(19)
(11)EP 3 269 393 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
11.12.2019 Bulletin 2019/50

(21)Application number: 15890589.3

(22)Date of filing:  14.10.2015
(51)International Patent Classification (IPC): 
A61K 47/64(2017.01)
A61K 31/337(2006.01)
A61P 35/00(2006.01)
A61K 38/08(2019.01)
A61K 31/704(2006.01)
(86)International application number:
PCT/CN2015/091888
(87)International publication number:
WO 2016/173214 (03.11.2016 Gazette  2016/44)

(54)

HSP90 INHIBITION PEPTIDE CONJUGATE AND APPLICATION THEREOF IN TREATING TUMOR

HSP90-HEMMENDES PEPTIDKONJUGAT UND ANWENDUNG DAVON IN DER TUMORBEHANDLUNG

CONJUGUÉ PEPTIDIQUE INHIBITEUR DE HSP90 ET APPLICATION DE CE DERNIER DANS LE TRAITEMENT D'UNE TUMEUR


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 27.04.2015 CN 201510203194

(43)Date of publication of application:
17.01.2018 Bulletin 2018/03

(73)Proprietor: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
Beijing 100005 (CN)

(72)Inventors:
  • LIU, Yanyong
    Beijing 100005 (CN)
  • YANG, Nan
    Beijing 100005 (CN)
  • ZUO, Pingping
    Beijing 100005 (CN)

(74)Representative: Prinz & Partner mbB 
Patent- und Rechtsanwälte Rundfunkplatz 2
80335 München
80335 München (DE)


(56)References cited: : 
WO-A1-2014/080251
CN-A- 103 342 735
WO-A2-2013/158644
CN-A- 104 800 858
  
  • CHRISTIANE FISCHER ET AL: "A Bombesin-Shepherdin Radioconjugate Designed for Combined Extra- and Intracellular Targeting", PHARMACEUTICALS, vol. 7, no. 6, 27 May 2014 (2014-05-27), pages 662-675, XP55464916, DOI: 10.3390/ph7060662
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Technical field



[0001] The present invention relates to targeting agents for tumor treatment, particularly, relating to the use of Heat Shock Protein 90 (HSP90) inhibitory peptide in preparing targeting therapeutic conjugates for treating tumors.

Background Art



[0002] Malignant tumor seriously threatens the human health and improving the present therapeutic efficacy is an important mission for the medical researchers. Most of the current available chemotherapeutics exhibits a poor targeting capability with only 5-10% of administrated dosage accumulated in the tumor sites. Meanwhile, severe adverse side effects of these drugs also affect the optimized dosage, leading to incomplete recovery, relapse and drug resistance.

[0003] Tremendous clinical advances achieved for the tyrosine kinase inhibitors aiming at the specific mutation of signaling pathway in tumor tissues. For the people carrying genetic mutated phenotype, the molecularly targeted drugs are obviously effective with low adverse effects. However, molecularly targeted cancer therapy remains challenged by an extremely small proportion of patients that can benefit and the occurrence of drug resistance 10-12 months later. Cancer cells often find ways to compensate or replace for the blockage of the pathway, therefore pinpointing a single pathway is unlikely to eradicate cancer cells. Researchers pay more attention to the signal converging point and trying to developing drugs that simultaneously attack multiple targets to improve the therapeutic efficacy and action spectrum. In this regard, heat shock protein 90 (Hsp90) represents an attractive cancer therapeutic target.

[0004] Hsp90 is an abundant and ubiquitous molecular chaperone in the eukaryotic cell that plays an important role in regulating correct folding, maturation and stabilization of numerous nascent client proteins including enzyme, receptor and signal molecular. The cancer cells initiate protective mechanisms to antagonize the challenge of chemotherapeutics, including the rapid up-regulation of HSP90, which constitute the basis for chemotherapy adaption and resistance. Accordingly, Hsp90 is constitutively expressed at 2-10 fold levels in cancer cells than their normal counterparts [1]. Moreover, HSP90 is also highly expressed on the cancer cell surface [2]. About tens of oncogenic components are the client protein of HSP90, which make it important oncogenic signal converging point [3]. Inhibiting Hsp90 function will lead to degradation of multiple oncogenic proteins. HSP90 also participates the response to chemotherapeutics by regulating the autophagy, therefore inhibiting HSP90 is facilitated to improve the antitumor efficacy.

