3 | FaDu |
| Cell extract | Intact cell | Cell extract | Intact cell | Intact cell |
| Suramine (μ M) | 2.8±1.5 | 1.4±1.1 | 1.6±0.7 | 2.3±0.9 | 1.7±1.0 |
| AZT (μM) | -a | Undetermined | -a | Undetermined | 2.1±1.3 |
aCan not determine that in the cell extract, described activation is the part that suppresses Telomerase because activation from AZT to the triphosphate occurs over just in the intact cell.
Suramine and AZT use the prolongation in the 7-15 week that Suramine or AZT carry out to handle the 34-55% telomere that causes in the FaDu cell to the influence of Telomerase length to shorten, and about 30% telomere shortens in the PC3 cell.
Conclusion: Suramine can suppress the activity of Telomerase effectively at the milli concentration level that rubs, and similar to the effect of known telomerase inhibitor AZT.
Embodiment 2
In negative knurl animal body, Suramine is effective telomerase inhibitor
Suramine suppresses Telomerase in vivo and shortens telomere length.Suramine is a telomerase inhibitor validity in vivo, is to assess by the telomere length in institute's implanted tumor cells in the immunosuppressant mouse body is measured.
Treatment process(it is subcutaneous that 0.51 * 106 cell/100ul) is transplanted in male BALB/cnu/nu mouse with the FaDu cell.This mouse is accepted 10 mg/kg Suramines through the repeated doses of tail intravenous injection.Initial dose should be after tumour transplatation be given and, twice repeat administration weekly afterwards immediately.Suramine treatment 2-6 collects tumor specimen after week.Adopt the fluorescence in situ hybridization method to analyze each intracellular telomere length in the frozen tissue section.The field of microscope of 10 400 times of amplifications of picked at random from each section, the tumour cell percentage of record telomere signal weakening or disappearance.Result and injecting normal saline but not the control group of Suramine compare.
Suramine is in vivo to the influence of Telomerase lengthMouse body tumor planting success ratio after Suramine or physiological saline processing is identical, is 100%, and body weight raises not obvious.The result of FISH shows prolongation in time, and the telomere length in the tumour cell shortens gradually.In the animal body that Suramine was handled, in first two week, the cell proportion of telomere signal weakening or disappearance is maintained at about 10% control group level; Being elevated to 40% cell in the 3rd week, is 75%, the five week to surpass for 80%, the six week 95% around the.Do not observe variation at the control animal cells in vivo that uses physiological saline to handle.
Conclusion: Suramine can shorten the telomere length of the tumour of growing in the body effectively.
Embodiment 3
PPS is effective telomerase inhibitor
PPS suppresses Telomerase and has studied and be exposed to the active inhibition of Telomerase in the FaDu cell behind the PPS.
Treatment processUse the cells contacting growth interface of processing in allowing culturing bottle of PPS to begin afterwards.Test the same day, substratum is shifted out, with the substratum displacement that contains inhibitor.Used drug level is respectively 0,0.1,1,5,10,50 little PPS that rub.Use the quantitative TRAP analysis of improvement that telomerase activation is measured.
To the active influence of TelomeraseSuppressed by PPS at FaDu and SKOV-3 cell Telomerase Activity, and concentration dependent is arranged.Cause 50% concentration that suppresses to be respectively 0.56 and 0.60 mcg/ml.Cause 80% concentration that suppresses to be less than 10 mcg/ml for two kinds of cells.Cause 90% concentration that suppresses to be less than 100 mcg/ml for two kinds of cells.
ConclusionPPS can effectively suppress intracellular telomerase activation.The concentration that produces the inhibition of 50% telomerase activation is lower than anti-freezing desired concn (about 1 mcg/ml).
Embodiment 4
HTR-antisense body suppresses TelomeraseAntisense body research comprises following two steps: (a) setting up has adopted body and an antisense body to human telomerase RNA part; (b), and (c) determine that hTR-antisense body suppresses telomerase activation and induces the ability that shortens telomere with hTR-antisense body (perhaps hTR-has the contrast of adopted body) stable transfected cells.Method has detailed description at prior art such as Mo etc. among the Cancer Res.2003. etc.
