CN109876000A - Application of the Pabuk former times benefit cloth in mucous membrane malignant mela noma - Google Patents
Application of the Pabuk former times benefit cloth in mucous membrane malignant mela noma Download PDFInfo
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- CN109876000A CN109876000A CN201811064575.4A CN201811064575A CN109876000A CN 109876000 A CN109876000 A CN 109876000A CN 201811064575 A CN201811064575 A CN 201811064575A CN 109876000 A CN109876000 A CN 109876000A
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Abstract
The invention discloses application of the Pabuk former times benefit cloth in mucous membrane malignant mela noma.Specifically, the present invention provides become known for treating therapeutic application of the Pabuk former times benefit cloth of ER+/HER2- postmenopausal women with advanced breast cancer in mucous membrane malignant mela noma.It is different from treatment common skin malignant mela noma, Pabuk former times benefit cloth is high to grade malignancy and the multi-source mucous membrane malignant mela noma of definite medicine there is no to have extraordinary tumor inhibitory effect, the blank of drug therapy has been filled up for mucous membrane malignant mela noma, and there is very big application prospect.
Description
Technical field
The invention belongs to biomedicine fields, in particular it relates to which Pabuk former times benefit cloth (Palbociclib) is being treated
Application in mucous membrane malignant mela noma.
Background technique
Malignant mela noma (Melanoma) is a kind of tumour originating from melanocyte, and melanoma site of pathological change is divided into
Skin, acra, the melanomas such as mucous membrane, genetic background are different.In American-European white people, malignant mela noma is apt to occur in
Skin, finger tip and mucous membrane malignant mela noma are relatively rare.Wherein malignant melanoma of skin often betides chronic sunlight damage
The trunk of harmful or non-chronic sunlight damage, four limbs, the positions such as leg are derived from epidermis normal melanocyte or original nevocytic
A kind of malignant tumour, normal irregular changes that be serrated, it is rough and normal coarse and with squama shape or sheet furfur.Sometimes have
Sepage or oozing of blood, lesion can be higher by surface, sport main feature with BRAF V600E/V600K, and acra and mucous membrane are pernicious black
Melanoma is apt to occur in asian population, and the cause of disease is unclear, and disliking BRAF V600E/V600K mutation common in black in skin is not its master
Science of heredity is wanted to change.Acral lentiginous melanoma original site is vola, toes, finger tips and the inferior position of first, relies primarily on hand
Art treatment.Originate from the vicious transformation of mucous membrane basal layer melanocyte for mucous membrane malignant mela noma part, and progress is fast, tumour
Cell has the characteristics that significantly to infiltrate into mucous membrane, and cervical lymph transfer is common, and primary treatments are with surgeon's knot combination
Based on treatment, but five year survival rate only 20-25%.Therefore, the melanoma of different parts has different clinical phenotypes and biology
Scholarship and moral conduct is.
In the past few decades, originating from the malignant mela noma of skin, quantum jump in clinical treatment, skin is disliked
Property melanoma molecule parting and targeted therapy (for example, BRAF V600E/V600K mutation as to Wei Luofeini react it is good
A kind of good mark) achieve revolutionary change.However melanoma hypotype most fatal in the crowd of East Asia-mucous membrane is disliked
Property melanoma (mucosal melanoma, MM), treatment means clinical at present and targeted drug are still very deficient.MM disease
The long-term prognosis of people is very poor, and five year survival rate only has 20%-25%.Compared to the driving specific cutaneous malignant melanin of gene
The molecule pathogenic mechanism of tumor, MM is not yet clear.In fact, there is a serious shortage in the supply for the drug of targeting MM clinically, uniquely targeted with MM
Treatment-related gene is KIT.Clinically it is for the most common target therapeutic agent of mucous membrane malignant mela noma of KIT mutation
As metastatic or the treatment of mucous membrane malignant mela noma can not be cut off for Imatinib and high dose interleukin-22 (IL-2)
Selection.The phenomenon that multinomial clinical test is recurred after showing current Imatinib in treating MM there are drug resistance or medication, greatly
Limit its clinical promotion and application.In addition, the application of various kinds of cell poison based chemotherapy drug, for example, Dacarbazine, cis-platinum,
Taxol etc. can not improve the five year survival rate of patient well.Therefore, significant in East Asia, especially Chinese population at present
High-incidence MM patient " no medicine is available ", this field continues to seek the targeted drug that can effectively treat MM, this is for alleviating MM patient
Slight illness improves its survival rate with important clinical significance.Meanwhile the clinical Transformation Application of novel targeted drug will fill up current MM
Great market blank present in treatment has huge commercial value and social effect.
Summary of the invention
The present invention provides the new applications of Pabuk former times benefit cloth, i.e. the application in mucous membrane malignant mela noma.
First aspect present invention provides a kind of Pabuk former times benefit cloth or the purposes of its pharmaceutically acceptable salt, for making
The pharmaceutical composition of standby treatment mucous membrane malignant mela noma.