[0005] As stated in authorized patent (Patent Number: ZL201310258476.0; Patent Title: A tumor specific targeting peptide and thereof use), a targeting peptide (Amino acid sequence: LPLTPLP, hereinafter referred to as P7) was screened by phage display technique, and was conjugated to the Docetaxel-loaded polylactic acid nanoparticles for the preparation of medicines for early diagnosis and treatment. The conjugated nanoparticle obtained from the targeting peptide and the Docetaxel-loaded polylactic acid was hereinafter referred to as targeting nanoparticle preparation (TN-DTX).

[0006] WO 2014/080251 A1 discloses antibody-drug conjugates and anticipates antiHsp90-antibodies as suitable targeting moieties.

[0007] Christiane Fischer et al., "A Bombesin-Shepherdin Radioconjugate Designed for Combined Extra- and Intracellular Targeting", Pharmaceuticals, vol. 7, no. 6, 27 May 2014, pages 662-675, disclose shepherdin linked to cytotoxic 99mTc.

[0008] WO 2013/158644 A2 is directed to pharmacological compounds including an effector moiety conjugated to a binding moiety that directs the effector moiety to a biological target of interest. The compounds can be described as a protein interacting binding moiety-drug conjugate (SDC-TRAP) compounds, which include a protein interacting binding moiety and an effector moiety. In certain embodiments directed to treating cancer, the SDC-TRAP can include an Hsp90 inhibitor conjugated to a cytotoxic agent as the effector moiety.

Description of the invention



[0009] The invention is directed to a conjugate according to claim 1, wherein the conjugate is prepared by conjugating HSP90 inhibitory peptide with cytotoxic agents via a linker, wherein the amino acid sequence of the HSP90 inhibitory peptide is LPLTPLP.

[0010] Cellular membrane proteins were extracted by the applicants and the binding proteins were separated using polyacrylamide gel electrophoresis. Then, the isolated binding protein of targeting peptide was identified as HSP90 with amino acid sequence of LPLTPLP (hereinafter referred to as targeting peptide) by mass spectrometry analysis. The impact of the targeting peptide on the expression of HSP90 was carried out by Western Blotting analysis, results showed that targeting peptide directly inhibited the expression of HSP90 after the exposure of tumor cell to the targeting peptide, indicating that the targeting peptide was a HSP90 inhibitory peptide.

[0011] As stated in the background art section, HSP90 was highly expressed in a variety of tumor cell to defense the external stress such as temperature, drug and radiation, playing a vital role in the recurrence and drug resistance. Applicants explored the combinative effect of targeting peptide with Docetaxel, which was referred to as DTX hereinafter.

[0012] Combining docetaxel with targeting peptide significantly affected the expression of the autophagy-associated protein Beclin 1 and LC3. Exposure to targeting peptide alone did not affect the expression of those two proteins. Following exposure to docetaxel, the expression of the autophagy-associated protein Beclin 1 increased and the conversion of LC3-I to LC3-II occurred, indicating an elevation of autophagy. Simultaneous administrating docetaxel with LPLTPLP led to a decrease in the expression of beclin 1 and the reversal of the LC3-I to LC3-II conversion compared with docetaxel alone. The obtained results showed that the targeting peptide significantly inhibited the autophagy induced by docetaxel.

[0013] As can be seen that in addition to targeting capability, targeting peptide also can enhance the switching of tumor cell from autophagy to apoptosis when combined with docetaxel, leading to enhanced antitumor effect. Based on this background, a conjugate capable of targeting delivery, autophagy inhibition and cancer killing was constructed by conjugating the targeting peptide with docetaxel (hereinafter referred to as DTX-P7).

[0014] As stated in vivo experiment in the invention, DTX-P7 significantly decreased the tumor volume compared with docetaxel and nanoparticle prepared based on the targeting peptide, exhibiting obvious cytotoxic effects to lung adenocarcinoma, breast cancer and melanoma cells.

[0015] The invention provides the use of employing the HSP90 inhibitory peptide for the preparing the cytotoxic agent conjugates. The HSP90 inhibitory peptide specifically binds to cancer cells and suppresses the intracellular expression of HSP90.

[0016] This invention further provides a conjugate which is constructed by conjugating HSP90 inhibitory peptide with cytotoxic agent through a linker. The amino acid sequence of the HSP90 inhibitory peptide is LPLTPLP. Preferably, there are 1-3 G linked at the amino terminal of the HSP90 inhibitory peptide, and S, SH or SHS linked at the carboxyl terminal of the HSP90 inhibitory peptide. Preferably, the cytotoxic agents are selected from docetaxel, paclitaxel and doxorubicin. Preferably, the general formula of described linker is -CO-(CH2CH2)n-CO-, where n is selected from 1,2,3,4,5,6,7,8,9,10. The preferable formula of the linker is -CO-CH2CH2-CO-.

