CN104784105A - Gel compound of monoclonal antibody drugs - Google Patents
Gel compound of monoclonal antibody drugs Download PDFInfo
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- CN104784105A CN104784105A CN201510185504.XA CN201510185504A CN104784105A CN 104784105 A CN104784105 A CN 104784105A CN 201510185504 A CN201510185504 A CN 201510185504A CN 104784105 A CN104784105 A CN 104784105A
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Abstract
The invention relates to a gel compound of monoclonal antibody drugs, which can be provided for patients to perform intradermal injection, surrounding-tumor injection or intratumor injection on clinic, and belongs to the technical field of medicinal preparation. The gel compound of the monoclonal antibody drugs comprises monoclonal antibodies, at least one kind of extracellular matrix degrading enzymes, such as collagenase or hyaluronidase, a temperature-sensitive gel material, menstruum, a buffering agent, a stabilizing agent, a nonionic surfactant and an osmotic regulating agent. The invention further provides a method and uses of the gel compound of the monoclonal antibody drugs. The gel compound of the monoclonal antibody drugs, which is disclosed by the invention, has the effects of prolonging the release of the monoclonal antibodies, and promoting the tissue osmosis of monoclonal antibodies or the osmosis of the monoclonal antibodies into tumors; the gel compound of the monoclonal antibody drugs has the advantage of reducing drug administration times and enhancing treatment effects.
Description
Technical field
The present invention relates to one and comprise monoclonal antibody gel combination, belong to technical field of medicine.
Background technology
Along with the development of biotechnology, take monoclonal antibody as the visual field that the bio-pharmaceutical of representative comes into people gradually, day by day become the main flow in treatment malignant tumor, autoimmune pathologies.Monoclonal antibody drug has high degree of specificity because of it when playing a role, and makes it can overcome common chemotherapy drug selectivity poor, the shortcomings such as toxicity is large.
In malignant tumor and autoimmune pathologies treatment, monoclonal antibody drug antigenic specificity corresponding with cell surface combines, affinity is high, mechanism of action is complicated, single or number of ways combined effect, reach the object of killing tumor cell, comprising 1. antibody direct effects: receptor blocking, agonist suppress, mediation autophagy.2. immune-mediated is killed and wounded: complement dependent cytotoxicity, antibody-dependent cytotoxicity.3. specific effect is in blood vessel and substrate.
Based on These characteristics, in malignant tumor field, compared with common chemotherapeutics, monoclonal antibody drug can killing tumor cell more exactly, drastically increases therapeutic effect, and decreases untoward reaction, thus extend the survival of patients time, quality of making the life better.
Nowadays, while making great progress, monoclonal antibody drug also also exists unavoidable shortcoming.Because it is the essence of protein drug, make monoclonal antibody drug difficulty in the processes such as extraction, purification comparatively large, less stable in storage, use procedure, biological activity is easily influenced, causes patient's use cost too high, and the accessibility of monoclonal antibody drug is low.Because of its protein essence, cause monoclonal antibody drug in oral process, very easily by gastrointestinal proteasome degradation, in addition its biomacromolecule (molecular weight is up to 150000Da) characteristic, be difficult to cross over the biological barriers such as small intestine epithelium, monoclonal antibody drug can not be orally used.In Clinical practice process, monoclonal antibody drug administering mode mostly is single intravenous drip, in instillation process, along with serious " infusion reaction ", and long-time use may be needed, such as: in Her-2 positive breast cancer therapeutic process, need patient weekly or intravenous drip in every three weeks once, treatment time reaches 52 weeks, brings great pain to patient, and compliance is poor.
The new route of administration of monoclonal antibody drug is also being explored as far as possible by pharmaceutical manufacturer.Publication number be US 20110044977 U.S. patents disclose a kind of intradermal administration mode for the treatment of Her-2 positive breast carcinoma monoclonal antibody, by restructuring hyaluronidase and Qu Tuozhu monoclonal antibody conbined usage, the reversible degradation skin hyaluronic acid that restructuring hyaluronidase is special, make Herceptin can enter body circulation smoothly, play therapeutical effect, reduce the toxic and side effects be administered systemically.Utilize this administering drug combinations technology, the Qu Tuozhu monoclonal antibody of percutaneous dosing and Rituximab have obtained the approval of European drug administration.