In another preferred example, the pharmaceutically acceptable salt of the Pabuk former times benefit cloth includes hydrochloride, sulfate, phosphorus
Hydrochlorate, sulfonate, carbonate, acetate, tartrate, formates, acetate or isethionate.
In another preferred example, described pharmaceutical composition contains the Pabuk former times benefit cloth of safe and effective amount, and can pharmaceutically connect
The carrier received.
In another preferred example, described pharmaceutical composition also contains tumor therapeutic agent.
In another preferred example, the tumor therapeutic agent is directed to malignant mela noma, it is preferable that is directed to mucous membrane maligna
Plain tumor.
In another preferred example, the tumor therapeutic agent includes Imatinib (imatinib), Wei Luofeini
(Vemurafenib), dabrafenib (Dabrafenib), auspicious rich XiLin (Ribociclib), glass Ma XiLin
(Abemaciclib)。
In another preferred example, the tumor therapeutic agent is Imatinib.
In another preferred example, described pharmaceutical composition contains Pabuk former times benefit cloth and Imatinib.
In another preferred example, the ratio between the Pabuk former times benefit cloth in described pharmaceutical composition and Imatinib dosage are 0.5-
1.5:0.5-1.5, it is preferable that it is 1:1.
In another preferred example, the pharmaceutical composition is unit dosage form.
In another preferred example, the pharmaceutical composition includes gastro-intestinal administration dosage form or parenteral dosage forms.
In another preferred example, the pharmaceutical composition include tablet, pill, powder, granule, capsule, syrup,
Emulsion, suspending agent, injection, patch, drops, paste, suppository or spray.
In another preferred example, the method for application of the pharmaceutical composition include oral, intramuscular injection, intravenous injection,
Intravenous drip, intratumor injection, bowel lavage, spraying, external application or intraperitoneal injection.
In another preferred example, the safe and effective amount refers to 50-1000mg/kg.
In another preferred example, the safe and effective amount includes 50-200mg/kg (mouse) or 500-1000mg/kg
(people).
In another preferred example, the safe and effective amount is 100-700mg/kg, more preferably 500-700mg/kg.
In another preferred example, the mucous membrane malignant mela noma includes the mucous membrane maligna element from the upper respiratory tract
Tumor.
In another preferred example, the upper respiratory tract mucous membrane includes the mucous membrane in oral cavity, nasopharynx or oropharynx position.
In another preferred example, the mucous membrane malignant mela noma derives from oral cavity.
In another preferred example, the mucous membrane malignant mela noma includes that mucous membrane malignant mela noma in situ or wettability are glutinous
Film malignant mela noma.
In another preferred example, the mucous membrane malignant mela noma includes a variety of pathology differentiation grading stages.
In another preferred example, the differentiation grading of the mucous membrane malignant mela noma includes: (GX) point that can not be assessed
Change good (G1), (G2) of moderate differentiation, poorly differentiated (G3) and/or undifferentiated (G4) pathological grading.
In another preferred example, the upper respiratory tract mucous membrane includes the mucous membrane at oral cavity, lip, nasopharynx or oropharynx position.
In another preferred example, the mucous membrane malignant mela noma derives from people.
Second aspect of the present invention provides a kind of method of external non-therapeutic inhibition mucous membrane malignant mela noma, including
Step: Pabuk former times benefit cloth or its pharmaceutically acceptable salt are added in the cell culture system of Xiang Hanyou mucous membrane malignant mela noma
Or the pharmaceutical composition containing it, to inhibit mucous membrane malignant melanoma cell.
In another preferred example, the concentration of the Pabuk former times benefit cloth is 0.001 μM -1000 μM, it is therefore preferable to 0.1uM-100
μM。
Third aspect present invention provides a kind of method for treating mucous membrane malignant mela noma, comprising steps of to needs
Object applies the Pabuk former times benefit cloth of safe and effective amount or the pharmaceutical composition containing it, to treat mucous membrane malignant mela noma.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and specifically described in below (e.g. embodiment)
It can be combined with each other between each technical characteristic, to form a new or preferred technical solution.As space is limited, not another herein
One tired states.
Detailed description of the invention
Fig. 1 is shown after it applied Pabuk former times benefit cloth, from the pernicious (Figure 1A: hard palate, figure of upper respiratory tract mucous membrane
1B: gum, Fig. 1 C: cheek mucous membrane, Fig. 1 D: oropharynx；Fig. 1 E, F: nasopharynx) melanoma Mice Body in inhibitory effect.
Fig. 2 shows Pabuk former times benefit cloth, Imatinib individually and drug combination and the tumor suppression comparison result that compares,
Middle Pabuk former times benefit cloth significant effect is better than Imatinib, and drug combination has significant synergy.