[0017] This invention provides the preparation of conjugates employing HSP90 inhibitory peptide and cytotoxic agents such as docetaxel, paclitaxel and doxorubicin, through esterification or amidation reaction mediated by the linker. Preferably, the amino terminal of the peptide is connected to the 2-OH of docetaxel and paclitaxel mediated by the linker to prepare the conjugate.

[0018] In other respect, this invention provides a conjugate by linking amino terminal of HSP90 inhibitory peptide to the 3-NH2 on the 10-lyxo-hexopyranosyl group of the doxorubicin mediated by the linker.

[0019] In another aspect, this invention provides a conjugate by linking amino terminal of HSP90 inhibitory peptide to the 8- glycolyl group of the doxorubicin mediated by the linker.

[0020] The present invention relates to the drugs using the conjugates mentioned above as active ingredient. The conjugates are combined with pharmaceutically acceptable solid or liquid excipients and /or supplements to prepare any formulations applicable to human or animal. Furthermore, this invention relates to the use of conjugates for preparing the cancer preventive and /or therapeutic drugs. The tumors refer to HSP90 highly-expressed cancer, preferably, comprising lung cancer, lung adenocarcinoma, melanoma, gastric cancer, breast cancer, renal carcinoma, liver cancer, oral epidermoid carcinoma, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, colorectal cancer and neural tumors, more preferably, lung adenocarcinoma, breast cancer and melanoma, most preferably, lung adenocarcinoma.

[0021] The conjugates in this invention can be administrated in way of unit dose. The administrated routes include intestinal and parenteral routes, such as oral administration, intravenous injection, intramuscular injection, subcutaneous injection and trans-nasal, oral mucosa, eye, pulmonary, dermal, vaginal and rectal administration, and so on.

[0022] The compositions can be administrated in a liquid, solid or semi-solid formulation. Liquid formulation comprises the solution (including true solution and colloidal solution), emulsion (o/w, w/o, or multiple emulsion), suspension, injection (including injection workshop, powder injection and infusion solution), eye-drops, nasal-drops, lotion, liniment and so on. Solid formulation includes tablet (including conventional, enteric-coated, buccal, dispersible, chewable, effervescent, orally disintegrating tablets), capsule (including hard, soft or enteric capsule), granules, powder, pellet, dropping pill, suppository, membranes, paster, aerosol or dry powder inhalations, spray and so on. Semisolid formulation comprises ointment, gel, cataplasm and so on.

[0023] The conjugate can be made into either commonly used preparations, or sustained release, controlled release, targeting preparation and diverse microparticle drug delivery system.

[0024] To prepare the tablet formulation, the widely-known excipients including diluent, adhesive, wetting agent, disintegrant, lubricant, glidant can be used together with the conjugate. The diluent includes starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, newtol, microcrystalline cellulose, calcium sulfate, calcium hydrophosphate, calcium carbonate, and so on. Wetting agent comprises water, ethanol, isopropanol, and so on. The adhesive includes starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, arabic gum, gelatin, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methyl cellulose, ethyecellulose, acrylic resin, Carbopol, polyvinylpyrrolidone, polyethylene glycol, and so on. The disintegrant includes the dry starch, microcrystalline cellulose, low substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, polyoxyethylene sorbitol aliphatic ester, sodium dodecyl sulfate and so on. Lubricant and glidant comprises talcum powder, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol and so on.

[0025] The tablet formulation can be further prepared to the coating tablet, such as sugar-coating, film-coated, enteric-coated, double-layer or multilayer tablet.

[0026] To prepare the composition into capsule formation, the conjugate can be mixed with diluent, glidant and directly packaged into hard or soft capsules. The conjugate can also be mixed with the diluent, adhesive, disintegrant to prepare the granule or pellet, and packaged into hard or soft capsules. The diluent, adhesive, wetting agent, disintegrant, glidant used to prepare the tablet can also be applicable to the capsule formulation.