In malignant tumor and other diseased tissue structures, extracellular matrix-cell has all played great function.Wherein, in extracellular matrix, collagen protein and hyaluronic acid be two kinds most important be also the constituent that content is maximum.In malignant tumor, fibrous two kinds of albumen can increase the compactness of tumor stroma, increase the interstitial pressure of tumor tissues, thus can enter tumor deep from tumor outside by blocking treatment medicine, destroy the therapeutical effect of medicine.Specific degraded tumor stroma can promote that medicine enters inside tumor effect, can further improve the therapeutic effect of medicine effectively.Mako Kato etc. report that intravenous injection collagenase can reduce mesenchyma stroma of tumors pressure, improve accumulation (the Kato et al of cationic-liposome in tumor, Collagenase-1injection improved tumor distribution and gene expression of cationic lipoplex, Int J Pharm.2012 Feb 28; 423 (2): 428-34).
Temperature-sensitive gel is by the molecular gel rubber system of amphipathic nature polyalcohol high score.Time low temperature (lower than critical gelation temperature), between polymeric, spacing is compared with large and active force is little, and water-wet side and hydrone active force are predominant intermolecular forces, make its form that is in a liquid state.Time high temperature (higher than critical gelation temperature), between polymeric, spacing reduces, and amphipathic block presents micelle form, and micelle is compressed further, and micelle intermolecular hydrophobic kernel active force is sharply strengthened, and makes it present solid-state form.Under the characteristic of polymeric creates its different temperatures, the characteristic of phase in version.
After thermosensitive in situ gel injection injects human body in liquid form, phase in version is there is rapidly in injection site, form solid gel, after bag medicine carrying thing, can reach topical effect, prolong drug, in the holdup time of agents area, realizes good slow releasing function, reach a shot, the effect of long-time treatment.Publication number is that the Chinese patent of CN 103239389 discloses a kind of Progesterone thermosensitive in situ gel based on poloxamer188, is used as the treatment of threatened abortion.
Based on above technical background, the object of the present invention is to provide a kind of gel combination comprising monoclonal antibody drug and extracellular matrix degrading enzymes, selective degradation matrix organization, strengthen medicine to permeate to deep, strengthen therapeutical effect, single-dose, the therapeutic effect of long-time onset.
Summary of the invention
The object of the present invention is to provide a kind of monoclonal antibody gel combination.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of monoclonal antibody gel combination, described monoclonal antibody gel combination comprises: monoclonal antibody, temperature-sensitive material, extracellular matrix degrading enzymes and solvent.
Monoclonal antibody gel combination provided by the present invention, wherein said extracellular matrix degrading enzymes comprise in collagenase, hyaluronidase, Bacillus polymyxa Neutral proteinase one or more.
Monoclonal antibody gel combination provided by the present invention, wherein said temperature-sensitive material comprises poloxamer, PLA-PEG-PLA, poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, polyethylene glycol-polylactic acid-Polyethylene Glycol, polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide-Polyethylene Glycol, PCL-PEG-PCL, NIPA polymer and Chitosan-phospholipid complex and neutralizes one or more.
Monoclonal antibody gel combination provided by the present invention, wherein said monoclonal antibody comprises: one or more in Herceptin, Cetuximab, Avastin, Rituximab, handkerchief trastuzumab, Buddhist nun's trastuzumab, infliximab, adalimumab, gemtuzumab, her monoclonal antibody.
Monoclonal antibody gel combination provided by the present invention, further preferably, described monoclonal antibody is Herceptin.
Monoclonal antibody gel combination provided by the present invention, further preferably, described temperature-sensitive material is poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, wherein the mol ratio of poly (glycolide-co-lactide) and Polyethylene Glycol is 3:1, molecular weight polyethylene glycol is 1500Da, and poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide total molecular weight is 5000Da.
Monoclonal antibody gel combination provided by the present invention, further preferably, described temperature-sensitive material is poloxamer188 or PLURONICS F87 or their mixture.
Monoclonal antibody gel combination provided by the present invention, further preferably, described extracellular matrix degrading enzymes is I-collagenase.
Monoclonal antibody gel combination provided by the present invention, described lyase is water, normal saline, the one in 5% D/W.
Monoclonal antibody gel combination provided by the present invention, wherein said compositions also comprises buffer agent, and buffer agent is the one in phosphate buffer, histidine hydrochloride buffer, and its pH is 5.5-7.0, in faintly acid, more preferably histidine hydrochloride buffer.