Antisense and adopted construct arrangedThe have adopted body and the antisense body surface of preparation htr rna part reach plasmid.Provide adopted body of having of 185 base pairs and antisense body sequence below, it is consistent with GenBank sequence (accession number NR 001566).Above-mentioned processing can obtain containing the segmental 5 kinds of clones of hTR.Sequential analysis shows that a kind of clone is for there being adopted body, and other four kinds are the antisense body.Having adopted body and antisense body surface to reach plasmid transfection these hTR goes into human because of the FaDu cancer cell.
Transfection methodBut IPTG-induction type mammalian expression system is adopted in the transfection of antisense constructs.Institute's DCRP is used for experiment.
The influence of hTR-antisense body cell growthTable 2 has been summed up the result, described result show with not with the transfection of antisense body, without transfection but handle through IPTG, by have adopted body transfection and handle through IPTG or transfection antisense body but (promptly contrast without the cell of IPTG mediation, + IPTG, + adopted body+IPTG arranged, and antisense body) compares, antisense body+IPTG cell (promptly through the transfection of hTR-antisense body, handling to induce the cell of hTR expression through the IPTG again) speed of growth is slower.
HTR-antisense body is to the influence of taxol cytotoxicityStudy two kinds of stable hTR-antisense body cells transfected clones.The cytotoxicity of taxol adopts the SRB method to carry out quantitative assay, and described method pair cell Tot Prot is measured.Use IPTG to handle 44 (clone #1) through hTR-antisense body cells transfected, used taxol treatment again 96 hours to 57 (clone #2) days.Summed up the gained result in the table 2, shown that hTR-antisense body can suppress telomerase activation; The IC of taxol from the transfectional cell of antisense body transfection50IC than other cellular control unit50Reduce, hTR-antisense body all can make the cytotoxic effect of taxol strengthen about twice in two clones as can be seen.
The influence of table 2 hTR antisense body cell growth, taxol cytotoxicity, telomere length and telomerase activation
| Effect | Contrast | +IPTG | + adopted body+IPTG arranged | + antisense body | + antisense body+IPTG |
| Doubling time, hr | 22 | 23 | 23 | 23 | 27 |
| The IC of taxol50, clone #1 | 2.04 | 2.42 | 2.56 | 2.46 | 1.30 |
| The IC of taxol50, nM, clone #2 | 2.05 | 2.53 | 2.21 | 2.87 | 1.33 |
| The end limit fragment, kb | 2.73 | 2.91 | 2.72 | 2.89 | 1.75 |
| Telomerase activation, the % of contrast | 100 | 98.9 | 98.2 | 96.5 | 27％ |
185bp antisense hTR sequence:
5′；
1cagctgacattttttgtttgctctagaatgaacggtggaaggcggcaggccgaggctttt
61ccgcccgctgaaagtcagcgagaaaaacagcgcgcggggagcaaaagcacggcgcctacg
121cccttctcagttagggttagacaaaaaatggccaccacccctcccaggcccaccctccgc
181aaccc
3’
185bp has adopted hTR sequence:
5’：
1gggttgcgga?gggtgggcct?gggaggggtg?gtggccattt?tttgtctaac?cctaactgag
61aagggcgtag?gcgccgtgct?tttgctcccc?gcgcgctgtt?tttctcgctg?actttcagcg
121ggcggaaaag?cctcggcctg?ccgccttcca?ccgttcattc?tagagcaaac?aaaaaatgtc
181agctg
3’
HTR antisense body is to the influence of telomere length and telomerase activationAdopt the TALA method that telomere length (being the end limit fragment) is measured.Adopt the TRAP method of improvement that telomerase activation is measured.The result who sums up in the table 2 shows that hTR antisense body can reduce telomere length and lower telomerase activation.
Conclusion: generally speaking, these results show and adopt hTR-antisense body that the human tumor cell is handled, and can suppress telomerase activation, loss telomere, cell growth inhibiting and strengthen the cytotoxicity of taxol.
Embodiment 5
The Suramine of use side granzyme amount of suppression can make the intravital gross tumor volume of animal reduce
Reducing of gross tumor volume appears use the low dosage Suramine in some animal afterMensuration has been transplanted in the immunosuppressant mouse of FaDu tumour the low dosage Suramine for the influence of gross tumor volume.