[0027] This conjugate can be used to prepare the injection formulation with water, ethanol, isopropanol, propanediol or the mixture thereof as solvent and adding routinely used solubilizer, cosolvent, pH regulator osmotic pressure regulator. The solubilizer or cosolvent includes poloxamer, phosphatidylcholine, hydroxypropyl-β-cyclodextrin and so on. The pH regulator comprises phosphate, acetate, hydrochloric acid, sodium hydroxide and so on. The osmotic pressure regulator is comprised of sodium chloride, mannitol, glucose, phosphate, acetate, and so on. To prepare the freeze-dried powder injection, mannitol and glucose can be used as propping agent.

[0028] Moreover, colorant, preservative, perfume, flavor and other additives can be supplemented if needed.

[0029] The composition may be administrated via any of widely known route, as long as it is able to reach a desired effect.

[0030] The conjugates in the invention may be used individually or combined with other therapeutics or expectant drugs.

[0031] The anti-tumor effect of the conjugates has been confirmed to treat the malignant cancer.

[0032] This invention discloses that conjugates prepared by coupling HSP90 with cytotoxic agents such as docetaxel, paclitaxel and doxorubicin, are promising and valuable to enhance the anti-tumor efficacy, meanwhile reduce the dosage, which can lessen the adverse effect caused by chemotherapeutics accumulation. The conjugates exhibit triple anti-tumor effects: targeting delivery, chemotherapy, and promoting apoptosis, holding a great potential to improve the clinical outcome of a variety of tumors and reverse the drug resistance.

Legend illustration:



[0033] 

Figure 1 shows that the binding protein of targeting peptide is HSP90.

Figure 2A-2F present the effects of targeting peptide on the HSP90 expression, autophagy and apoptosis. Figure 2A shows the Western blotting results, indicating that targeting peptide significantly inhibited the expression of HSP90 in a dose-dependent manner. Results in Figure 2B-2D indicated that targeting peptide obviously inhibited the autophagy induced by docetaxel. Figure 2E showed that targeting peptide promoted the apoptosis induced by docetaxel by Annexin V analysis. Figure 2F showed that the apoptosis-enhancing effect was further confirmed by transmission electron microscope observation.

Figure 3 refers to the mass spectrometry of DTX-P7.

Figure 4 refers to the body weight changes of nude mice in the different groups.

Figure 5 refers to the tumor tissues resected from DTX, DTX-P7, TN-DTX treated groups.

Figure 6 refers to the relative volume of the tumors from DTX, DTX-P7, TN-DTX treated groups.



[0034] Herein, *: referring to comparison with control group; # referring to comparison with DTX and TN-DTX groups.

Embodiments



[0035] The following embodiments furthermore illustrate the content of this invention, but understandably should not be interpreted as in any way limiting its scope. It will be understood by those skilled in the art that the present invention may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. The scope of the invention is defined in the claims. Unless specialized, the employed techniques in the present invention are conventional methods for those skilled in the art.

Embodiment 1: Binding protein of targeting peptide and its effect on the HSP90 expression



[0036] The protein was extracted with protein extraction kit and subjected to SDS-Page electrophoresis. The interesting protein was recovered and digested with trypsin, then analyzed by mass spectrometry. As shown in Figure 1, molecular weight of band A was 53Kd, which was consistent with that of HSP90kDα. Molecular weight of band B was 48Kd, consistent with that of HSP90 kDβ. Therefore, the target of peptide is identified as HSP90 (Figure 1).

Embodiment 2: Effect of targeting peptide on the expression of HSP90



[0037] 
  1. (1) lung adenocarcinoma A549 cells were trypsinized and then centrifuged at 800×g for 5min. The precipitate was re-suspended with PBS and adjusted to 1×106 cells/ml.
  2. (2) the cells were seeded into 6-well plate and 2 ml culture medium was added. Then the cells were incubated at 37 °C, 5% CO2 overnight.
  3. (3) The peptide was diluted with medium and 300 µl solution was added to each well with a final concentration of 1×10-4 M. Equal volume of medium was added to the negative well. Triple wells were set for each group and experiment was repeated three times. The plate was incubated at 37 °C, 5% CO2 for 48 h. Protein was extracted and subjected to Western blotting analysis.


[0038] Results showed that targeting peptide effectively down regulated the expression of HSP90 in a dose-dependent manner (Figure 2A), indicating the peptide is a HSP90 inhibitory peptide.