Specific embodiment
The present inventor after extensive and in-depth study, for the first time it was unexpectedly observed that for treating the new drug in breast cancer advanced stage
Pabuk former times benefit cloth has very effective tumor inhibitory effect, and the effect and its known target for mucous membrane malignant mela noma
Point is function (such as CDK4/6) and uncorrelated.It is disliked by the screening of massive tumor inhibitor candidate compound and to from mucous membrane
The cross validation of the transplantable tumor mouse model of property melanoma, Pabuk former times benefit cloth can mucous membrane malignas to multiple location source
Plain tumor all has significant tumor inhibitory effect, and the volume of tumour is substantially reduced in mouse tumor model.Pabuk former times benefit cloth as a result,
The bottleneck that there is no specific treatment agent at present is broken in the strong breach that can be used as the treatment of mucous membrane malignant mela noma.And
As marketed drug, Pabuk former times benefit cloth is easily obtained and mature preparation process, can carry out opening for a variety of suitable preparations based on this
Hair.On this basis, the present invention is completed.
Pabuk former times benefit cloth (Palbociclib, CAS:571190-30-2, also referred to as Pa Boxini)
Pabuk former times benefit cloth is (to have shifted) breast cancer treatment in the advanced stage of Pfizer's acquisition FDA approval on 2 3rd, 2015
Agent is first approved cell cycle protein dependent kinase 4/6 (CDK4/6) inhibitor, chemical name: 6- acetyl group -8-
7 (8H) -one of cyclopenta -5- methyl -2- (5- (piperazine -1- base) pyridine -2- base amino) pyridine [2,3-d] pyrimidine.
The synthesis of Pabuk former times benefit cloth can obtain according to the method in the prior art, such as WO1998033798A2.
Pabuk former times benefit cloth for use in the present invention includes Pabuk former times benefit cloth compound itself or derivatives thereof, the derivative
The example of object has the polymorphic or its pharmaceutically acceptable salt of Pabuk former times benefit cloth, such as hydrochloride, the sulfuric acid of Pabuk former times benefit cloth
Salt, phosphate, sulfonate, carbonate, acetate, tartrate, formates, acetate or isethionate etc..These spread out
The preparation of biology can usually be obtained by conventional technical means, and its effect and effect can be according to cell experiment or equivalences
It is obtained compared with Pabuk former times benefit cloth in experiment, it will be appreciated by those skilled in the art that and in expected range.It is a kind of preferred
Pabuk former times benefit cloth be Pabuk former times benefit cloth hydrochloride.
Pabuk former times benefit cloth of the present invention or derivatives thereof is used directly for required object, or with pharmaceutically acceptable load
Body mixes or after preparing, and is administered in the form of pharmaceutical composition.Pabuk former times benefit cloth for use in the present invention or its derivative
The administration dosage of object is usually 5-150mg/kg/d, such as 50-120mg/kg/d, preferably 80-90mg/kg/d.And the application
Dosage in the expectable range of clinician or lab assistant, such as can be by validity, security test, to its agent
Amount carries out adjustment appropriate, to obtain optimal administration dosage.
Mucosal melanoma
Mucosal melanoma is a kind of hypotype of melanoma, and disease incidence is lower in the crowd of mucosal melanoma west, is accounted for
The total ratio of malignant mela noma less than 1.3%, but it is high-incidence in the crowd of East Asia (account for malignant mela noma it is black sum about
33.3%).Pharynx nasalis (23%) of the common site of pathological change for incidence, oral cavity (15%), including gum, hard palate, cheek etc.,
Very low 5 years overall survivals are 27%.Malignant melanoma of skin is mostly sun damage type, and prominent with BRAF, CDKN2A, NRAS
Become main feature, and mutational load is significantly higher than mucous membrane and dislikes black, it is pernicious relative to mucous membrane that the skin of early detection dislikes black prognosis
Melanoma is higher.Five year survival rate is 80%.Mucosal melanoma is different from malignant melanoma of skin, the cause of disease with it is ultraviolet
Line irradiation is unrelated, but the specific cause of disease and mechanism are unclear, and mucous membrane malignant mela noma is come relative to malignant melanoma of skin
It says, progress rapidly, since position is hidden, is not easy to detect discovery, no significant discomfort symptom is first medical more with Lymph Node Metastasis
See, five year survival rate is extremely low.For science of heredity angle, mucous membrane is disliked black compared to skin evil black, mutational load and driven nature base
The point mutation frequency of cause is lower, and there are more Gene copy numbers to change and chromosomal structural variation (missing, duplication, string
Connection duplication).Meanwhile mucous membrane dislikes visible a large amount of breaking point aggregation in black chromosome, this prompted mucous membrane dislike it is black there may be
More chromosomal rearrangements, it may be possible to as caused by the science of heredity event-driven of some complexity.In terms of key gene mutation,
Mucous membrane dislikes the black skin that lacks and dislikes BRAF relatively conventional in black, RAS family, NF1, TP53, PTEN, CDKN2A etc. well-known driving
Property gene high frequency mutation；And there are a large amount of key gene copy numbers to change.For example the region 12q14 (includes well-known former cancer
Gene C DK4) there are high-frequency gene magnifications by and 5p15 (including well-known proto-oncogene TERT), and this has been prompted in science of heredity
In terms of basis and pathogenic mechanism, mucous membrane, which is disliked, black dislikes that black there are notable differences with skin.