Treatment processThigh district immunosuppressant mouse implants 5 * 10 through subcutaneous injection5The FaDu cell.Suramine (10 mg/kg) is through intraperitoneal injection, weekly twice totally 6 week.Tumor cell inoculation began the administration Suramine same day.Processing changes the Suramine solution of injection into physiological saline, and is in full accord for other processing of control animals.Observe the tumour size twice weekly.
The low dosage Suramine is to the influence of tumor growth6 animals received the Suramine treatment.In 5 animals, gross tumor volume increases in time.Other one tumour of merely hitting is still growth originally, reaches 4 millimeters after 2 weeks, reduces gradually afterwards, in the 6th all completely dissolves.
Conclusion: handle through long telomerase inhibitor Suramine, can make the intravital tumour completely dissolve of some host.
Embodiment 6
Use the low dosage Suramine to carry out pre-treatment, can strengthen chemotherapeutical anti-tumor activity in the negative knurl animal
This example is described the anti-knurl effect enhancing of chemotherapeutics after the prolongation pre-treatment of telomerase inhibitor.The processing of telomerase inhibitor is still hanged down at the knurl load and just begun in the time of can not touching, and is similar to the situation of the small tumour that maybe can not detect.
Treatment process: in the female nude mouse thigh district in age in 6-8 week through subcutaneous injection 5 * 105Great-hearted FaDu cell.Suramine (10 mg/kg) is then through intraperitoneal injection, weekly twice totally 6 week.Tumor cell inoculation same day, tumour be hour (representing minimal change (minimal disease)) very, beginning administration Suramine.Except changing injection solvent into physiological saline by Suramine solution, in full accord for other processing of control animal.After 6 all pre-treatment period, interrupt Suramine and handle, all animals are all accepted semiweekly 10 mg/kg paclitaxel treatments, totally three weeks.Twice observation and record tumour size weekly in three weeks of paclitaxel treatment.
The pretreated effect of low dosage SuramineAs shown in table 3, with the control animals in salt 6 weeks of water pretreatment, 3 week the back show that gross tumor volumes increase, accept the Suramine pre-treated animal and gross tumor volume then occurs and dwindle.
Table 3 in the mouse body that uses taxol to treat the Suramine pre-treatment to the influence of gross tumor volume
| Handle | The tumour size of different time after taxol treatment begins (% initial tumor size, mean value ± SD) |
| 0 | 0.5 week | 1 week | 1.5 week | 2 weeks | 2.5 week | 3 weeks |
| Salt solution → taxol | 100±0 | 148±23 | ?169±46 | 196±47 | ?188±46 | 116±113 | 143±126 |
| Suramine → taxol | 100±0 | 105±56 | ?75±106 | 84±119 | ?69±98 | 66±93 | 38±53 |
Embodiment 7
Keeping Suramine low, Telomerase inhibition concentration can promote tumour to dwindle, delay tumor growth, prolong the human cancer survival time of patients
Suffer from that pathology is made a definite diagnosis, progressive stage, transitivity, be to use taxol, carboplatin and Suramine to treat by the human patients of IIIB, the nonsmall-cell lung cancer of IIV phase by stages.Treat and give once in per three weeks.The load of Suramine (load) dosage is about 240 milligrams/square metre, and the mathematical equation of the dosage applicant research and development of back is calculated (PCT patent No. PCT/US02/30210).These Suramine dosage make after 21 days plasma concentration reach about 2 at least and rub to about 90 millis.As shown in Example 1, these concentration are enough to suppress Telomerase.Totally 54 patients have accepted treatment.6 patients that at first receive treatment have accepted the treatment of single dose Suramine, and other patients have accepted the treatment of 24 hours the separate doses in interval.Find any toxicity that the use Suramine causes.49 patient's data can be assessed.The total reaction rate is 40.8% and (comprises 6% complete reaction, corresponding no detectable tumour; 34.8% partial reaction, corresponding tumour dwindles at least 50%) the progression of disease time (time to disease progression) is (TTP) above 6 months, median survival time (median survival time) is (MST) above 12 months (Villalona-Calero, et al., Clin.Cancer Res., 9:3303-3311,2003; Villalona-Calero, et al., MSLC Meeting, Vancouver, August, 2003).