Embodiment 3: Effect of targeting peptide on the apoptosis



[0039] The cells were seeded and cultured. Targeting peptide was diluted with culture medium. Appropriate concentration of DTX was also prepared for the seeded cells. Equivalent culture medium was added as negative control group, 1×10-9 M targeting peptide solution added as Peptide group, 10-9 M DTX added as DTX group, 10-9 M targeting peptide and 10-4 M DTX added as combination group (DTX+P). The cells were incubated at 37 °C, 5% CO2 for 6 h to analyze the apoptosis by Annexin V method. The protein was extracted to measure the expression of apoptosis related proteins.

[0040] Results of annexin V showed that targeting peptide induced apoptosis and further promoted apoptosis when combined with DTX.

[0041] Western blotting analysis showed that targeting peptide induce caspase-3 cleavage and the cleavage was further enhanced when combined with DTX compared with DTX alone. Targeting peptide promoted apoptosis and enhanced the apoptosis induced by DTX (Figure 2E, F)

Embodiment 4: effect of targeting peptide on the autophagy



[0042] The cells were seeded and cultured. Targeting peptide was diluted with culture medium. Appropriate concentration of DTX was also prepared for the seeded cells. Equivalent culture medium was added as negative control group; 1×10-9 M targeting peptide solution was added as Peptide group; 10-9 M DTX was added as DTX group; 10-9 M targeting peptide and 10-4 M DTX were added as combination group (DTX+P). The cells were incubated at 37 °C, 5% CO2 for 6 h to analyze the autophagosome. Protein was extracted to measured expression of autophagy related proteins.

[0043] Figure 2B- Figure 2D showed that the expressions of autophagic Beclin1 and LC3 were changed by DTX and targeting peptide treatment. Targeting peptide treatment alone did not affect the expression of these proteins. DTX enhanced the expression the Beclin 1 and conversion of LC3-I to LC3-II, indicating an elevated increase in autophagy. Compared with the DTX treatment only, Beclin1 expression and conversion of LC3-I to LC3-II decreased when DTX and targeting peptide were simultaneously added, suggesting that targeting peptide significantly inhibited the autophagy induced by DTX.

Embodiment 5: Preparation of DTX and HSP 90 inhibitory peptide Conjugates (DTX-P7) Synthesis of DTX -suc



[0044] DTX (1.5 g) and succinic anhydride (abbreviated as Suc, 0.5 g) were dissolved in CH2Cl2 (20 mL). After pyridine (0.5 ml) was added, the mixture was stirred for 1 week at room temperature. The reaction mixture was concentrated in a vacuum to remove the CH2Cl2. 50 ml 5% citric acid solution was added and then extracted with 50 ml ethyl acetate for 3 times. Then obtained organic phase was distillated to remove ethyl acetate. The crude DTX-Suc was further subjected to C18 preparative chromatography for purification and freeze. Preparative chromatography method: mobile phase: 40-70 % acetonitrile (60-30 % water) /0-30 min, flow rate of 20 ml/min. Analytical chromatography method: mobile phase: 0-50 % acetonitrile (100-50 % water) /0-50 min, flow rate of 1ml/min.

Synthesis of DTX-P7



[0045] DTX-P7 conjugates were prepared using the solid-phase peptide synthesis method. The amino acid was successively linked onto the 0.83g Fmoc-Pro-Trt resin (substitution degree of the resin was 0.6 mmol/g). After the peptide bond constructed, DTX-Suc was linked to the N terminal of the peptide chain. The crude conjugates dissociated from the resin was purified with preparative C18 column. The targeted fraction was collected and lyophilized to obtain the DTX-P7, and subsequently submitted to further HPLC and mass spectrometry analysis. Preparative chromatography method: mobile phase: 40-70 % acetonitrile (60-30 % water) /0-30 min, flow rate of 20 ml/min. Analytical chromatography method: mobile phase: 0-50 % acetonitrile (100-50 % water) /0-50 min, flow rate of 1ml/min. The mass spectrometry analysis of DTX-P7 was shown in Figure 3.



[0046] Similar method was employed to prepare the PTX-P7 conjugate (Structure 2) by coupling paclitaxel with peptide, DOX-P7 (Structure 3) by coupling peptide at NH2 of doxorubicin, and DOX-OH-P7 (Structure 4) at hydroxide radical.






Embodiment 6: Establishment of human non-small cell lung cancer xenograft nude mice model



[0047] A549 cell, a non-small cell lung cancer cell line, growing in logarithmic phase were collected and gently pipetted with 1 ml F-12K media containing 10% PBS to prepare the single cell suspension, which was consequently counted with appropriate dilution. The cell suspension was mixed with Matrigel (1:3) under ice bath. The concentration of the cell suspension was adjusted to 8×106 cells /ml and stored at 0°C before transplantation.