Mainly it is with immunization therapy and targeted therapy for transfer or unresectable cutaneous melanoma, therapeutic modality
It is main.Such as the patient for carrying BRAF V600 activated mutant, dabrafenib/Trimetinib (Dabrafenib/ is used
) or Wei Luofeini trametinib/examine than combining targeted therapy for Buddhist nun (Vemurafenib/cobimetinib).In recent years black
The immunization therapy risen in melanoma oncotherapy, the monoclonal antibody treatment of predominantly anti-PD1, including pyridine aldoxime methyliodide (PAM) monoclonal antibody
(Pembrolizumab) it either receives Wu Dankang (Nivolumab), also shows good therapeutic effect among the patients.
And the treatment for mucosal melanoma, operation excision is generally included, and it is insensitive to radiation and chemotherapy.In the past 30 years, FDA
DTIC and high dose IL-2 treatment advanced stage MM is only had approved, but efficient low, life cycle cannot be obviously prolonged.And other targeting medicines
Curative effect also urgently observe, and Bu Tong effectively situation is different with ethnic group and catastrophe.The target of mucous membrane malignant mela noma
To drug other than imatinib, without more effective targeted therapy mode, but Imatinib (imatinib) clinical response rate is low
Status need we have found that new targeted drug is directed to mucous membrane malignant mela noma.
Mucosal melanoma for use in the present invention, main includes the mucosal melanoma of incidence, such as from oral cavity,
The mucosal melanoma at the positions such as oropharynx, nasopharynx.
Mucosal melanoma is usually the wild type distributed, can also be the mucosal melanoma with genic mutation type.?
The mucosal melanoma with mutated gene known includes KIT gene mutation, BRAF gene mutation etc..
It can include various mucosal melanomas by stages with the mucosal melanoma that Pabuk former times benefit cloth of the present invention is treated,
It is preferred that the III phase (T3).Common are by currently the only generally acknowledged mucous membrane malignant mela noma by stages be AJCC on by stages.
I.e. once making a definite diagnosis as T3, further according to whether being to there is Lymph Node Metastasis or DISTANT METASTASES IN to classify.Mucosal melanoma:
The III phase (T3, N0, M0),
IV A phase (T4a, N0, M0, T3-T4a, N1, MO),
IV B phase (T4b, any N, M0), IVC (any T, N, M1)
Wherein, N represents lymphatic metastasis；M represents DISTANT METASTASES IN；T represents infiltration degree:
T3: non-cancer lesion
T4a: mid-term lesion, lesion involve deep tissues, bone or cartilage
T4b: advanced lesions, lesion involve brain, endocranium, basis cranii, afterwards organize cranial nerve (Ⅸ, Ⅹ, Ⅺ, Ⅻ), masseter gap,
Internal carotid, prevertebral space or mediastinal structures.
About mucous membrane malignant mela noma, there is only the pathological gradings carried out according to pathological observation cellular morphology at present.Such as:
(G1) of (GX) well differentiated that can not be assessed, (G2) of moderate differentiation, poorly differentiated (G3) and/or undifferentiated (G4) disease
Reason classification.
Pharmaceutical composition
As used herein, term " pharmaceutical composition " refers to the composition that will be administered for a specific purpose.
As used herein, term " active constituent " refers to Pabuk former times benefit cloth or derivatives thereof.
For the object of the invention, pharmaceutical composition contains Pabuk former times benefit cloth or derivatives thereof as active constituent and medicine
Acceptable carrier and the substance for treating mucous membrane malignant mela noma on.Term " pharmaceutically acceptable " refers to government
Medicine administrative organ or what is gone out and approve in pharmacopeia can be used for vertebrate, the substance particularly for the mankind.In general, medicine
Acceptable carrier refers to one or more biocompatible solids or liquid filler or gelatinous mass on, they, which are suitable for people, makes
With and it is necessary to there are enough purity and sufficiently low toxicity.Each component energy and sheet in " compatibility " referred to herein as composition
The active constituent of invention and they between mutually admix, and significantly reduce the drug effect of active constituent.Can pharmaceutically it receive
Carrier part example have cellulose and its derivates (such as sodium carboxymethylcellulose, ethyl cellulose sodium, cellulose ethanoate
Deng), gelatin, talcum, solid lubricant (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soya-bean oil, sesame oil, peanut
Oil, olive oil etc.), polyalcohol (such as propylene glycol, glycerol, mannitol, sorbierite), emulsifier (such as), wetting agent (such as
Lauryl sodium sulfate), colorant, flavoring agent, stabilizer, antioxidant, preservative, apirogen water etc..
The method of application of inventive compound or pharmaceutical composition is not particularly limited, and representative method of application includes
(but being not limited to): oral, intramuscular injection, intravenous injection, intravenous drip, intratumor injection, bowel lavage, spraying, external application or abdominal cavity note
It penetrates.