Monoclonal antibody gel combination provided by the present invention, wherein said compositions also comprises stabilizing agent, and its stabilizing agent is the one of lactose, trehalose, galactose kind, more preferably trehalose.
Monoclonal antibody gel combination provided by the present invention, wherein said compositions also comprises and non-ionic surface active agent, and its non-ionic surface active agent is the one in polysorbas20 or Tween 80, more preferably polysorbas20.
Monoclonal antibody gel combination provided by the present invention, wherein the weight ratio of monoclonal antibody and extracellular matrix degrading enzymes is 1:40-40:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-2:1.
Monoclonal antibody gel combination provided by the present invention, wherein the weight ratio of monoclonal antibody and extracellular matrix degrading enzymes is 1:20-20:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-1:1.
Further preferably, monoclonal antibody drug gel combination provided by the invention, wherein, the weight of described monoclonal antibody drug and extracellular matrix degrading enzymes accounts for thermo-sensitive gel composition percentage by weight and is:
A.0.01%-5% monoclonal antibody drug,
B.0.01%-5% extracellular matrix degrading enzymes.
Monoclonal antibody drug gel combination provided by the invention, the content of various composition is:
Wherein, described percentage ratio is weight percentage.
Preferred further, the content of various composition is
Monoclonal antibody drug gel combination provided by the invention, the content of various composition is:
Monoclonal antibody gel combination of the present invention, its preparation method is as follows:
A. solvent and temperature-sensitive material is selected to prepare blank thermosensitive in situ gel;
B. blank thermosensitive in situ gel and monoclonal antibody and extracellular matrix degrading enzymes are fully mixed, dissolve, obtain monoclonal antibody gel combination.
Preferably, monoclonal antibody gel combination of the present invention, its preparation method is as follows:
A. take 250mg poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, add 1000mg water, slowly stir under ice bath and obtain blank thermosensitive in situ gel.
B. take collagenase 20mg, Herceptin 7.5mg, add blank thermosensitive in situ gel, under ice bath, slowly stir, obtain monoclonal antibody drug and tumor stroma specific drug gel preparation.
The gel preparation comprising monoclonal antibody drug and tumor stroma specific drug provided by the invention, wherein, can be used for intratumor injection, the injection of tumor week, intradermal injection.
A kind of monoclonal antibody drug gel combination provided by the invention has the following advantages:
1. reduce the frequency of utilization of monoclonal antibody drug, instil with clinical vein and contrast, preparation in vivo the holdup time long, single administration, long-time onset, raising patient compliance.
2. strengthen monoclonal antibody drug curative effect, compared with using monoclonal antibody drug, separately monoclonal antibody drug thermosensitive in situ gel, separate cell epimatrix digestive enzyme thermosensitive in situ gel with intravenous injection, the gel preparation comprising monoclonal antibody drug and extracellular matrix degrading enzymes significantly can strengthen the therapeutical effect of monoclonal antibody.
Through research, we find, present stage, and the pharmaceutical preparation research for monoclonal antibody drug is less, are that its biodegradability of thermosensitive in situ gel and the safety of representative limits its application with PNIPAAm.
The present invention is directed to the shortcoming that existing gel preparation safety is not good, adopt safety and the good gel rubber material of biocompatibility, ensure its safety.Share with extracellular matrix degrading enzymes, strengthen its therapeutic effect.
Tumor extracellular matrix primarily of collagen protein formation can increase the compactness of tumor stroma, causes medicine to be difficult to cross over substrate and enters tumor deep, reduce therapeutic effect.By tumor extracellular matrix digestive enzyme of the present invention and monoclonal antibody conbined usage, tumor extracellular matrix digestive enzyme selective degradation tumor extracellular matrix, what increase monoclonal antibody enters tumor dose, strengthens its therapeutic effect, produces good synergism.
In the present invention, the ratio of monoclonal antibody drug is controlled at 0.01%-5%, under the prerequisite strengthening therapeutic effect can be ensured again, reduce the use amount of expensive monoclonal antibody to greatest extent, reduce patient burden.
Accompanying drawing illustrates:
Bovine serum albumin thermosensitive in situ gel In-vitro release curves is comprised in Fig. 1 embodiment 14.