These clinical test results are in the similar patient who suffers from progressive stage, transitivity, IIIB/IV phase nonsmall-cell lung cancer, taxol/carboplatin is carried out early stage clinical test results relatively demonstration, add Suramine the anti-tumor activity of taxol/carboplatin is significantly improved.For example, 290 patients' that finish in the recent period experiment shows, the general reaction rate is about 17% (only has＜1% patient obtained reaction completely), TTP is 3.1 months, and MST is 8.1 months (Schiller et al., New Eng J Med, 346:92-98,2002).
Conclusion: long-term (as 12 to 30 weeks) keep Telomerase inhibition Suramine plasma concentration, can strengthen the anti-tumor activity of taxol and carboplatin, improve reactivity, delay tumour progression, prolong the human cancer survival time of patients.
Embodiment 8
Suramine is longer intravital plasma half-life dog
The intravital Suramine pharmacokinetics of dogUse four beasle dogs (beaele dog) that the blood plasma pharmacokinetics of Suramine is studied.
Treatment processUse four beasle dogs, body weight is 11.4 ± 0.4 kilograms.Intubate in the cephalic vein of animal two forelegs.6.75 mg/kg Suramines were gone into a lateral vein through venoclysis in 30 minutes, take a blood sample from the opposite side vein.Suramine is with the form administration of Naphuride salt brine solution.At 5 minutes, 30 minutes, 1,2,4,6,9,12, take a blood sample in the time of 24,48 and 72 hours, 7,14 and 21 days, inject in the pipe of heparinization centrifugal preparation blood plasma.With the plasma concentration of high effective liquid chromatography for measuring Suramine, existing describe in detail (Kassack, et al., J Chromatogr.BBiomed.Appl., 686:275-284,1996), front.There is not compartment (Non-compartmental) pharmacokinetic analysis (Gibaldi, etal., Pharmacokinetics., 1982) with standard manner.
The result: in the dog body, Suramine is eliminated slowly, and total body clearance is 2.1 ± 0.2 milliliters/hour/kilograms, and the final transformation period is 13.0 ± 3.8 days.
Conclusion: Suramine is eliminated in the dog body slowly, and it is especially long that it eliminates the transformation period, reaches 13 days.
Embodiment 9
PPS has strengthened the anti-tumor activity of chemotherapy
Present embodiment instruction second kind of telomerase inhibitor poly-pentose poly sulfuric ester (PPS), it has strengthened the anti-tumor activity of cell toxicity medicament in the primary culture of tumour cell of cultivating and tumour patient.It is between 10 to 100 mcg/ml time that the chemotherapy of PPS has adopted body effect to occur in Telomerase inhibition concentration, be lower than respectively the PPS concentration that produces anti-tumor activity＞10 times and＞100 times.(Wellstein etc., J.Natl.Cancer Inst., 83:716-720,1991; Zugmaier, etc., Ann.NYAcad.Sciences, 886:243-248,1999).
Use two kinds of renal cell carcinoma cells (RCC45 and RCC54) to study.5 FU 5 fluorouracil (fluorouracil) is as chemotherapeutics.Cell was handled 96 hours through 5 FU 5 fluorouracil merging or nonjoinder PPS, used ELISA to measure drug effect, and it is as mixing DNA precursor (bromodeoxyribouridine (bromodeoxyuridine), restraining effect BrdU).The result shows that PPS is at 10 to 100 grams per milliliter no cytotoxicities; As single medicine, the IC of PPS50It is 1,400 grams per milliliter.Table 4 has shown the IC of 5 FU 5 fluorouracil50Value (promptly producing 50% drug level that suppresses) lowers with the adding of PPS.The result of table 4 also shows the second kind of telomerase inhibitor Suramine (30 little rubbing) that adds the Telomerase inhibition concentration, can further lower the IC of 5 FU 5 fluorouracil50But the chemotherapy sensitizing effect of this explanation telomerase inhibitor is addition.
The PPS of the non-toxic concentration of table 4 strengthens the activity of 5 Fluracils
| Clone | The IC50 of 5 FU 5 fluorouracil, μ M |
| No PPS | Add 10 μ g/ml PPS | Add 100 μ g/ml PPS | Add 10 μ g/ml PPS and add 30 μ M Suramines |
| RCC 45 | 7.58 | 5.15 | Non-availability | Non-availability |
| RCC 54 | 7.27 | 4.65 | 3.58 | 2.34 |
Embodiment 10
Very the telomerase inhibitor AZT of lower concentration has strengthened the anti-tumor in vivo effect of chemotherapeutics
Present embodiment has been described in the mouse body of the immune deficiency of carrying the number of people and neck cancer FaDu heterograft, and telomerase inhibitor AZT is to the enhancement of chemotherapeutics (for example taxol) antitumor action.