Solid dosage forms for oral administration includes tablet, pill, powder, granule or capsule.In these solid formulations
In type, active constituent is mixed at least one conventional inert excipients (or carrier), such as sodium citrate or Dicalcium Phosphate, or under
Ingredient mixing: (a) filler or expanding material is stated, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid；(b) adhesive,
For example, hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum；(c) moisturizer, for example,
Glycerol；(d) disintegrating agent, for example, agar, calcium carbonate, potato starch or tapioca, alginic acid, certain composition silicates and carbon
Sour sodium；(e) retarding solvent, such as paraffin；(f) absorbsion accelerator, for example, quaternary ammonium compound；(g) wetting agent, for example, cetanol and
Glycerin monostearate；(h) adsorbent, for example, kaolin；(i) lubricant, for example, talcum, calcium stearate, magnesium stearate,
Or mixtures thereof solid polyethylene glycol, lauryl sodium sulfate,.In capsule, tablet and pill, dosage form also may include buffering
Agent.
Coating and shell material preparation can be used in solid dosage forms such as tablet, sugar-pill, capsule, pill and granule, such as casing and
Other materials well known in the art.They may include opacifying agent, also, active constituent or compound are released in this composition
Put to discharge in certain a part in the digestive tract in a delayed fashion.It is adoptable embedding component example be polymeric material and
Wax material.When necessary, active constituent can also be with one of above-mentioned excipient or a variety of formation microencapsulation forms.
Liquid formulation for oral administration includes pharmaceutically acceptable emulsion, solution, suspending agent, syrup or tincture.
Other than active constituent, liquid dosage form may include the inert diluent routinely used in this field, such as water or other solvents, solubilising
Agent and emulsifier, example know, ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-BDO, dimethylformamide
And oil, the especially mixture of cottonseed oil, peanut oil, maize germ, olive oil, castor oil and sesame oil or these substances
Deng.
Other than these inert diluents, composition also may include auxiliary agent, such as wetting agent, emulsifier and suspending agent, sweet taste
Agent, corrigent and fragrance.
Other than active constituent, suspension may include suspending agent, for example, ethoxylation isooctadecane alcohol, polyoxyethylene mountain
The pure and mild Isosorbide Dinitrate of pears, microcrystalline cellulose, aluminium methoxide and agar or the mixture of these substances etc..
Composition for parenteral injection may include physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid,
Suspension or lotion, and the aseptic powdery for re-dissolving into sterile Injectable solution or dispersion liquid.It is suitable aqueous and
Nonaqueous carrier, diluent, solvent or excipient include water, ethyl alcohol, polyalcohol and its suitable mixture.
The dosage form of the compounds of this invention for local administration includes ointment, powder, patch, spray and inhalant.
Active constituent aseptically with physiologically acceptable carrier and any preservative, buffer, or when necessary may need
Propellant be mixed together.
The compounds of this invention can be administered alone, or be administered in combination with other pharmaceutically acceptable compounds.
It is the mammal that the compounds of this invention of safe and effective amount is applicable to treatment when using pharmaceutical composition
(such as people), wherein dosage is the effective dosage pharmaceutically thought when application, for the people of 60kg weight, day is to medicament
Amount is usually 1~2000mg, preferably 6~600mg.Certainly, specific dosage be also contemplated that administration route, patient health situation etc. because
Element, within the scope of these are all skilled practitioners technical ability.Usually wherein " safe and effective amount " refers to: the amount of compound is enough bright
It is aobvious to improve the state of an illness, and be unlikely to generate serious side effect.
In addition, pharmaceutical composition of the present invention or active constituent can also be applied together with other tumor therapeutic agents, such as
The malignant mela noma target spot inhibitor for knowing target spot, such as Imatinib (imatinib), reach Wei Luofeini (Vemurafenib)
La Feini (Dabrafenib), auspicious rich XiLin (Ribociclib), glass Ma XiLin (Abemaciclib).
Using
The present invention provides a kind of clinical new applications of Pabuk former times benefit cloth, i.e., have been difficult to definite medicine so far for treating
The mucous membrane malignant mela noma of object.Due to the evening by stages of mucous membrane malignant mela noma, grade malignancy height, poor prognosis, so far not
There can be an effective therapeutic agent, the therapeutic agent of general black element tumor is almost to no effect.And present inventors have surprisingly found that, pa
Cloth former times benefit cloth has very excellent inhibitory effect, and this effect conduct with itself to mucous membrane malignant mela noma
The mechanism of CDK4/6 inhibitor is not quite similar.
And when being used for cell experiment, inventive compound can be also used for inhibiting the mucous membrane maligna in cell culture
The proliferation of plain tumor has established new basis for the foundation of cell model.In general, in cell experiment, added present invention activity
The concentration of ingredient is about 0.001 μM -1000 μM, it is therefore preferable to which 0.01-100 μM, which can be according to the required effect reached
Fruit is further adjusted and is verified to the concentration of addition, this is in the range of those skilled in the art can routinely obtain.