[0048] Thirty BALB/c nu/nu female nude mice (16g) were acclimated to the environment for 1 week before the formal experiment. When cell transplanted, A549 cell (8×105cells/100µl) was subcutaneously (s.c.) injected into the right side of the axilla of the animal after carefully disinfected with iodophor. State of the mice were observed before they were returned to rearing cage.

Embodiment 7: Nude mice grouping and drug administration



[0049] The mice were frequently observed and tumor volume was measured with a Vernier caliper, the volume was calculated by the equation (long diameter x short diameter2) (1/2), When tumor reaching a size of 100 mm3, mice were divided into 4 groups: (1) Saline (control), (2) DTX, (3) TN-DTX and (4) DTX-P7.

[0050] Next day after animal grouping, agents were administrated via intraperitoneal injection once a week for a total of four injections. The first administrated day was recorded as Day 0 and the other administrated days were respectively recorded as Day 7, Day 14, Day 21. The dosages are uniform 10 mg/kg based on the mass of DTX.

[0051] The mice were measured and weighted every two times a week. Tumor dimensions were measured using calipers. Tumor volume (V) and relative tumor volume (RTV) were calculated with the following equations:

(where a represents long diameter, b represents short diameter)
RTV=Vt/V0 (where V0 represents the tumor volume measured at first drug administration, Vt represents the tumor volume measured at t day)

[0052] The first day of the fifth week after the first injection, (28 day), the mice were sacrificed with an intraperitoneal injection of 0.5 % pentobarbital sodium solution (50 mg/kg). The tumor tissues were resected and weighed. A part of tumor tissue was stored in 4% paraformaldehyde solution at 4 °C and the remaining part was stored at -80 °C.

[0053] The body weight was also monitored during the in vivo studies and there was no significant difference in body weight among four groups indicating a good safety (Figure 4) for all DTX preparation. The changes of tumor volumes of each group were showed in Figure 5 and further presented as curve graph in Figure 6. The results showed that all the DTX, DTX-P7, TN-DTX exerted obvious inhibitory effects on the tumor growth. DTX-P7 obviously inhibited the tumor growth. Particularly, DTX-P7 exhibited robust tumor regression with a significant difference compared with the CON group from the 4th day (p < 0.05). The efficacy of DTX-P7 was superior to that of DTX, and the targeting nanodrug TN-DTX which was publicized in the inventor's previous patent (p<0.01). These data suggested that the inhibitory effects of DTX-P7 was not a simple superposition of DTX and targeting peptide, but a surprising suppressing effect. The inhibitory effects of DTX-P7 was remarkable compared with the previously publicized TN-DTX. Results showed that conjugate of HSP90 inhibitory peptide conjugating with DTX via linker present remarkable advantage for suppressing tumor growth when compared with the previously publicized nanodrug TN-DTX.

Embodiment 8: cytotoxicity of four conjugates for diverse cancer cells



[0054] Lung adenocarcinoma A549, breast cancer MCF-7S and melanoma A375 cells were diluted respectively to 3× 104 cells/ ml and pipetted to 96-well plate with 100µl in each well. Twenty-four hours later, 5, 20, 80 or 320 nM DTX-P7, Paclitaxel-heptapeptide conjugate (PTX-P7), Doxorubicin-NH2- heptapeptide conjugate (DOX-P7) and Doxorubicin-OH-heptapeptide conjugate (DOX-OH-P7) were added to the wells and incubated for 48h. Then 10µl CCK-8 was added and measured 1.5 h later at 450nm to calculate the viability and median inhibitory concentration IC50. Results in Table 1 showed that the four conjugates exerted excellent inhibitory effects to diverse tumor cells indicating a potential for a variety of cancer treatment.
Table 1. the cytotoxicity of four conjugates for diverse tumor cells (IC50, nM)
Tumor CellDTX-P7PTX-P7DOX-P7DOX-OH-P7
Lung A549 4.5 7.2 6.5 6.2
Breast cancer MCF-7S 11.7 16.2 23.3 18.1
Melanoma A375 16 20.6 30.6 28.4

References



[0055] 
  1. 1. Ferrarini M, Heltai S, Zocchi MR, Rugarli C. Unusual expression and localization of heat-shock proteins in human tumor cells. Int J Cancer 1992;51:613-9.
  2. 2. Becker B, Multhoff G, Farkas B, Wild PJ, Landthaler M, Stolz W, Vogt T., Induction of HSP90 protein expression in malignant melanomas and melanoma metastases. Exp. Dermatol. 2004;13: 27-32.
  3. 3. Zhao R, Davey M, Hsu YC, Kaplanek P, Tong A, Parsons AB, Krogan N, Cagney G, Mai D, Greenblatt J, Boone C, Emili A, Houry WA. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the HSP90 chaperone. Cell 2005; 120: 715-727.