Mouse (male Balb/c nu/nu mouse in the immune deficiency of carrying the number of people and neck cancer FaDu heterograft, age in 6-8 week) in the body, be with or without under the situation of AZT, the assessment taxol activity, the formation of heterograft is as follows: in left and right side rib wall district in subcutaneous injection 0.1 ml physiological saline 106Great-hearted tumour cell began the pre-neoplastic size in pharmacological agent after growing about 14 days and surpasses 15mm3Four treatment groups are: saline control group, AZT group, taxol group, taxol+AZT group.To saline control group pump pickle 200 microlitres continuous 5 day every day; The injection continuous 5 day every day of taxol group is dissolved in 10 mg/kg taxols (being Taxol), 200 microlitres in Cremophor and the ethanol; AZT organizes through the Alzet of subdermal implantation micropump with speed 200 nanograms/hour 7 days AZT infusions of acceptance.Taxol+AZT winding is subjected to the combined therapy of taxol group and AZT group, and wherein the infusion of AZT began one day in advance than the taxol injection.Paclitaxel treatment begins the back and measured the weight of animals and tumour size on the the 1st, 3,6,8 and 10 day.
The antitumous effect of pharmacological agent has following three kinds of methods to measure.First kind is to measure dwindling of gross tumor volume.Gross tumor volume is measured at first and is given prominence to (protruding) tumor model (mold) (this model forming is rapid) by preparation Jeltrate, prepares afterwards and the recovery model (countermold) of weighing.Secondly, measure the apoptosis effect.The 10th day,, obtain tumor specimen and it is soaked in the formalin animal euthanasia.Make the tissue slice of 5 micron thickness and dye with h and E.Tumor biopsy is carried out the morphology evaluation, assessment tumorous cellular density and density of apoptotic cells.Because apoptotic cell disappears in time, so the density of non-apoptotic cell can be used as apoptotic second index.Cell density is by using image analytical method, and the counting cell number is measured (Song, et al., Proc.Natl.Acad.Sci.USA, 97:8658-8663,2000) in the field of microscope of selected four 400 times of amplifications at random.The 3rd, measure the ability that medicine prolongs the survival time.The monitoring animal is after 100 days or instruct moribund condition (moribundity) (referring to that length of tumor surpasses 10 centimetres).The plasma concentration of AZT is measured in parallel test, uses three mouse at subcutaneous implantation Alzet 1002 perviousness micropumps.Medicine be infused in samples of animal blood before carried out four days.Long-time infusion like this guarantees to obtain constant steady state Suramine plasma concentration.The elisa assay method mensuration AZT plasma concentration that employing can be buied (Neogen, Lexington, KY).
The result who sums up in the table five shows, AZT has strengthened the anti-tumor in vivo effect of taxol, at first, the combined therapy of AZT and taxol makes that gross tumor volume reduces in 10 days follow-up period, has then observed the increase of height to 4 times gross tumor volume in control group, taxol group, AZT group.In the time of the tenth day, the gross tumor volume of accepting the animal body of taxol and AZT combination therapy is significantly less than (p＜0.001, ANOVA, the replication) of other all dosage groups.The second, the evaluation of tumour form shows, accepts the animal of taxol and AZT combination therapy, and the density of apoptotic cells of its tumour is higher than other dosage groups 2-4 doubly, and the density of non-apoptotic cell is lower than other dosage groups 2.6-4 doubly.The 3rd, existence (survival) time series analysis (Kaplan Meier analysiss) shows that the middle bit time reach moribund condition is 21-26 days for control group and AZT group, and has been increased to 42 days and is 49 days for the combination therapy group for the taxol group.The tumor free survivor of taxol group, and the combination therapy group has two routine tumor free survivors (2/12,16%).The survival rate of group of accepting taxol and AZT combination therapy is statistically apparently higher than every other group (p＜0.01, logarithm order (log rank) check).