Beneficial effect of the present invention
The present invention provides completely new approach for treatment mucous membrane malignant mela noma, enriches controlling for mucous membrane malignant melanoma
Treatment means have established new basis to improve 5 year life cycle of mucous membrane malignant mela noma.Further, since drug it is known that
Clinical application will be relatively beneficial on new application and drug safety.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
Versatile material
1640 culture medium of RPMI, fetal calf serum (fetal bovine serum, FBS) are purchased from Gibco company.
CCK-8 is purchased from Biotool company.
Selected 150 kinds of drug candidates are the micromolecular inhibitor for being used to treat malignant mela noma that NCCN guide is recommended
And the small molecule targeted drug of cytotoxic drug and some other kinds of tumor therapy target, cytotoxic drug and
Small molecule targeting anti-tumor preparation is purchased from selleck company.
1 PDC of embodiment (patient derived cell) screens sensibility candidate compound
Inclusion criteria: the malignant mela noma primary lesion of mucous membrane is betided.Mucous membrane malignant mela noma, which there is no, clearly divides
Type.By stages according to AJCC.
The mucous membrane malignant mela noma sample of acquisition is subjected to PDC model discrimination sensibility inhibitor, the method is as follows:
1) it collects the PDC cell of logarithmic growth phase and prepares cell suspension, cell count, by cell concentration after pancreatin digestion
It is adjusted to 2 × 104A/ml；
2) it seeds cells into 96 orifice plates, 3 secondary orifices are at least arranged in every hole 100ul cell suspension, each drug concentration；
3) cell is placed in 37 DEG C, overnight incubation in 5% carbon dioxide incubator；
4) second day after cell inoculation, by drug concentration gradient 0nM, 10-3NM, 10-2NM, 10-1NM, 1nM, 10nM,
100nM, 1000nM, 10uM are added in 96 orifice plates；
5) cell is put into incubator, continues culture 72 hours；
6) CCK-8 reagent and culture medium are configured to mixed liquor according to 1:10, piping and druming uniformly, sucks former culture in 96 orifice plates
100 microlitres of CCK-8 dilution is added in base, every hole.One group of blank control is set, i.e., is added in one group of blank well for not having cell
3 multiple holes are equally at least arranged in 100 microlitres of CCK8 dilutions.96 orifice plates are slightly shaken after dosing, keep CCK-8 and side wall residual
Liquid mixes well in the liquid and plate stayed；
7) it is added after CCK-8 and cell is continued into culture 3 hours；
8) microplate reader is set into wavelength 450nm, measures the light absorption value (OD value) in every hole, and obtain IC50Value.
It is as follows to the screening list of medications of nearly 150 kinds of target drugs of 5 plants of cells progress:
MDM2:nutlin3 MX69, YH239-EE, NVP-CGM097, Idasanutlin
TERT:BIBR1532, costunolide (Costunolide)
Her2: how are Lapatinib (Lapatinib), Afatinib (Afatinib), linatinib (Neratinib), card
Buddhist nun (Mubritinib), AC480, TAK-285, Irbinitinib, song are replaced for Buddhist nun (Canertinib), Sapitinib, wood benefit
Trastuzumab Trastuzumab, handkerchief trastuzumab (Pertuzumab).
CDK:Roscovitin, enlightening that former times benefit cloth (Dinaciclib), AZD5438, MK-8776, PHA-793887, PHA-
767491 R457、XL413、P276-00、NU6027、Ro-3306、Kenpaullone、Senexin A、Purvalanol A、
ML167, TG003, SU9516, Pabuk former times benefit cloth (Palbociclib)
PARP: olaparib (Olaparib), Veliparib, Rucaparib, Talazoparib, Iniparib,
PJ34HCL, UPF1069, AZD2461, Niraparib, E7449, NU1025, benzamide (Benzamide), NMS-P118,
Picolinamide (Picolinamide)
Kit: Dasatinib (Dasatinib), Sutent (sunitinib)
PDGFR: pa is received for Buddhist nun (Ponatinib), Avapritinib
EGFR: Erlotinib (Erlotinib), Gefitinib (gefitinib), AG-490
VEGFR: Sorafenib (Sorafenib), Sutent (sunitinib), Cabozantinib, Foretinib,
Fan Deta replaces Buddhist nun (Vandetanib), Nintedanib (Nintedanib), Rui Gefeini (Regorafenib), pazopanib
(Pazopanib), Si Dinibu (Cediranib), PD173074, more Weis replace Buddhist nun (Dovitinib), Linifanib, Wa Tala
Buddhist nun (Vatalanib), Tivozanib, pleasure are cut down for Buddhist nun (Lenvatinib), Bu Linibu (Brivanib)
C-MET: gram azoles for Buddhist nun (Crizotinib), Su11274, PHA-665752, SGX-523, BMS-777607,
Tivantinib, Glesatinib, Capmatinib, Tepotinib, Merestinib, Norcantharidin
(norcantharidin).