The gel preparation of monoclonal antibody drug and extracellular matrix degrading enzymes is comprised at body pharmacodynamic study tumor growth curve in Fig. 2 embodiment 15.
The gel preparation of monoclonal antibody drug and extracellular matrix degrading enzymes is comprised at body pharmacodynamic study body weight change curve in Fig. 3 embodiment 15.
The gel preparation of monoclonal antibody drug and extracellular matrix degrading enzymes is comprised at body pharmacodynamic study tumor biopsy apoptosis fluorescence intensity figure in Fig. 4 embodiment 15.
The gel preparation of monoclonal antibody drug and extracellular matrix degrading enzymes is comprised at body pharmacodynamic study tumor biopsy collagen protein fluorescence intensity figure in Fig. 5 embodiment 15.
Detailed description of the invention:
Further illustrate below by way of specific embodiment and explain the present invention, but not as restriction of the present invention.
Embodiment 1, comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel: precision takes Herceptin 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising Herceptin and collagenase thermosensitive in situ gel preparation.
Embodiment 2, comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poloxamer188 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtains blank thermosensitive in situ gel.
2. comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel: precision takes Herceptin 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising Herceptin and I-collagenase thermosensitive in situ gel preparation.
Embodiment 3, comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel: precision takes Trastuzumab 1mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising Herceptin and I-collagenase thermosensitive in situ gel preparation.
Embodiment 4, comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Herceptin and I-collagenase thermosensitive in situ gel: precision takes Herceptin 1600mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising Herceptin and I-collagenase thermosensitive in situ gel preparation.
Embodiment 5, comprise the preparation of Herceptin and hyaluronidase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Herceptin and hyaluronidase thermosensitive in situ gel: precision takes Herceptin 15mg, hyaluronidase (205u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising Herceptin and I-collagenase thermosensitive in situ gel preparation.
Embodiment 6, comprise the preparation of Rituximab and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Cetuximab and collagenase thermosensitive in situ gel: precision takes rituximab monoclonal antibody 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising Rituximab and I-collagenase thermosensitive in situ gel preparation.
Embodiment 7, comprise the preparation of handkerchief trastuzumab and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Avastin and collagenase thermosensitive in situ gel: precision takes handkerchief trastuzumab 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising handkerchief trastuzumab and I-collagenase thermosensitive in situ gel preparation.
Embodiment 8, comprise the preparation of Buddhist nun's trastuzumab and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Rituximab and collagenase thermosensitive in situ gel: precision takes Buddhist nun's trastuzumab 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising Buddhist nun's trastuzumab and I-collagenase thermosensitive in situ gel preparation.
Embodiment 9, comprise the preparation of infliximab and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of handkerchief trastuzumab and collagenase thermosensitive in situ gel: precision takes infliximab 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising infliximab and I-collagenase thermosensitive in situ gel preparation.
Embodiment 10, comprise the preparation of adalimumab and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of Buddhist nun's trastuzumab and collagenase thermosensitive in situ gel: precision takes adalimumab 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising adalimumab and I-collagenase thermosensitive in situ gel preparation.
Embodiment 11, comprise the preparation of gemtuzumab and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of gemtuzumab and collagenase thermosensitive in situ gel: precision takes handkerchief trastuzumab 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising gemtuzumab and I-collagenase thermosensitive in situ gel preparation.
Embodiment 12, comprise the preparation of her monoclonal antibody and I-collagenase thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of her monoclonal antibody and collagenase thermosensitive in situ gel: precision takes her a monoclonal antibody 15mg, I-collagenase (201u/mg) 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains comprising her monoclonal antibody and I-collagenase thermosensitive in situ gel preparation.
Embodiment 13, comprise the preparation of bovine serum albumin thermosensitive in situ gel
1. blank thermosensitive in situ gel preparation: precision takes poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide 500mg, adds 2000mg deionized water, slowly stirs 48h under ice bath, until completely dissolved, obtain blank thermosensitive in situ gel.
2. comprise the preparation of bovine serum albumin thermosensitive in situ gel: accurate bovine serum albumin 40mg, slowly rock mixing, add the blank thermosensitive in situ gel of above-mentioned preparation, slowly stir 10 minutes under ice bath, mix homogeneously, obtains the thermosensitive in situ gel preparation comprising bovine serum albumin.