The treatment of single medicine (taxol or AZT) produces minimum toxicity, and the nontoxicity associated death takes place, with the amplitude minimum (＜3%) that loses weight of weight ratio before the treatment.Add AZT and can not increase the weight of alleviating of body weight in taxol, prompting AZT can not increase the host toxicity of taxol.The AZT plasma concentration that adopts the ELISA method to measure is 9.6 to receive and rub.This concentration is starkly lower than and suppresses Telomerase desired concn (2 little rubbing see Table 1 in the FaDu cell) or bring out the required concentration of cytotoxicity (the 20 little FaDu of rubbing cells; Mo etc., Cancer Res.63:579-585,2003).The result of this research is amazing.
Table 5 AZT is to the strengthening effect of taxol antitumor action
| Treatment (n) | Non-apoptotic cell number/400 * visual field | Apoptotic cell number/400 * visual field | Apoptotic cell per-cent/tumour | Body weight during termination test, the % pretreatment values |
| Physiological saline contrast (11) | 235±39 | 31±7 | 12±2％ | 106±3 |
| AZT(10) | 249±36 | 28±7 | 10±3％ | 105±6 |
| Taxol (12) | 168±53a | 66±34a | 30±17％a | 97±6a |
| Taxol+AZT (12) | 64±68b | 129±36b | 72±26％b | 99±4a |
aCompare with control group and AZT group p＜0.05
bCompare with every other group p＜0.05.
cMean value ± the SD of four groups of independent experiments of data representation.Cell density and level of apoptosis utilization are measured the successive zone with 4 in each tumour at random in image analysis.
Conclusion: the result shows that use can obtain receiving rub the extremely low AZT dosage of grade AZT plasma concentration and treat, and can strengthen the anti-tumor activity of chemotherapy.
Embodiment 11
The AZT of administration Telomerase amount of suppression can cause the intravital gross tumor volume of animal to dwindle
The intravital gross tumor volume of animal reduces behind administration AZTThe influence of the immunosuppressant mouse assay repeat administration single medicine AZT of FaDu cell to gross tumor volume transplanted in use.
Treatment processIn the mouse two side rib abdomen districts of immune deficiency through subcutaneous injection 106A FaDu cell/side rib abdomen.Adopt Alzet perviousness micropump by continuous h inf administration AZT, injection speed is 5 micrograms/mouse/sky.This AZT dosage causes the stable plasma concentration of about 10 nanograms/milliliter.Inoculate back 14 days beginning administration AZT, continue 14 days.For control animals, change the Suramine solution of injecting into physiological saline, other processing are in full accord.Observe gross tumor volume twice weekly.
The AZT treatment is to the effect of tumor growthAll 16 transplanting have the animal of tumour cell all to show the tumour that can measure before accepting the AZT treatment.In 2 animals wherein, tumour stops growing when beginning to accept the AZT treatment, and volume reduces gradually subsequently.Tumour was untouchable in the 38th day, and postmortem in the 59th day confirms the tumour completely dissolve.These two animal initial tumor average-volumes are 35 cubic millimeters of (scopes: the 18-80 cubic millimeter), similar to the gross tumor volume in all the other animals.
ConclusionLife-time service can obtain receiving low dosage list medicine AZT (telomerase inhibitor) treatment of grade plasma concentration of rubbing, can be so that the tumour completely dissolve.
Embodiment 12
Expection Telomerase suppression therapy combination tumor volume-dwindle treatment can improve result of treatment
Present embodiment has shown that when cell being subtracted the treatment of going out with life-time service telomerase inhibitor combined utilization, cell subtracts the expection of the antitumous effect of the treatment of going out and strengthens.The use of telomerase inhibitor can cell subtract go out before, subtract to go out simultaneously or subtract to go out with cell and carry out after finishing at cell.
Use the long-term treatment of telomerase inhibitor and the effect that cell subtracts the treatment coupling of going outIn order to make telomerase inhibitor can suppress cell proliferation effectively, or apoptosis-induced and cell aging, the intravital tumor load of host must be less, before making below the critical level of extremely inhibition propagation of telomerase inhibitor reduction telomere length and apoptosis-induced and cell aging, tumor load can not reach deadly level.This can subtract the treatment of going out by the employing cell and realize.Therefore, suppress combined utilization if the cell of certain form subtracts the treatment of going out with secular telomerase activation, the telomere length in the tumour cell will shorten in time so, final cell death inducing or cell aging.