Stat3:SH-4-54, NT157, WHI-P154, Napabucasin, Nifuroxazide (Nifuroxazide),
Ochromyclnone, HJC0152, niclosamidum (Niclosamide), C188-9
PI3K:Dactolisib, Pictilisib, Idelalisib, Buparlisib, Tanalisib, Autophinib
BCL2:ABT-737, Navitoclax, Obatoclax, methanesulfonic acid (mesylate), TW-37, Venetoclax,
BTSA1、S63845、WEHI-539、FX1、AT101、HA14-1、Sabutoclax.
WNT/PACTIN:LGK-974, WNT-C59, XAV-939, ICG-001, IWR-1-ENDO, isoquercitrin
(Isoquercitrin)、ICRT3、GNF-6231、IWP-L6、IWP-2、KY02111
JAK: tropsch imatinib (Tofacitinib), momelotinib, FM-381, PF-06651600, AT9283,
Fedratinib、AZD1480、AZ960、CEP-33779、Gandotinib、baricitinib、Filgotinib、
Peficitnib、Cerdulatinib、Oclacitinib
HADC:Vorinistat, entinostat, pabishta (panobinostat), Trichostatin A
(Trichostatin A), Mocetinostat, WT161, darcy department spy (Dacinostat), Givinostat.
Chemotherapeutic: cis-platinum (Cisplatin), Temozolomide (Temozolomide), Dacarbazine (Dacarbazine),
Taxol (Paclitaxel), carboplatin (Carboplatin)
NCCN- melanoma recommends drug: Wei Mofeini (Vemurafenib), dabrafenib (Dabrafenib), Sibutramine Hydrochloride
For Buddhist nun (Trametinib), Cobimetinib, Imatinib (Imatinib)
After screening, these compounds are obtained for the experimental result of separate sources mucous membrane malignant mela noma.It is therein
Part the selection result is as shown in table 1 below.
Table 1
Wherein, 002#, 006#, 010#, 011#, 014# are respectively to come from oropharynx, gum, nasopharynx, hard palate, the generation of cheek mucous membrane
Table mucous membrane malignant mela noma, this five plants of cell Proliferations are most fast, and five plants of cells are thus respectively used to subsequent transplantation tumor mould
The building of type.
And as can be seen from result of Table 1, Pabuk former times benefit cloth for mucous membrane malignant mela noma have preferable effect, and from
The concentration of 100nM starts have effect.A variety of known common skin melanoma therapeutic agent Vemurafenib,
Dabrafenib, Selumetinib, Tasisulam etc. be not inhibited to mucous membrane malignant mela noma, and it is a variety of
The similar CDK4/6 inhibitor of the cytotoxic drug and Pabuk former times benefit cloth known is also pernicious black to the mucous membrane in various position sources
Melanoma does not have inhibiting effect equally.Pabuk former times benefit cloth has been prompted to may be by new unknown role approach to glutinous as a result,
Film malignant mela noma is inhibited.
The building of 2 Transplanted tumor model of embodiment and the tumor inhibition effect of Pabuk former times benefit cloth
In order to tissue of patient source tumour biology and genetics characteristics preferably restored, will be in embodiment 1
The sample of acquisition equally carries out the building of mice-transplanted tumor model, and a step of going forward side by side demonstrate,proves the inhibiting effect of Pabuk former times benefit cloth.
1) by sterile petri dish, surgical kit is put into delivery bin and carries out disinfection by ultraviolet light 30 minutes；
2) mouse is weighed, and asepsis injector extracts 10% chloraldurate, according to mouse weight based on 0.004ml/g
Dosage used is calculated, maximum dose is no more than 0.1ml, is injected into mouse peritoneal.
Anaesthetize points for attention: slowly injection, while muscle tone is observed, it is anesthesia when mobility obviously weakens or disappears
Success；
It should be noted holding body temperature after anesthesia, take Insulation, using heating sheet, desk lamp irradiation；
Depth of anesthesia can not suitably add anesthetic completely, add dosage no more than standard metering 20%
3) it is cleaned tumor tissues 2 times with containing 1% dual anti-PBS, it is 5-10 minutes each, tumor is trimmed in PBS with eye scissors
Body removes necrotic tissue, tissue is divided into 3mm × 3mm × 3mm tissue block.
4) it is sterilized in the back of mice skin anaesthetized with cotton ball soaked in alcohol, isolated tissue block is filled in into sleeve needle, will be covered
Cylinder needle shifts mouse dorsal sc onto, and flat mouse height moves under water length about 2cm, slowly pushes away sleeve needle end for tissue block and releases sleeve
Needle, the slow withdraw of the needle.Every mouse injects a position and determines Mice Inoculated quantity according to the tumor tissues amount of collection, general every
A tumor sample is inoculated with 3-5 mouse, and first generation mouse is denoted as P1 generation (tumor patient P0), and remaining tumor tissues are for conventional
Paraffin embedding.