Embodiment 14, comprise bovine serum albumin thermosensitive in situ gel In-vitro release curves
That to get in 2ml Samples EXAMPLE 13 preparation comprises bovine serum albumin thermosensitive in situ gel, under 4 degree, bovine serum albumin thermosensitive in situ gel liquid 300 microlitre will be comprised and be added to Transwell (Corning, 24 millimeters, 3 microns) in cell, be placed in rapidly in the gas bath isothermal vibration device (ZHWY-100C, intelligence city, Shanghai analytical tool) being set to 37 degree in advance, form solid gel.By being placed in the 50ml centrifuge tube (Corning) containing 10ml 37 degree of deionized waters containing solid gel Transwell cell, in gas bath isothermal vibration device, 37 degree, 100 revs/min, interval time samples, and adds the blank release medium of same volume.The release liquid taken out, according to BCA protein quantification test kit (ancient cooking vessel state is prosperous) rule of operation, carries out assay, and calculates the accumulation drug release total amount of each time point.
Release profiles is shown in Fig. 1.Result is visible, and the thermosensitive in situ gel that the present invention relates to bovine serum albumin cumulative release amount of 9 days under 37 degree is less than 80%.
Embodiment 15, comprise the gel preparation of monoclonal antibody and extracellular matrix degrading enzymes at body pharmacodynamic study
Choose female Nu/nu nude mice (18-22g, Beijing dimension tonneau China laboratory animal), in right side axil intradermal vaccination 1 × 10
6breast carcinoma BT474 cell, inoculate administration 150 microlitre after 15 days, according to pharmaceutical compositions in specific embodiment 1, administration component is 6 groups, often organize 7, be respectively: matched group, Blank gel group, Herceptin gel group, single intravenous group, vein be group, collagenase Herceptin gel group repeatedly.
Matched group: tumor-side injection, single-dose;
Blank gel group: tumor-side injection, single-dose;
Trastuzumab gel group: tumor-side injection, single-dose, accumulated dose 30mg/kg;
Single intravenous group: tail vein injection, single-dose, accumulated dose 30mg/kg;
Vein is group repeatedly: tail vein injection, Per-Hop behavior once, totally four times, accumulated dose 30mg/kg;
Collagenase Herceptin gel group: tumor-side injection, single-dose, accumulated dose Herceptin 30mg/kg, collagenase 15mg/kg
(1) tumor growth curve: use major diameter (L) minor axis (r) of vernier caliper measurement group nude mouse tumor in after administration every 5 days, calculate gross tumor volume V (V=[L × (r)
2]/2), draw tumor growth curve, the results are shown in Figure 3.Fig. 3 result shows, and Blank gel does not have Tumor suppression growth result, and collagenase Herceptin gel group drug effect is best, and Herceptin gel takes second place, but is better than intravenously administrable group.
(2) body weight change curve: after administration, every 5 angels weigh with scale nude mice body weight.As the display of Fig. 3 result, in each group, nude mice body weight change is not obvious, does not have significant difference, illustrates that preliminary safety is good.
(3) tumor tissues apoptosis experiment (TUNEL): after administration terminates observation, often group picks out a nude mice, tumor is cut and prepares frozen section, other each tissue preparation paraffin sections.Tumor frozen section carries out apoptosis dyeing with reference to TUNEL test kit to section, uses confocal microscopy fluorescence, and adds up fluorescence intensity, the results are shown in Figure 4.Fluorescence intensity is larger, shows that the apoptotic cell quantity in tumor tissues is more, also represents that drug effect is better from another one side.Fig. 4 result shows, and collagenase Herceptin gel group fluorescence intensity is the strongest, illustrates that its apoptosis is maximum, consistent with pharmacodynamic results.
(4) tumor tissues collagen protein imaging: after administration terminates observation, often group picks out a nude mice, tumor is cut and prepares frozen section, other each tissue preparation paraffin sections.Tumor frozen section collagen antibody labelling, confocal microscopy collagen protein changes, and the results are shown in Figure 5.Fluorescence intensity is larger, shows that tumor tissues collagen content is more, undesirable from another one side illustration pharmacodynamic results.Fig. 5 result shows, and contained by the bent appropriate pearl of collagenase single gram of anti-gel group, collagen protein is minimum, and illustrate that collagenase is by collagen degradation in tumor tissues, facilitate monoclonal antibody drug and enter tumor tissues deep, drug effect is best.