Treatment processIts optimal treatment method is the cancer patients that the cell of certain form subtracts the treatment of going out property, will accept selected cell and subtract the treatment of going out.For subtracting the treatment of going out, the selected cell of individual patient depends on patient's cancer, healthy state, age, former treatment history and other factors of in treatment plan, considering usually.After cell subtracts going out property treatment plan and finishes, if think that the patient still cures or disease may recur, then the patient will accept to utilize the continuation of telomerase inhibitor to treat, and used dosage is treated in described continuation is enough to bring out the Telomerase retarding effect.Should continue at least 2 weeks the course of treatment of continuing treatment, and preferably be longer than February, or more preferably be longer than 6 months, or more preferably until thinking described patient's recovery from illness.Preferred so optional treatment plan, wherein telomerase inhibitor treatment beginning or begin simultaneously before cell subtracts the treatment of going out with it, and after continuing to cell and subtracting the treatment of going out and finish, this is owing to making tumour be exposed to longer for some time of Telomerase restraining effect.
ConclusionExpection subtracts the treatment of going out property with Telomerase suppression therapy and conventional cell and unites use, can make the Telomerase suppression therapy more effective, also will solve a long-standing problem, is exactly preferentially to utilize the Telomerase restraining effect to treat the method for tumour patient.
Embodiment 13
The topical Suramine can cause in the target organ but not the Telomerase inhibition concentration in the blood plasma
Present embodiment is described the topical telomerase inhibitor, can be implemented in to reach Telomerase inhibition concentration in target tissue or the organ, and have low and invalid Telomerase inhibition concentration in blood plasma.
Suramine The bladder wall after the regional delivery and system's concentrationSuramine solution is slowly splashed in the bladder cavity of dog, and to the The bladder wall concentration (bladder wallconcentrations as a function distance from the bladder cavity) of conduct with the function of the distance of bladder cavity, and systemic plasma concentration is studied.
Treatment processUse 5 beasle dogs of 9.0 ± 0.4 kilograms of body weight.Intubate is used for administration narcotic in the cephalic vein of animal, and intubate is used for blood sampling in jugular vein.Insert urethral catheter and be used for quantitative instillation (dose instillation) and the tolerant sampling of intravesical.Through the intravesical administration animal Suramine aqueous solution (20 ml waters contain 6 mg/ml Suramines).Be interrupted the 120 minutes concentration (concentrations of the bladder contents and systemicplasma were sampled at frequent intervals for 120 minutes) in the tolerant and systemic blood plasma of frequent selective examination intravesical, put to death animal and collect bladder body this moment.The about 2 centimetres x2 centimetre tissue slice of surface-area is taken from The bladder wall, and is freezing fast in dry ice refrigerative stainless steel square position (flat stainless steel plate).Outer rim to tissue sample is repaired, in order to avoid polluted by drip liquid, the frozen tissue piece is cut into and 40 microns parallel sections of urothelial surface.Utilize HPLC assay determination organized layer, blood plasma and the intravesical Suramine concentration in tolerant.
The result: for the animal of accepting Suramine 6 mg/ml through intravesical, 3.6 mg/ml when 6 mg/ml of the concentration during intravesical is tolerant during from 0 minute are reduced to 120 minutes.The plasma concentration of all time points all is lower than 0.1 mcg/ml.(0 millimeter) successively decreases to serosa surface (4-5 millimeter) the in-house drug level of The bladder wall gradually from the urethra surface.The tissue concentration at 0 millimeter place is approximately about 80 microgram/grams, successively decreases until about 2 millimeters places with logarithm-linearity (log-linear) pattern, and the concentration at this place reaches about 3 microgram/grams.Concentration display density in the The bladder wall resistates is about 3 microgram/grams hardly with change in depth.
Conclusion: these results are presented at the drug level that the administration of organ inner compartment can be provided at the Telomerase inhibition in the purpose organ or tissue, and then much lower times of systemic plasma concentration is not enough to produce Telomerase inhibition effect.
Embodiment 14
Cell culture system
The cell culture assays test is swallowed the FaDu cell to human prostate PC3 tumour cell, people's mammary gland MCF7 cell or people and is carried out.If the needs any according to the invention in three kinds of cells of this paper, the selection and the dosage of telomerase inhibitor are suitable for the present invention.The preferred PC3 cell that uses.