Nude nude mice and NOD-SCID severe combined immunodeficiency Mice Mice are dynamic purchased from the western Poole-Bi Kai experiment in Shanghai
Object Co., Ltd
Transplanted tumor model is named as PDX002# (MM002), PDX006# (MM006), PDX010# (MM0010), PDX011#
(MM011), PDX014# (MM0014) is numbered on the tumor inoculation date with patient.After transplanting successfully, when gross tumor volume reaches
Average 200-300mm3When (every group of 5-15 mouse), the mouse of load transplantable tumor is randomly divided into control group and test group.It is right
According to group and test group give respectively 28 days processing plans physiological saline (oral, daily) and Pabuk former times benefit cloth (30,60,90,
120mg/kg takes orally, daily).After measuring transplantable tumor volume 2 times, medication 28 days weekly, nude mice is anaesthetized, is taken pictures, by tumor group
Taking-up is knitted, is operated on ice, tumour is divided into multiple fritters, a part embedding, a part freezes in liquid nitrogen.Gross tumor volume=L
×W2× 0.5, wherein L represents maximum gauge, and W represents tumour minimum diameter.Tumor growth inhibition (tumor growth
Inhibition, TGI) TGI=[1- (TVf, treated-TVi, treated)/(TVf, control-TVi, control)] ×
100%, wherein TVf represents tumor average volume in experimental endpoints group, and TVi represents tumor average volume in experiment starting point group.
As a result, it has been found that the Pabuk former times benefit cloth of 60-90mg/kg/d is shown significantly in PDX011# and PDX014# model
Anti-tumor effect (TGI=46.02%-76.08% (60-90mg/kg/d), 62.21% (90mg/kg/d), P < 0.01), and
Mouse model has been resistant to drug well.(weight changes < 15% in 30 days processing time).
In order to further supplement anti-tumor effect of the Pabuk former times benefit cloth in PDX011# model, will extend to treatment time
53 days, it is found that in PDX011# model, 60.30% when the TGI level that drug concentration relies on is from 60mg/kg is risen to
The 87.05% of 90mg/kg.More importantly.Using the mouse model for the Pabuk former times benefit cloth that concentration is 90mg/kg or 120mg/kg
Apparent toxic effect is not observed, shows tumor suppression continual and steady in 53 days drug exposure times sections instead and makees
With.(see Fig. 1)
In order to compare Pabuk former times benefit cloth and Imatinib (in U.S.'s NCCN guide, to there are the MM of KIT gene mutation recommendations
Unique target therapeutic agent) Different therapeutical effect in PDX model, be arranged simultaneously in the PDX014# model there are KIT gene
Imatinib and Pabuk former times benefit cloth are individually and drug combination curative effect evaluation.Pabuk former times benefit cloth is shown compared with Imatinib as the result is shown
Higher tumor inhibitory effect (TGI=62.21%vs TGI=TGI=25.57%), it is notable that Pabuk former times benefit
The most significantly collaboration tumor inhibitory effect (TGI=82.14%) that the drug combination of cloth and Imatinib are shown, is shown in Fig. 2.
3 genome sequencing of embodiment
Genome sequencing is carried out to the five plants of cells filtered out in embodiment 1, sequencing finds these mucous membrane malignas
In plain tumor, CDK4 and TERT, MDM2, FRS2, KIT, BRAF, the well-known tumour driving gene such as CCND1, which exists, obviously to be expanded.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (9)
1. the purposes of Pabuk former times benefit cloth or its pharmaceutically acceptable salt, which is characterized in that it is pernicious black to be used to prepare treatment mucous membrane
The pharmaceutical composition of melanoma.
2. purposes as described in claim 1, which is characterized in that described pharmaceutical composition contains the Pabuk former times benefit of safe and effective amount
Cloth and pharmaceutically acceptable carrier.
3. purposes as described in claim 1, which is characterized in that described pharmaceutical composition also contains tumor therapeutic agent.
4. purposes as described in claim 1, which is characterized in that the safe and effective amount refers to 50-1000mg/kg.
5. purposes as described in claim 1, which is characterized in that the mucous membrane malignant mela noma includes deriving from the upper respiratory tract
The mucous membrane malignant mela noma of mucous membrane.
6. purposes as claimed in claim 5, which is characterized in that the upper respiratory tract mucous membrane include oral cavity, lip, nasopharynx or
The mucous membrane at oropharynx position.
7. purposes as described in claim 1, which is characterized in that the mucous membrane malignant mela noma derives from people.
8. a kind of method that external non-therapeutic inhibits mucous membrane malignant mela noma, which is characterized in that comprising steps of to containing glutinous
Pabuk former times benefit cloth or its pharmaceutically acceptable salt or the medicine containing it are added in the cell culture system of film malignant mela noma
Compositions, to inhibit mucous membrane malignant melanoma cell.
9. method according to claim 8, which is characterized in that the concentration of the Pabuk former times benefit cloth is 0.1uM-100 μM.
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