Claims (10)
1. a monoclonal antibody gel combination, comprise: monoclonal antibody, temperature-sensitive material, extracellular matrix degrading enzymes and solvent, the weight ratio of monoclonal antibody and extracellular matrix degrading enzymes is 1:40-40:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-2:1.
2. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, the weight ratio of monoclonal antibody and extracellular matrix degrading enzymes is 1:20-20:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-1:1.
3. gel combination as claimed in claim 1, it is characterized in that, the percentage by weight that monoclonal antibody and extracellular matrix degrading enzymes account for monoclonal antibody gel combination is respectively: 0.01%-5% and 0.01%-5%.
4. gel combination as claimed in claim 1, it is characterized in that, the amount ratio of various composition is as follows:
5. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, described monoclonal antibody, comprising: one or more in Herceptin, Cetuximab, Avastin, Rituximab, handkerchief trastuzumab, Buddhist nun's trastuzumab, infliximab, adalimumab, gemtuzumab, her monoclonal antibody; Be preferably Herceptin;
Described temperature-sensitive material, comprise: poloxamer, PLA-PEG-PLA, poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, polyethylene glycol-polylactic acid-Polyethylene Glycol, polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide-Polyethylene Glycol, PCL-PEG-PCL, NIPA polymer and Chitosan-phospholipid complex neutralize one or more, be preferably poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, poloxamer188.
6. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, described extracellular matrix degrading enzymes, comprising: one or more in collagenase, hyaluronidase, Bacillus polymyxa Neutral proteinase, is preferably collagenase,
Described lyase is selected from: water, normal saline, the one in 5% D/W.
7. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, described compositions also comprises: buffer agent, stabilizing agent and non-ionic surface active agent.
8. the preparation method of monoclonal antibody gel combination according to claim 1, comprises the steps:
A. solvent and temperature-sensitive material is selected to prepare blank thermosensitive in situ gel;
B. blank thermosensitive in situ gel and monoclonal antibody and extracellular matrix degrading enzymes are fully mixed, dissolve, obtain monoclonal antibody gel combination.
9. the preparation method of monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, step is as follows:
A. take 250mg poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, add 1000mg water, under ice bath, be slowly stirred to abundant dissolving, obtain blank thermosensitive in situ gel;
B. take collagenase 20mg, Herceptin 7.5mg, join blank thermosensitive in situ gel respectively, under ice bath, slowly stir and collagenase and the appropriate pearl Dan Kekang of song are fully dissolved, obtain monoclonal antibody gel combination.
10. monoclonal antibody gel combination as claimed in claim 1, is characterized in that, in tumor, tumor week or intradermal injection.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107964535A (en) * | 2016-12-23 | 2018-04-27 | 浙江海隆生物科技有限公司 | Screening method and application of CHO monoclonal cell strain |
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| CN110638745A (en) * | 2019-06-27 | 2020-01-03 | 复旦大学 | HER-2 humanized monoclonal antibody long-acting sustained-release preparation and preparation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107964535A (en) * | 2016-12-23 | 2018-04-27 | 浙江海隆生物科技有限公司 | Screening method and application of CHO monoclonal cell strain |
| CN110167594A (en) * | 2017-01-17 | 2019-08-23 | 基因泰克公司 | Subcutaneous HER2 antibody formulations |
| US11654105B2 (en) | 2017-01-17 | 2023-05-23 | Genentech, Inc. | Subcutaneous HER2 antibody formulations |
| CN110167594B (en) * | 2017-01-17 | 2023-11-21 | 豪夫迈·罗氏有限公司 | Subcutaneous HER2 antibody formulations |
| CN110404077A (en) * | 2018-04-28 | 2019-11-05 | 苏州康聚生物科技有限公司 | Tumour ECM degradation and/or inhibitor and its complete kit and application |
| CN110404077B (en) * | 2018-04-28 | 2024-02-02 | 苏州康聚生物科技有限公司 | Tumor ECM degradation and/or inhibitor, kit and application thereof |
| CN109364253A (en) * | 2018-11-21 | 2019-02-22 | 南开大学 | One kind is for improving infiltrative nanoparticle of tumor tissues and its preparation method and application |
| CN110638745A (en) * | 2019-06-27 | 2020-01-03 | 复旦大学 | HER-2 humanized monoclonal antibody long-acting sustained-release preparation and preparation method thereof |
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