Human benign prostatic PC3 tumour cell, mammary gland MCF7 cell or pharynx FaDu cell can (American Type Culture Collection) obtain from the U.S. common culture collection center.The doubling time of these three kinds of clones is about 24 hours.All three kinds of clones should be as monolayer culture in containing 5%CO2, 95% air wet environment, 37 ℃ in.PC3Cell should remain in RPMI 1640 substratum; The MCF7 cell remains in RPMI 1640 or the minimum essential medium (MEM); The FaDu cell remains among the MEM.All substratum should be supplemented with 9% heat-inactivated foetal calf serum, 2 millis rub 1-glutamine, 0.1%10 milli rub non-essential amino acid, 90 mcg/ml gentamicins and 90 mcg/ml cefotaxime sodiums (cefotaxime sodium).Use trypsinase to expire the culture harvested cell at the end from the Asia, before bed board, be suspended from the fresh culture again.According to trypan blue exclusion method (trypan blueexclusion), the cell of viability＞90% is used to estimate the cytotoxicity of telomerase inhibitor such as Suramine.Cell is laid on 96 orifice plates, and density makes and can not reach the full end in latter stage in pharmacological agent stage.By in no pharmaceutical culture medium, allowing cell attachment in the surface of described plate in 20-24 hour.Afterwards, cell and the substratum that contains FGF antagonist (embodiment uses 0.2 milliliter of Suramine) together are incubated, the concentration of described FGF antagonist is striden at least at 4 logarithm scopes (spanning at least 4 logscales).With the inhibition of the evaluation of pesticide effectiveness for BrdU is mixed, for example can be according to Cell ProliferationELISA BrdU (Boehringer Mannheim).
ConclusionThe present invention describes with reference to various embodiments and illustrates with it, and those skilled in the art can find to do various improvement, and its composition can use its Equivalent to substitute, and does not depart from scope of the present invention.In addition, can carry out multiple modification according to instruction of the present invention and not depart from base region of the present invention to be suitable for particular case or material.Use normal experiment, those skilled in the art will recognize that the many equivalents that maybe can affirm the specifically described specific embodiments of the present invention.Described equivalent comprises within the scope of the claims.Therefore, the present invention is not the specific embodiments that best mode of the present invention is implemented in intention limit and conduct, but the present invention will comprise that all fall into the embodiment in the claim scope.In this application, all units are a meter system, and all are measured and per-cent is all represented by weight, except as otherwise noted.Simultaneously, all documents that the application quoted are included in herein as a reference.In unless otherwise indicated, only use conventional test method, here the Equivalent of a lot of special applications embodiment of the present invention of special narration.These Equivalents all are included in the rights statement scope described later.Therefore, it is emphasized that the present invention is not restricted to that those are special as some of the mode of best practice the present invention expection, in this application, what all units used is a meter system, and all countings and per-cent are weight.And, all show the reference source behind all references here.
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Sequence table
＜110〉Jesse .L.S. Austria (Au, Jessie L.-S.)
Wientjes，Guilliame
＜120〉method and composition of use Suramine, pentosan polysulfate ester, telomerase antisense body and telomerase inhibitor
＜130〉60/44,061, on January 31st, 2003 submitted to
<150>US?60/444,061
<151>2003-01-31
<160>2
<170>PatentIn?version?3.1
<210>1
<211>185
<212>DNA
＜213〉people (Homo sapiens)
<400>1
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ccgcccgctg?aaagtcagcg?agaaaaacag?cgcgcgggga?gcaaaagcac?ggcgcctacg 120
cccttctcag?ttagggttag?acaaaaaatg?gccaccaccc?ctcccaggcc?caccctccgc 180
aaccc 185
<210>2
<211>185
<212>DNA
＜213〉people (Homo sapiens)
<400>2
gggttgcgga?gggtgggcct?gggaggggtg?gtggccattt?tttgtctaac?cctaactgag 60
aagggcgtag?gcgccgtgct?tttgctcccc?gcgcgctgtt?tttctcgctg?actttcagcg 120
ggcggaaaag?cctcggcctg?ccgccttcca?ccgttcattc?tagagcaaac?aaaaaatgtc 180
agctg 185