Claims

1. A conjugate, characterized in that the conjugate is prepared by conjugating HSP90 inhibitory peptide with cytotoxic agents via a linker, wherein the amino acid sequence of the HSP90 inhibitory peptide is LPLTPLP.
 
2. The conjugate of claim 1, wherein the amino terminal of the HSP90 inhibitory peptide is linked to 1-3 G(s), and the carboxyl terminal of the HSP90 inhibitory peptide is linked to S, SH or SHS.
 
3. The conjugate according to claim 1 or 2, wherein the cytotoxic agents are selected from docetaxel, paclitaxel and doxorubicin.
 
4. The conjugate according to claim 1 or 2, wherein the linker has the following general formula: -CO-(CH2CH2)n-CO-, wherein n being selected from 1,2,3,4,5,6,7,8,9,10;
the linker preferably being -CO-CH2CH2-CO-.
 
5. The conjugate according to claim 1 or 2, wherein the amino terminal of the HSP90 inhibitory peptide being connected to 2-OH of docetaxel or paclitaxel via the linker, or the amino terminal of the HSP90 inhibitory peptide being connected to -3-NH2 on the 10-lyxo-hexopyranosyl group of the doxorubicin via the linker, or the amino terminal of the HSP90 inhibitory peptide being connected to 8- glycolyl group of the doxorubicin via the linker.
 
6. The conjugates according to claim 5, being selected from conjugates having the following chemical structures:







wherein the amino terminal of the HSP90 inhibitory peptide is at L in all Structures 1 to 4, and PLPTLPL in Structures 1 and 2 represents the HSP90 inhibitory peptide in C->N direction
 
7. The conjugate according to any one of claims 1-6 for use in a drug for preventing and / or treating tumor.
 
8. The conjugate for use according to claim 7, wherein the tumor is HSP90 highly-expressed tumor, preferably being selected from lung cancer, lung adenocarcinoma, melanoma, gastric cancer, breast cancer, renal carcinoma, liver cancer, oral epidermoid carcinoma, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, colorectal cancer and neural tumor, more preferably from lung adenocarcinoma, breast cancer and melanoma, further preferably being lung adenocarcinoma.
 
9. A tumor targeting agent for inhibiting tumor progress, comprising the conjugate according to any one of claims 1 to 6 and pharmaceutically acceptable excipient.
 


Ansprüche

1. Konjugat, dadurch gekennzeichnet, dass das Konjugat durch Konjugieren eines HSP90-inhibitorischen Peptids mit zytotoxischen Agenzien über einen Linker hergestellt wird, wobei die Aminosäuresequenz des HSP90-inhibitorischen Peptids LPLTPLP ist.
 
2. Konjugat nach Anspruch 1, wobei das Aminoende des HSP90-inhibitorischen Peptids mit 1-3-G(s) verknüpft ist und das Carboxylende des HSP90-inhibitorischen Peptids mit S, SH oder SHS verknüpft ist.
 
3. Konjugat nach Anspruch 1 oder 2, wobei die zytotoxischen Agenzien aus Docetaxel, Paclitaxel und Doxorubicin ausgewählt sind.
 
4. Konjugat nach Anspruch 1 oder 2, wobei der Linker die folgende allgemeine Formel: -CO-(CH2CH2)n-CO- aufweist, wobei n aus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ausgewählt ist;
wobei der Linker vorzugsweise -CO-CH2CH2-CO- ist.
 
5. Konjugat nach Anspruch 1 oder 2, wobei das Aminoende des HSP90-inhibitorischen Peptids mit 2-OH von Docetaxel oder Paclitaxel über den Linker verbunden ist oder das Aminoende des HSP90-inhibitorischen Peptids mit -3-NH2 auf der 10-Lyxohexopyranosyl-Gruppe des Doxorubicins über den Linker verbunden ist oder das Aminoende des HSP90-inhibitorischen Peptids mit der 8-Glycolyl-Gruppe des Doxorubicins über den Linker verbunden ist.
 
6. Konjugate nach Anspruch 5, ausgewählt aus Konjugaten mit den folgenden chemischen Strukturen:







wobei sich das Aminoende des HSP90-inhibitorischen Peptids in allen Strukturen 1 bis 4 bei L befindet und PLPTLPL in den Strukturen 1 und 2 das HSP90-inhibitorische Peptid in C>N-Richtung darstellt.
 
7. Konjugat nach einem der Ansprüche 1-6 zur Verwendung in einem Arzneimittel zur Verhinderung und/oder Behandlung eines Tumors.
 
8. Konjugat zur Verwendung nach Anspruch 7, wobei der Tumor ein HSP90 hochexprimierender Tumor ist, vorzugsweise ausgewählt aus Lungenkrebs, Lungenadenokarzinom, Melanom, Magenkrebs, Brustkrebs, Nierenkarzinom, Leberkrebs, oralem Epidermoidkarzinom, Gebärmutterhalskrebs, Eierstockkrebs, Bauchspeicheldrüsenkrebs, Prostatakrebs, Kolorektalkrebs und einem neuralen Tumor, noch bevorzugter aus Lungenadenokarzinom, Brustkrebs und Melanom, weiter bevorzugt ein Lungenadenokarzinom ist.
 
9. Auf einen Tumor abzielendes Agens zum Inhibieren der Tumorprogredienz, umfassend das Konjugat nach einem der Ansprüche 1 bis 6 und einen pharmazeutisch akzeptablen Arzneistoffträger.
 


Revendications

1. Conjugué, caractérisé en ce que le conjugué est préparé en conjuguant un peptide inhibiteur de HSP90 avec des agents cytotoxiques par l'intermédiaire d'un lieur, la séquence en acides aminés du peptide inhibiteur de HSP90 étant LPLTPLP.
 
2. Conjugué selon la revendication 1, la terminaison amine du peptide inhibiteur de HSP90 étant liée à 1-3 G(s), et la terminaison carboxyle du peptide inhibiteur de HSP90 étant liée à S, SH ou SHS.
 
3. Conjugué selon la revendication 1 ou 2, les agents cytotoxiques étant choisis parmi le docétaxel, le paclitaxel et la doxorubicine.
 
4. Conjugué selon la revendication 1 ou 2, le lieur présentant la formule générale suivante : -CO-(CH2CH2)n-CO-, n étant choisi parmi 1, 2, 3, 4 ,5, 6, 7, 8, 9, 10 ;
le lieur étant de préférence -CO-CH2CH2-CO-.
 
5. Conjugué selon la revendication 1 ou 2, la terminaison amine du peptide inhibiteur de HSP90 étant reliée à 2-OH du docétaxel ou du paclitaxel par l'intermédiaire du lieur, ou la terminaison amine du peptide inhibiteur de HSP90 étant reliée à -3-NH2 sur le groupe 10-lyxo-hexopyranosyle de la doxorubicine par l'intermédiaire du lieur, ou la terminaison amine du peptide inhibiteur de HSP90 étant reliée à un groupe 8-glycolyle de la doxorubicine par l'intermédiaire du lieur.
 
6. Conjugués selon la revendication 5, choisis parmi des conjugués présentant les structures chimiques suivantes :







la terminaison amine du peptide inhibiteur de HSP90 se trouvant au niveau de L dans toutes les structures, et PLPTLPL dans les structures 1 et 2 représentant le peptide inhibiteur de HSP90 dans le sens C->N.
 
7. Conjugué selon l'une des revendications 1 à 6 pour l'utilisation dans un médicament pour la prévention et/ou le traitement d'une tumeur.
 
8. Conjugué pour l'utilisation selon la revendication 7, la tumeur étant une tumeur à HSP90 fortement exprimé, de préférence choisie parmi le cancer du poumon, l'adénocarcinome du poumon, le mélanome, le cancer de l'estomac, le cancer du sein, le carcinome rénal, le cancer du foie, le carcinome épidermoïde oral, le cancer du col utérin, le cancer de l'ovaire, le cancer du pancréas, le cancer de la prostate, le cancer colorectal et le cancer neural, de manière plus préférée parmi l'adénocarcinome du poumon, le cancer du sein et le mélanome, étant de manière encore plus préférée un adénocarcinome du poumon.
 
9. Agent de ciblage de tumeurs pour l'inhibition de la progression tumorale, comprenant le conjugué selon l'une des revendications 1 à 6 et un excipient pharmaceutiquement acceptable.
 




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